U.S. patent application number 16/585789 was filed with the patent office on 2020-04-02 for system and method useful for sarcomere imaging via objective-based microscopy.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Scott L. Delp, Michael E. Llewellyn, Gabriel Nestor Sanchez, Mark J. Schnitzer.
Application Number | 20200100659 16/585789 |
Document ID | / |
Family ID | 40222018 |
Filed Date | 2020-04-02 |
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United States Patent
Application |
20200100659 |
Kind Code |
A1 |
Sanchez; Gabriel Nestor ; et
al. |
April 2, 2020 |
SYSTEM AND METHOD USEFUL FOR SARCOMERE IMAGING VIA OBJECTIVE-BASED
MICROSCOPY
Abstract
Biological tissue such as skeletal and cardiac muscle can be
imaged by using an objective-based probe in the tissue and scanning
at a sufficiently fast rate to mitigate motion artifacts due to
physiological motion. According to one example embodiment, such a
probe is part of a system that is capable of reverse-direction
high-resolution imaging without needing to stain or otherwise
introduce a foreign element used to generate or otherwise increase
the sensed light. The probe can include a light generator for
generating light pulses that are directed towards structures
located within the thick tissue. The system can additionally
include aspects that lessen adverse image-quality degradation.
Further, the system can additionally be constructed as a hand-held
device.
Inventors: |
Sanchez; Gabriel Nestor;
(Stanford, CA) ; Delp; Scott L.; (Stanford,
CA) ; Schnitzer; Mark J.; (Palo Alto, CA) ;
Llewellyn; Michael E.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
40222018 |
Appl. No.: |
16/585789 |
Filed: |
September 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14546085 |
Nov 18, 2014 |
10499797 |
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16585789 |
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13305390 |
Nov 28, 2011 |
8897858 |
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14546085 |
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12165977 |
Jul 1, 2008 |
8068899 |
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13305390 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 5/4519 20130101;
A61B 5/0084 20130101; A61B 1/00172 20130101; A61B 5/726 20130101;
G01N 21/6458 20130101; A61B 5/0071 20130101; A61B 5/0086 20130101;
A61B 5/0068 20130101; A61B 5/0062 20130101; A61B 1/043 20130101;
A61B 1/313 20130101; G01N 21/6486 20130101; A61B 1/0638 20130101;
A61B 1/00165 20130101; A61B 5/7207 20130101; A61B 1/00045 20130101;
A61B 5/6848 20130101; A61B 5/0044 20130101; A61B 5/4528
20130101 |
International
Class: |
A61B 1/00 20060101
A61B001/00; A61B 5/00 20060101 A61B005/00; A61B 1/313 20060101
A61B001/313 |
Claims
1. A method for analyzing a biological tissue of a subject,
comprising: (a) providing at least one light source in optical
communication with said biological tissue; (b) using said at least
one light source to deliver light pulses to said biological tissue
to cause one or more signals intrinsic to a property of said
biological tissue to be generated, wherein during delivery of said
light pulses to said biological tissue, an optical unit comprising
an afocal lens arrangement shifts a focal plane of said light
pulses in said biological tissue; and (c) collecting at least a
subset of said one or more signals intrinsic to said property of
said biological tissue.
2. The method of claim 1, further comprising using a scanning
mirror to provide line scanning of said light pulses.
3. The method of claim 2, wherein said afocal lens arrangement is
disposed between said at least one light source and said scanning
mirror.
4. The method of claim 1, wherein said afocal lens arrangement is
disposed between said at least one light source and an optical
probe that focuses said light pulses.
5. The method of claim 4, further comprising maintaining a
resolution of said one or more signals during focal plane changes
by providing light pulses that overfill a back aperture of said
optical probe.
6. The method of claim 4, wherein said optical probe comprises an
objective, and wherein said objective focuses said light pulses at
said biological tissue.
7. The method of claim 6, wherein said objective is
telecentric.
8. The method of claim 6, wherein said objective provides
substantially constant magnification of said light pulses during
focal plane changes.
9. The method of claim 6, wherein said objective is not translated
for focusing said light pulses.
10. The method of claim 1, wherein said afocal lens arrangement
maintains constant power of said light pulses generated from said
at least one light source to maintain a maximum resolution of said
light pulses.
11. The method of claim 1, wherein said afocal lens arrangement
comprises a mobile lens that shifts said focal plane in said
biological tissue.
12. The method of claim 11, wherein said afocal lens arrangement
further comprises a fixed lens that maintains a substantially
constant beam waist of said light pulses leaving said afocal lens
arrangement.
13. The method of claim 12, wherein a scanning mirror is disposed
in a focal plane of said fixed lens, and wherein said scanning
mirror provides line scanning of said light pulses.
14. The method of claim 1, wherein said afocal lens arrangement
provides convergence or divergence to said light pulses, thereby
shifting said focal plane in said biological tissue.
15. The method of claim 1, further comprising using said at least
subset of said one or more signals to generate an image of said
biological tissue.
16. The method of claim 1, wherein said one or more signals
comprise second-harmonic generation signals (SHG) or
autofluorescence signals.
17. The method of claim 16, further comprising using said SHG or
autofluorescence signals to generate an image of said biological
tissue.
18. A method for analyzing a biological tissue of a subject,
comprising: (a) providing at least one light source in optical
communication with said biological tissue; (b) using said at least
one light source to deliver light pulses to said biological tissue
to cause one or more signals intrinsic to a property of said
biological tissue to be generated, wherein said light pulses are
delivered to said biological tissue at a line resolution rate
sufficient to reduce motion artifacts; and (c) collecting at least
a subset of said one or more signals intrinsic to said property of
said biological tissue.
19. The method of claim 18, wherein said one or more signals
comprise second-harmonic generation signals (SHG) or
autofluorescence signals.
20. The method of claim 18, wherein said line resolution rate is at
least about 1 kiloHertz (kHz).
Description
RELATED PATENT DOCUMENTS
[0001] This patent document is a continuation under 35 U.S.C.
.sctn. 120 of U.S. patent application Ser. No. 14/546,085 filed on
Nov. 18, 2014, which is a divisional of U.S. patent application
Ser. No. 13/305,390 filed on Nov. 28, 2011 (U.S. Pat. No.
8,897,858), which is a continuation-in-part of U.S. patent
application Ser. No. 12/165,977 filed on Jul. 1, 2008 (U.S. Pat.
No. 8,068,899), which claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Patent Application Ser. No. 60/947,769
filed on Jul. 3, 2007; these patent documents, including the
Appendix filed in the underlying provisional patent application,
are fully incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to biomedical
cellular-level imaging systems and methods and more specifically to
minimally-invasive systems and methods for characterizing
biological thick tissue as a function of properties that are
intrinsic to the tissue.
BACKGROUND
[0003] Biomedical-engineering advancements have provided a variety
of tools to explore the detailed structure and behavior of
biological tissues. Traditional equipment in this area has provided
images and other data by way of x-rays, sound waves, and visible
and infrared (IR) light to characterize the structure and behavior
of certain tissues. Although generally successful, the image
quality provided by such conventional equipment is limited and not
applicable to all types of biological tissues. As examples, x-ray
equipment typically transmits relatively low-level radiation and is
used to characterize the location of the tissue as a function of
its periphery, and visible/IR light imaging tools are used for
characterizing transparent and semi-transparent tissue but are
ineffective for imaging optically-dense ("thick") tissue.
[0004] Conventional approaches for high-resolution images of thick
tissue have not been widely implemented due to approach-specific
issues. Generally, these approaches can be categorized as
"transmission-mode" (a.k.a., "forward-direction") systems and
"reverse-direction" systems. Transmission-mode systems radiate
energy at the tissue from one side and use a nearby sensing device
on the opposite side of the tissue to sense the radiated energy
after it is impacted by the tissue. One form of forward-direction
imaging relies on SHG (second harmonic generation) which is known
to be a forward-directed nonlinear optical process. In SHG, a light
source directs photons at a target material for interacting and
combining into higher-energy photons. The higher-energy photons are
predominantly forwardly-directed at a sensing device on the
opposite side of the tissue. While useful for many in vitro
applications, this transmission-mode approach can be extremely
invasive due to the need for a sensing device on the opposite side
of the tissue. In more tissue-sensitive applications such as in
vivo examinations and in vitro investigations where the integrity
of the tissue is to be maintained after examination, this approach
would be unacceptable due to the placement of the sensing device
deep within the subject under examination.
[0005] Reverse-direction systems radiate energy at the tissue from
one side and use a sensing device on the same side of the tissue to
sense energy radiated in response. Unlike transmission-mode
systems, these systems do not require placement of a sensing device
on the opposite side of the tissue and therefore could be
considered less invasive for in vivo applications. For
high-resolution imaging of thick tissue, however, these systems
require relatively strong signals and can require pre-treatment of
the tissue with a foreign matter (e.g., dye, exogenous gene or
protein) in order to enhance signals responding to excitation of
the tissue by light. Such pre-treatment is undesirable for reasons
concerning the invasiveness of the foreign matter and its
alteration of the cells under examination.
[0006] Recent attempts to use reverse-direction systems have not
been widely adopted. These attempts have relied on back-directed
SHG or on endogenous (or native) fluorescence for tissue
characterization for a variety of reasons. These approaches are
burdened by insufficient signal strengths and/or the need to
physically mitigate physiological motions associated with blood
flow and respiratory activity. For imaging skeletal and/or cardiac
muscle tissues, motions associated with sarcomere contractions
further perturb image quality.
SUMMARY
[0007] The present invention is directed to methods for using and
arrangements involving an optical probe for characterizing
biological thick tissue. Certain applications of the present
invention are directed to overcoming the above-mentioned
limitations and addressing other issues as may become apparent in
view of the description herein.
[0008] The present invention provides significant bio-medical
high-resolution imaging advancements with minimally-invasive
optical-probe implementations that produce high-resolution images
of biological thick tissues using predominantly intrinsic
biocellular sources. One example embodiment of the present
invention uses a microendoscopic probe inserted, like a needle, as
part of a minimally-invasive imaging procedure for stimulating
structures intrinsic to the thick tissue. The probe is also used to
collect the resulting signal for characterization of the tissue
structure. The optical probe scans the thick tissue at a line
resolution rate that is sufficiently-fast to mitigate motion
artifacts due to contractile motion and/or physiological motion. In
this context, a high-resolution imaging application produces images
at sarcomere-level with subcellular detail and subcellular detail
of other structures, while mitigating motion artifacts due to
contractile motion and/or physiological motion such as respiration
and blood flow. Certain example embodiments are implemented in
vivo.
[0009] According to a particular embodiment of the present
invention, the bio-medical imaging involves a reverse-direction
operation. Such implementations, in accordance with the present
invention, produce high-resolution images of biological thick
tissues using a minimally-invasive optical probe to sense relevant
intrinsic signals, thereby avoiding problems associated with
pre-treatment of the tissue with a foreign matter, such as
fluorescent dye, exogenous gene or protein.
[0010] According to more specific embodiments, the characterization
can be in any of various forms which are sometimes
application-dependent and/or dependent on the tissue. For instance,
in one specific application, an optical probe is inserted into
skeletal muscle. Light pulses transmitted by the probe stimulate
the generation of signals, such as fluorescence and/or SHG, from
intrinsic properties of certain tissue structures. These signals
are collected and then processed to provide information such as
sarcomere lengths, mobility, and indications of tissue
dysfunctionality. This information can be provided in forms
including displayed forms, for example, reports, units of measure
and biological reproduction images, as well as non-displayed forms
such as stored electronic data useful for latent processing.
[0011] According to certain example embodiments of the present
invention, a system is implemented for visualizing sarcomeres in
vivo. The system includes an optical probe having a light-pulse
generator to send light pulses from the optical probe to certain
targeted structure in the tissue. A photosensor senses, in response
to the light pulses, selected signals generated from the sarcomere
tissue and predominantly present due to properties intrinsic to the
targeted structure. A signal processor is communicatively coupled
to the optical probe to characterize the sarcomere tissue based on
the sensed selected intrinsic signals.
[0012] In more specific embodiments, the light pulses from the
light-pulse generator are tuned to a wavelength that interacts with
the properties intrinsic to the tissue structure. The selected
signals are generated from fluorescent mitochondrial molecules or
from SHG.
[0013] The above summary is not intended to describe each
illustrated embodiment or every implementation of the present
invention. The figures and detailed description that follow more
particularly exemplify these embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The invention may be more completely understood in
consideration of the detailed description of various embodiments of
the invention that follows in connection with the accompanying
drawings, in which:
[0015] FIG. 1A illustrates an endoscopic imaging system adapted to
excite structure(s) with optical signals (e.g., fluorescent NADH
(nicotinamide adenine dinucleotide) or SHG signal) within certain
thick tissue structures and to collect resulting intrinsic signals
in response, according to an example embodiment of the present
invention;
[0016] FIGS. 1B and 1C are images of animal tissue obtained in vivo
via a microendoscopic probe according to an example embodiment of
the present invention;
[0017] FIG. 2 shows a flow diagram depicting a method for use with
an endoscopic imaging system adapted to excite optical signals
based on intrinsic properties of thick tissue structures and to
collect intrinsic signals in response, according to another example
embodiment of the present invention;
[0018] FIG. 3 shows an example implementation of the present
invention for specific application for high-resolution imaging
cardiac sarcomeres, with an optional stimulus step being provided
for altering a condition of the heart and repeating certain steps
to provide additional imaging assessing the biological tissue;
[0019] FIG. 4A-4D show lead channels that simultaneously deliver
electrical leads (commonly used for cardiac stimulus) along with
multiple (send/receive) optical fibers, according to various
example implementations of the present invention;
[0020] FIG. 5 is another endoscopic imaging system in accordance
with the present invention;
[0021] FIGS. 6A-6D are representations of images showing the
dynamics of sarcomere contradictions, also consistent with the
present invention;
[0022] FIG. 7 is an optical pathway of a microscope, consistent
with the instant disclosure;
[0023] FIG. 8 shows a focus lens arrangement for three different
image planes in an example embodiment consistent with the instant
disclosure;
[0024] FIGS. 9A-C show a constant beam waist at the back aperture
of a GRIN endoscope, consistent with the present disclosure;
[0025] FIG. 10A shows beam scanning, consistent with an embodiment
of the instant disclosure, at a location slightly above the back
aperture of the GRIN endoscope relay;
[0026] FIG. 10B shows that the central "chief rays" of the same
embodiment of FIG. 10A are parallel to the axis of the
endoscope;
[0027] FIG. 11 shows only the chief rays of the same scan angles in
FIG. 10;
[0028] FIG. 12 shows an example machined handheld device and
integrated endoscope, in accordance with the instant
disclosure;
[0029] FIGS. 13A-B show example embodiments of a handheld device,
and components thereof, consistent with the instant disclosure;
[0030] FIG. 14 shows a light path through an example embodiment of
a handheld device, and the light paths relation to a photodiode for
light power sensing;
[0031] FIGS. 15A-B show example embodiments of an integrated
endoscope in accordance with the instant disclosure;
[0032] FIG. 16 shows a light path through a needle package and a
GRIN-based endoscope in an example embodiment;
[0033] FIG. 17A shows an example imaging arrangement of the
integrated endoscope relative to a set of muscle sarcomeres;
[0034] FIG. 17B shows a side view of the example imaging
arrangement of FIG. 17A;
[0035] FIG. 18 shows a constructed needle, in accordance with the
instant disclosure, having a pocket channel for GRIN optics;
[0036] FIG. 19 shows a tri-facet geometry of an example embodiment
of a needle;
[0037] FIG. 20 shows suction line and GRIN optics placement in a
needle in an example embodiment of the instant disclosure;
[0038] FIG. 21 shows suction inlet geometry of an example
embodiment of the integrated endoscope;
[0039] FIG. 22 shows a signal wire connected to a needle
arrangement of an integrated endoscope in accordance with an
example embodiment of the instant disclosure;
[0040] FIG. 23A shows an example probe clamping mechanism for
securing an integrated endoscope to a handheld device based on the
instant disclosure;
[0041] FIG. 23B shows a perspective view of FIG. 23A;
[0042] FIG. 24 shows an integrated endoscope alignment based on the
instant disclosure;
[0043] FIGS. 25A-D show an example procedure, in accordance with
the instant disclosure, for clamping an integrated endoscope to a
handheld device;
[0044] FIG. 26 shows a spring loaded trigger injector, in an
example embodiment, that is used to deliver the integrated
endoscope to a sample beneath the skin;
[0045] FIG. 27A-B show an example embodiment of a dual movement
locking mechanism that prevents an integrated endoscope form being
released from an injector;
[0046] FIGS. 28A-C show an example procedure for attaching a
handheld device and integrated endoscope, consistent with the
instant disclosure; to a human subject, and
[0047] FIG. 29 shows an example muscle image achieved through use
of a handheld device and integrated endoscope, consistent with the
instant disclosure, of a tibialis anterior muscle of a human
subject.
[0048] While the invention is amenable to various modifications and
alternative forms, specifics thereof have been shown by way of
example in the drawings and will be described in detail. It should
be understood, however, that the intention is not to limit the
invention to the particular embodiments described. On the contrary,
the intention is to cover all modifications, equivalents, and
alternatives falling within the spirit and scope of the
invention.
DETAILED DESCRIPTION
[0049] The present invention is believed to be useful for a variety
of different thick-tissue evaluation applications, and the
invention has been found to be particularly suited for use in
minimally-invasive medical applications, arrangements and methods
which are benefited from high-resolution details of the thick
tissue under examination. As discussed in the background above,
these biomedical approaches include, but are also not necessarily
limited to, in vivo examinations. Various aspects of the invention
may be appreciated through a discussion of examples using this
context.
[0050] In connection with various aspects of the present invention,
it has been discovered that motion artifacts can be avoided by
increasing the line scan rate as a function of the sarcomere
dynamics and physiological motion, and the ability to collect the
responsive signals that are predominantly present due to properties
intrinsic to the structure. As these signals are typically weak and
extremely difficult to detect, even by the most sophisticated of
microendoscopes, controlling the line scan rate can be important
for high resolution images of biological structure in and around
sarcomere. Consistent with an important aspect of various
embodiments of the present invention, unexpected high resolution
images of this sarcomere-related structure are obtained by setting
the scan rate to substantially minimize motion artifacts while
permitting for the collection of these weak intrinsic-based
signals.
[0051] In a more specific embodiment, images of sarcomeres and
sub-cellular structures are obtained by using the optical probe to
send light pulses toward structure in the biological thick tissue
at a sufficiently fast line-resolution rate to mitigate motion
artifacts due to sarcomere dynamics and physiological motion. This
rate being sufficiently fast to cause (in response to the light
pulses) signals to be generated from and across a sufficient
portion of the structure to span a sarcomere length, and to collect
selected aspects of the generated signals that are predominantly
present due to properties intrinsic to the structure.
[0052] Consistent with one example embodiment of the present
invention, an apparatus permits for very-high resolution
characterization of cellular-level structure in thick tissue in a
manner that is minimally invasive. The apparatus is capable of
reverse direction imaging without staining or otherwise introducing
a foreign element used to generate or otherwise increase the sensed
light. The apparatus has a probe that includes a light generator
for generating light pulses that are directed towards certain
structures located within the thick tissue. The light pulses
interact with intrinsic characteristics of the tissue structures to
generate a signal. An emitted-light collector collects light (e.g.,
excited light or light based on SHG) used to characterize aspects
of the thick tissue. Reliance on intrinsic characteristics of the
tissue structures is particularly useful for applications in which
the introduction of foreign substances to the thick tissue is
undesirable, such as in human imaging.
[0053] Consistent with another embodiment of the present invention,
the light generator and sensor are used in vivo using a probe, such
as a needle or similar injection device, to minimize invasiveness
while collecting sufficient cellular-level information for detailed
visualizations. In vivo applications can be subject to image
artifacts resulting from movement of the target animal. The light
is directed from the aperture of the probe to the targeted thick
tissue. The probe includes a light collector with a light-directing
lens arrangement designed to provide a probe diameter that is
sufficiently-small (in a specific example embodiment, about 1 mm to
about 0.35 mm) to permit for needle-like insertion of the probe
into the targeted thick tissue. For non-SHG applications, the probe
has an objective with a numerical aperture (NA) and other
attributes adequate for collecting intrinsic-based signals.
[0054] In connection with the present invention, certain
embodiments use a probe to capture relatively low-intensity,
intrinsically-based signals for a bright image with fine details of
tissue structure, such as sarcomeres, while being
sufficiently-narrow to be implemented in needle-like dimensions for
microendoscopic applications performed in vivo.
[0055] According to experimental example embodiments, the
above-described scopes have been implemented using three example
sizes of gradient refractive index (GRIN) lenses: 1000, 500 and 350
microns O.D. To fit inside a needle for endoscopic delivery, the
following commercially-available needles can be used:
TABLE-US-00001 endo needle needle ID needle OD 1000 16-18 Ga
1070-1270 micron 1270-1650 microns 500 21-23 Ga 510-570 micron
640-820 microns 350 24-25 Ga 370-410 micron 510-570 microns
Such needles are available, for example, from Popper & Sons,
New Hyde Park, N.Y.
[0056] In some applications, two-photon fluorescence microendoscope
probes are implemented with minimally invasive compound GRIN lenses
with flexible fiber-optic technology.
[0057] In another instance, the present invention is implemented in
a reverse-direction system using a light generator and sensor
located in close proximity. Proximity can be measured as either a
spatial distance or as an angle relative to the direction of the
light generated by the light generator. In one particular instance,
the sensed signal is the result of fluorescence generated from
excitation of the cell structure (e.g., from the mitochondria).
Because fluorescence is an isotropic phenomena, the light is
equally dispersed. Accordingly, the angle of the sensor relative to
the light pulses need not be a critical consideration.
[0058] In another instance, the sensed light is the result of the
light pulses passing through the cell structure and creating an SHG
signal. The SHG signal is dependent on the light pulses, and the
direction of the light pulses is relevant to the direction of the
SHG signal. It has been discovered that an SHG signal can sometimes
be classified into three components including forward directed,
backscattered and backward directed. A forward directed SHG signal
includes the signal components that continue in the direction of
the light pulses. A backscattered SHG signal includes the signal
components resulting from scattering of a forward directed SHG
signal such that the SHG signal travels towards the light generator
and sensor. A backward directed SHG signal includes the signal
components that are directed opposite the direction of the
originating light pulses without scattering. Thus, the placement of
the sensor affects the relative collection efficiencies of the SHG
signal components that are received. For instance, the placement of
the sensor in the path of the backward directed SHG signal
component can be particularly useful in reverse-direction systems
(e.g., by facilitating the sensing of both the backward directed
SHG signal and the backscattering SHG signal).
[0059] Turning now to the figures, FIG. 1A illustrates an
endoscopic imaging system adapted to excite optical signals based
on intrinsic thick tissue structures and to collect intrinsic
signals in response. The system facilitates high-resolution imaging
of thick tissue in vivo. Femtosecond laser pulses (e.g., 80-150 fs)
are generated by a laser 210. In a particular instance, the laser
is a Ti-sapphire laser generating light with a wavelength around
700 nm to 1000 nm. The particular wavelength can be selected
depending upon the application. Due to the frequency-doubling
characteristic of intrinsic SHG signals, the frequency of the SHG
signal is directly proportional to the frequency of the excitation
light (generated pulses). Thus, the generated pulses can be
selected to minimize tissue absorption and scattering of the SHG
signal for in vivo applications. Microendoscopic probe 112 acts to
both direct the generated pulses and collect the intrinsic signals.
This means that for fluorescent and SHG signals, the imaging
process relies primarily upon backward directed/scattered
(low-energy) light.
[0060] For intrinsic fluorescent and SHG signals, the probe can be
inserted in close proximity to the targeted tissue or, as shown by
muscle fiber 114, inserted into the targeted tissue. For isotropic
light, the amount of light collected by a given collector changes
relative to the distance from the source. Moreover, absorption and
scattering of light from surrounding tissue increases as the
distance from the source increases (the scattering length is about
five times shorter than the absorption length). For approaches
directed to SHG signal generation, it has been discovered that the
SHG signal collected by a probe aligned with the excitation light
generator (e.g., collecting backward SHG signals) and in close
proximity to the thick tissue is surprisingly strong. This is
particularly useful for non-invasive and minimally-invasive in vivo
imaging. The imaging time can also be increased to increase the
total amount of light received; however, increased imaging time can
lead to increased susceptibility to physiological motion resulting
in unwanted motion artifacts in the image.
[0061] Physiological motion, such as respiration and blood flow,
are compensated for using a number of techniques. Using one such
technique, microendoscopic probe 112 is inserted into the thick
tissue. The probe 112 is small enough to allow physiological motion
of the thick tissue to cause corresponding motion in the probe 112,
while still capturing the imaging signals as discussed above. Thus,
the effects of such physiological motion are mitigated by
corresponding motion in the probe 112. The amount of allowable
physiological motion can be estimated from the desired image
resolution. For example, subresolution physiological motion would
minimally affect subsequent image quality. The relevant amount of
physiological motion is dependent upon the resolution of the
desired image. For instance, images having a resolution on the
order of a few micrometers are not substantially affected by
physiological motion where the imaging is directed over a smaller
span, e.g., much less than about 1-2 micrometers. Other factors
that would affect the correlation between physiological motion and
probe motion include the depth of the thick tissue, the length of
the probe, and the stiffness of the optical fibers and the physical
properties of the thick tissue.
[0062] In one embodiment, microendoscopic probe 112 is connected to
control arrangement 102 using fiber cables and control lines for
scanning, whereas in other embodiments physical separation is not
provided (e.g., by fiber cables). Control arrangement 102 includes
various light generation and detecting components controlled by a
processor/computer 106. In a particular instance, light generation
block 210 (e.g., a Ti:Sapphire laser) produces light pulses that
are transmitted to probe 112 using fiber optics, and
photomultiplier tube (PMT) block 108 receives light collected by
probe 112 using fiber optics. The use of a flexible light-transport
medium is useful because probe 112 can be moved independently
relative to the position of light generation block 210 and PMT
block 108. Scanning mirrors 228 provide directional control over
the light pulses. Endoscope optics 230 directs both the transmitted
light pulses and the corresponding collected light.
[0063] As discussed above, to minimize the size of the probe, the
probe should be sufficiently small and the objective and related
optical properties of the probe should be able to capture the
intrinsic-based signals. In a specific example, the probe is a
gradient refractive index (GRIN)-lens microendoscopic probe used to
provide a minimally-invasive mechanism for imaging such signals in
thick tissue in vivo. In another more specific embodiment, rather
than securing both the probe and the subject, the probe can be
allowed to move with the thick tissue. This freedom of movement is
particularly useful for reducing motion artifacts due to
physiological motion. For many applications, it should be
appreciated that the first priority is to be able to collect the
intrinsic signals, and the second priority is to make the probe
small enough without compromising the first priority; however,
there are applications, such as where the target tissue is
pre-exposed, that may be performed that do not necessarily have the
small size requirement.
[0064] In one instance, the probe scans the thick tissue using a
scanning device to direct light pulses toward the thick tissue. One
such scanning device is a micro-electro-mechanical systems (MEMS)
mirror. Scanning mirror control 104 provides signals to control the
scanning device. The size of the scanning device is also a
component of the overall size of the probe.
[0065] In one specific example application involving visualization
of dynamic sarcomeres, a system as illustrated in connection with
FIG. 1A is used to obtain images based on line-scan speeds of about
2 kHz, with each line being 256 pixels long and a dwell time of 2
.mu.sec. During imaging, each line is approximately 0.512 msec. To
prevent damage to the tissue by the laser, the power is limited to
less than 50 mW incident at the sample. In other embodiments, the
SHG signal is boosted by increasing power, but too much power might
damage the tissue. With a limit on the signal being generated at
the sample and using desired (or optimized) optics for practically
collecting the largest fraction of that signal, the signal-to-noise
ratio (SNR) becomes a limiting factor. In one instance, the line
scan is 2 KHz while still maintaining a reasonable SNR. These
parameters are adequate for providing images of skeletal
sarcomeres. In order to image faster, the noise is decreased or the
fraction of the SHG signal (that is collected) is increased. When
using a PMT, which has a relatively low noise, decreasing the noise
further is not practical. To increase the fraction of the signal
being collected, the NA of the GRIN lenses can be increased, e.g.,
from an NA of 0.46 to an NA of about 0.7; such an increase
increases the fraction of SHG collected by a factor of about two.
In another application, the dwell time is reduced to about 1
.mu.sec and the scan rate is increased to about 4 kHz.
[0066] Other physiologic movements are caused by the heart beat and
breathing which are about 1 Hz and 0.2 Hz respectively. By imaging
at about 2 kHz, embodiments of the present invention allows for the
collection of an entire image of 256 lines (0.13 sec) during a time
when the animal is between heartbeats or breathing, thus
practically eliminating motion artifacts. Assuming the same
magnitude of signal is being generated from the skeletal muscle;
applications of the present invention are useful for imaging a
beating heart or organs in the thorax and abdomen.
[0067] FIG. 2 shows a flow diagram of a method for use with an
endoscopic imaging system adapted to excite optical signals based
on intrinsic thick tissue structures and to collect resulting
intrinsic signals. The flow diagram shows two main paths, one
beginning with the Ti:Sapphire pulsed laser 210 and the other
beginning with the stabilized subject 212 (e.g., animal or human,
typically in vivo). These paths show the operation of the imaging
probe and the preparation of the subject, respectively.
[0068] Referring to the path beginning with block 212, stabilizing
the subject to some degree can be important and useful for limiting
movement that would interfere with medical oversight during the
procedure as well as with the production of high-resolution images.
In accordance with surgical procedures used as part of the present
invention, such physical restraint optionally includes sedation
(214) and/or conventional physical restraints (216) for limiting or
controlling motion. At more detailed levels of tissue
characterization, physical restraints can include conventional
restraints to physiological motion such as by limiting blood flow
(218-219) and respiration (220). In other embodiments (alone or in
combination with those discussed herein), respiration compensation
can be accomplished by sedation, holding one's breath or through
forced ventilation timed so that pauses in the ventilation occur
during the imaging process. Also consistent with various ones of
the above embodiments, block 222 shows use of a microendoscope that
is sufficiently small so that when it is inserted into the thick
tissue, the microendoscope moves with the tissue thereby mitigating
the effect of the motion.
[0069] As will be discussed below, certain embodiments of the
present invention produce high-resolution images of significant
thick tissue structure without requiring significant compensation
for such motion. Consistent with the above-discussed aspects of the
present invention and as depicted by blocks 224 and 226 of FIG. 2,
such motion artifacts can be avoided by increasing the line scan
rate as a function of the sarcomere dynamics and physiological
motion, and the ability to collect the responsive signals that are
predominantly present due to properties intrinsic to the structure.
In this manner, sufficient intrinsic-based signals can be picked up
by a commercial microendoscope for producing high-resolution images
of structure in and around sarcomere, while mitigating artifacts
caused by the sarcomere and related physiological motion.
[0070] In a more specific embodiment also according to the present
invention, motion artifacts are mitigated as needed for the
application at hand. In this manner, a computer-based digital
imaging application uses conventional (e.g., standard-deviation)
calibration techniques to discern the quality of the process.
Should the data processing indicate that the sarcomere lengths
cannot be discerned (i.e., insufficient resolution), the images are
rejected as having unacceptable degrees of motion artifacts. The
sarcomere length within an image is found by a computer algorithm,
e.g., Fourier transform, wavelet transform or fitted sine-wave,
such that a confidence interval is also generated for the
measurements. In one application, an example threshold for an
acceptable standard-deviation is about 5% (confidence interval is
compared to an arbitrary value; +/-5% is commonly used, to
discriminate between images that contain measurable sarcomere
lengths from those that do not). Data-capture adjustments, whether
automatically by the computer or manually by the system user, are
then made and/or further imaging efforts are repeated.
[0071] As examples of such detailed images obtained according to
these embodiments of the present invention, FIGS. 1B and 1C are
respective reproductions of images of sarcomere and sub-cellular
structure. FIG. 1B is an image taken from in vivo mouse lateral
gastrocnemius using a 350 .mu.m endoscope. The scale bar indicates
25 .mu.m. FIG. 1C is a three-dimensional reconstruction of lateral
gastrocnemius muscle in a living mouse. The model was created from
a stack of 1 .mu.m thick images taken with a 350 .mu.m endoscope in
a living mouse using SHG. The scale bar indicates 10 .mu.m.
[0072] With the microendoscope inserted in the subject 212, the
laser 210 initiates the methodology by sending pulses through the
microendoscope 230 while using a GRIN endoscope and excitation
pre-chirping and optical signal processing as is conventional, as
depicted at 215. Scanning mirrors 228 direct the pulses at the
relevant tissue site, and dichroic mirrors are used to separate the
excitation light from the emission light (block 231). The
line-resolution rate is set sufficiently fast relative to the
motion artifacts expected due to factors such as sarcomere movement
and physiological motion. In a particular instance, the pulses are
directed such that they capture the entire length of the sarcomere
being imaged. Through the various techniques discussed herein,
signals are generated in response to the pulsed light. These
signals are collected by the probe and used as part of the data for
creating the desired image. This process may be repeated as
desired. For example, the multiple line scans may be used to
generate larger imaging sections or to capture sequential images of
the same sarcomere under different conditions. In a particular
instance, the effectiveness of a form of therapy may be evaluated
using images captured both before and after therapy is provided for
the patient.
[0073] The path beginning with the pulsed laser 210 shows two
approaches. A first approach (left path) involves fiber optics
attached to a microendoscopic probe and a second approach (right
path) is implemented without fiber optics. The fiber approach
involves a first step of excitation pre-chirping to compensate for
transmission over optical fiber (e.g., to reduce
group-velocity-dispersion). The signal is then converted to the
desired shape and path using conditioning optics and routed to
polarization rotating optics. Upon polarization, the resulting
femtosecond laser pulses are passed through an optical excitation
fiber 232, such as a photonic crystal fiber. The second, non-fiber
optics, approach operates much the same as the first approach
without the need to compensate for transmitting the pulses through
an optical fiber.
[0074] Once the optical pulses reach the microendoscopic probe, a
scanning device (e.g., MEMS mirror) directs the pulses. A dichroic
mirror allows light of a certain wavelength to pass, while
reflecting light of another. Thus, the dichroic mirror separates
the laser pulses (excitation light) from the intrinsic signals
(emission light). The laser pulses are directed through the GRIN
microendoscope and to the thick tissue. The laser/excitation pulses
striking the thick tissue result in intrinsic signals (240A for
intrinsic fluorescence (TPF) or 240B for SHG). The GRIN
microendoscope 230 collects the intrinsic signals passing them to
the dichroic mirror. The dichroic mirror routes the collected
signals towards emission filters 246. The collected signals pass
through emission fiber 250 and routing optics 252 (or fiber optics)
to a photo-detector 254. The photo-detector 254 receives and
detects the collected signal. A processor 260 running customizable
software processes the information for producing the data 264 in
response to the photo-detector and thereby permitting for structure
visualization. In one instance, the software compensates for motion
artifacts and an image of the thick tissue is then generated for
viewing.
[0075] In one particular embodiment, a microscope objective focuses
ultrashort pulsed laser illumination onto the face of a gradient
refractive index (GRIN) microendoscope. The microendoscope
demagnifies and refocuses the laser beam within the muscle and
returns emitted light signals, which reflect off a dichroic mirror
before detection by a photomultiplier tube (PMT). A
350-.mu.m-diameter GRIN microendoscope clad in stainless steel can
be used for minimally invasive imaging in the arm of a human
subject.
[0076] For static imaging of individual sarcomeres, another
embodiment provides images of a single mouse muscle fiber in
culture, acquired using epi-detection of two-photon excited
autofluorescence and band-pass filtered to highlight sarcomeres. As
a variation, a band-pass filtered image of the same fiber can be
obtained using trans-detection of second-harmonic generation (SHG).
As an enhancement, overlaying the above two image types reveals
that autofluorescence signals, thought to arise from mitochondria
located mainly at the Z-discs of sarcomeres, interdigitate with the
SHG signal thought to arise in myosin tails.
[0077] Optical probe systems described herein can be implemented as
a microendoscope probing approach, according to the present
invention, by using very small lens systems having an acceptable
objective lens and overall diameters as described above. For
instance, such microscopic endoscopes can be implemented using lens
technology described in U.S. Pat. No. 5,161,063 and as described in
other references including, but not limited to, technology that is
commercially-available from a variety of manufacturers. One such
manufacturer is Olympus (as cited in U.S. Pat. No. 5,161,063) which
markets such scopes having diameters at about 700 microns; other
acceptable microscopic endoscopes can be similarly constructed
using miniature-sized lens. For further information regarding such
systems, reference may be made to, "In Vivo Imaging of Mammalian
Cochlear Blood Flow Using Fluorescence Microendoscope", Otology and
Neurotology, 27:144-152, 2006, "In Vivo Brain Imaging Using a
Portable 3.9 Gram Two-photon Fluorescence Microendoscope", Optics
Letters, Vol. 30, No. 17, Sep. 1, 2005, and the following U.S.
Patent Publications: No. 2004/0260148 entitled "Multi-photon
endoscopic imaging system"; No. 2004/0143190 entitled "Mapping
neural and muscular electrical activity"; No. 2003/0118305 entitled
"Grin fiber lenses"; No. 2003/0117715 entitled "Graded-index lens
microscopes"; No. 2003/0031410 entitled "Multi-photon endoscopy";
No. 20020146202 entitled "GRIN fiber lenses"; and No. 2002/0141714
entitled "Grin-fiber lens based optical endoscopes".
[0078] In certain systems and applications of the present
invention, embodiments described herein include optical fiber
arrangements, and in some applications, a bundle of optical fibers.
Various example embodiments are directed to the use of optical
fibers such as those described in the following U.S. Patent
Publications: No. 2005/0157981 entitled "Miniaturized focusing
optical head in particular for endoscope" (to Berier et al.), No.
2005/0207668 entitled "Method for processing an image acquired
through a guide consisting of a plurality of optical fibers" (to
Perchant et al.), No. 2005/0242298 entitled "Method and equipment
for fiber optic high-resolution, in particular confocal,
fluorescence imaging" (to Genet et al.) and No. 2003/0103262
entitled "Multimodal miniature microscope" (to Richards-Kortum et
al.); and as those described in the following U.S. Pat. No.
6,766,184 (Utzinger et al.) entitled "Methods and apparatus for
diagnostic multispectral digital imaging," U.S. Pat. No. 6,639,674
(Sokolov et al.) entitled "Methods and apparatus for polarized
reflectance spectroscopy," U.S. Pat. No. 6,571,118 (Utzinger et
al.) entitled "Combined fluorescence and reflectance spectroscopy,"
and U.S. Pat. No. 5,929,985 (Sandison et al.) entitled
"Multispectral imaging probe". Each of these above-cited documents
is fully incorporated herein by reference.
[0079] Various embodiments of the present invention are
specifically directed to measurement of sarcomere lengths in
healthy subjects and in individuals with neuromuscular diseases,
allowing discovery of the mechanisms leading to disabling muscle
weakness. For example, in the clinic, the device is used as a
diagnostic tool to determine the cause of weakness.
[0080] The capacity of muscles to generate forces is highly
sensitive to sarcomere length. Muscles generate their maximum force
at a sarcomere length of approximately 3 .mu.m, but generate almost
no force at lengths of 2 .mu.m or 4 .mu.m. In some instances,
profound weakness in patients with neuromuscular diseases, such as
cerebral palsy, may be caused by altered sarcomere lengths. The
ability to confirm this, in a variety of patient populations,
enables important studies that examine the mechanisms of muscle
weakness in persons with neuromuscular diseases.
[0081] In another example, implementations of the present invention
are applied during surgery wherein this technology is used to set
sarcomeres to the right length. The microendoscope is inserted into
muscle in order to visualize and measure the sarcomere lengths and
the related muscle-attachment points to provide maximum muscle
strength following musculoskeletal surgeries. This approach is used
to improve the outcome of tendon lengthenings, tendon transfers,
joint reconstructions and other musculoskeletal
reconstructions.
[0082] Another application is directed to cardiac health. Cardiac
health is dependent on contraction of cardiac muscle cells and
imaging of sarcomeres in a manner consistent with the above enables
distinction of healthy and diseased or damaged cardiac tissue. The
response to drugs may increase or decrease contractility, and
imaging sarcomere dynamics, as enabled here, allows these
assessments in living subjects and in vitro. In accordance with the
present invention, the following discussion is illustrative of
cardiac uses and applications.
[0083] FIG. 3 depicts a heart 300 having a channel (or lead) 320
introduced to the heart for the purpose of altering a condition of
the heart, according to one embodiment of the invention. Lead 320
includes electrodes 310-316 for delivering electrical stimulus or
for sensing electrical properties of the heart. Lead 320 may be
introduced to the heart using a number of different techniques. In
this instance, lead 320 is shown as being introduced through the
coronary artery. In one instance, a pacemaker device, located
external to the heart, controls the electrical stimulus provided by
electrodes 310-316. Images may be taken of myocardial sarcomeres
using the various methods, systems and devices described herein.
These images may include myocardium sarcomere as accessed via the
endocardium or epicardium. In these applications, the
above-discussed microendoscopes can be used to obtain such images
via myocardium tissue accessible from areas outside the heart or,
as with lead 322 and optical probe 324, areas within the heart. In
the latter application, the same access port (e.g., the coronary
artery) or another access port may be used.
[0084] Using the above approach for cardiac stimulating/monitoring
with related cardiac imaging, various specific applications are
realized. In a particular instance, images taken of the myocardial
sarcomere without stimulus from the electrodes 310-316 are compared
to images taken of the myocardial sarcomere with stimulus from
electrodes 310-316. This may be particularly useful in assessing
the effectiveness of a particular cardiac treatment. In another
instance, images of various cardiac treatments can be compared. For
instance, the effects of dual (atrial and ventral) stimulus may be
compared against ventral only stimulus. In another instance, the
location, voltage and pulse duration of the electrical stimulus may
be varied to allow for a comparison of the respective myocardial
sarcomere images. In other instances, damaged cardiac tissue can be
imaged to ascertain the extent of the damage or to assess the
effectiveness of a treatment of the damaged tissue.
[0085] In one embodiment, an input component is used to trigger the
imaging time. Such an input component may originate from a number
of sources. For instance, QRS signals of the heart, such as those
captured by an electrocardiograph, may be used to trigger the
imaging and/or as part of the system (e.g., using an EKG system
concurrent with the imaging approach illustrated in FIG. 1A). In
another example, signals originating from the pacemaker device can
be used to trigger the imaging and/or capture the myocardium (via a
pacing signal) while imaging the sarcomere and monitoring the
effectiveness of the treatment. Such image-timing techniques can be
useful for capturing images of myocardial sarcomere that correspond
to natural heart function, captured heart contractions (e.g.,
electrode induced), and the like.
[0086] In other embodiments, the heart may be altered using other
techniques and combinations of techniques. For instance, electrical
stimulus need not be administered using the electrode/lead
configuration displayed in FIG. 3. Instead, any number of
techniques may be employed. Other heart altering therapies, such as
drug induced alterations, may also benefit from the imaging of the
myocardial sarcomere. For background discussion, reference can be
made to any number of U.S. patents directed to cardiac monitoring
and cardiac therapy.
[0087] In another cardiac-imaging application, a specific
embodiment of the present invention is directed to the system shown
in FIG. 1A modified to include a lead within the microendoscope
probe to provide myocardium stimulus that is used concurrent with
the above-described dynamic sarcomere imaging. For example, such
microendoscope lead(s) can be combined with or within a single lead
channel in which multiple signals are transmitted via the same lead
channel for stimulation and monitoring purposes such as by
modifying embodiments illustrated in U.S. Pat. No. 6,208,886,
entitled "Non-linear Optical Tomography of Turbid Media" (e.g., see
FIG. 9 showing multiple (send/receive) fibers in same channel).
[0088] Accordingly, this specific embodiment includes the system of
FIG. 1A modified such that a lead channel simultaneously delivers
the electrical leads (commonly used for cardiac stimulus) along
with multiple (send/receive) optical fibers. The microendoscope
probes are then used as described in connection with the above
embodiments to provide myocardium stimulus and/or capture
concurrently with the dynamic sarcomere imaging. By varying the
timing, phases and power parameters of the myocardium stimulus,
suspect (diseased) cardiac sarcomere can be viewed at detailed
levels not previously recognized and thereby permitting
patient-customized cardiac monitoring, therapy and/or pace-signal
control for overall cardiac management.
[0089] FIGS. 4A-4D illustrate such example approaches in accordance
with the present invention. In each instance, a lead channel
simultaneously delivers the electrical leads (commonly used for
cardiac stimulus) along with multiple (send/receive) optical
fibers. As shown in FIG. 4A, the channel 402 includes multiple
nodes 404-408 at which electrodes 414 (such as 312 of FIG. 3)
and/or microendoscopic probes (such as 112 of FIG. 1) access the
myocardium. Where a node 404 includes both an electrode 410 and a
microendoscopic probe 412 (i.e., the end of probe 112 of FIG. 1),
the electrode can be implemented as a conductive terminal at or
immediately adjacent to the probe. By using multiple ones of such
nodes, control circuitry (for the electrodes and/or the optics) can
be selectively enabled so as to explore and access different areas
of the myocardium tissue without necessarily repositioning the
channel 402. FIGS. 4B-4D illustrate various configurations for the
channel 402 and the corresponding location of the nodes 410-434,
which may be suitable for different applications. For further
discussion relating to different configurations of the channel,
reference may be made to U.S. Pat. No. 5,181,511 entitled
"Apparatus and Method for Antitachycardia Pacing Using a Virtual
Electrode" (e.g., see FIGS. 5 and 6A-6F), which is hereby fully
incorporated by reference.
Additional Experimental Efforts and Related Embodiments
[0090] Here, we report direct visualization of individual
sarcomeres and their dynamical length variations using minimally
invasive optical microendoscopy to observe second harmonic
frequencies of light generated in the muscle fibers of live mice
and humans. We imaged individual sarcomeres in both passive and
activated muscle. Our measurements permit in vivo characterization
of sarcomere length changes that occur with alterations in body
posture and visualization of local variations in sarcomere length
not apparent in aggregate length determinations. High-speed data
acquisition enabled observation of sarcomere contractile dynamics
with millisecond-scale resolution. These experiments evince in vivo
imaging to demonstrate how sarcomere performance varies with
physical conditioning and physiological state, as well as imaging
diagnostics revealing how neuromuscular diseases affect contractile
dynamics. Further, with such in vivo measurements of individual
sarcomeres, we learn precisely the normal operating range or
variability of sarcomere length, how physiological regulation may
adjust sarcomere lengths, and/or how sarcomere lengths are
disrupted in disease.
[0091] In specific experimental embodiments, we use an optical
microendoscope having gradient refractive index (GRIN) microlenses
(350-1000 .mu.m diameter), to enter tissue in a minimally invasive
manner and provide micron-scale imaging resolution. To facilitate
studies in humans, certain embodiments avoid use of exogenous
labels and rather explore the potential for microendoscopy to
detect two intrinsic optical signals. The first of these signals
represents autofluorescence from nicotinamide adenine dinucleotide
(NADH) and flavoproteins, which are concentrated in mitochondria
along sarcomere Z-discs. The other signal represents
second-harmonic generation (SHG), coherent frequency-doubling of
incident light, which occurs within myosin rod domains. Our
instrumentation has used an upright laser-scanning microscope
adapted to permit addition of a microendoscope 512 for deep tissue
imaging (FIG. 5). A microscope objective 516 coupled the beam from
an ultrashort-pulsed Ti:Sapphire laser into the microendoscope, to
allow generation of two-photon excited autofluorescence and
second-harmonic signals. In both cases signal photons generated in
thick tissue returned back through the microendoscope 512 and were
separated from the excitation beam based on wavelength (FIG.
2).
[0092] We started investigations by imaging autofluorescence and
second-harmonic signals simultaneously from cultured muscle cells.
The two signals were distinguishable by wavelength, the partial
polarization of SHG signals and their dependence on incident light
polarization, and the predominance of trans- (forward-propagating)
over epi-detected (backward-propagating) SHG signals (Methods).
With <30 mW of incident laser power, sarcomeres were readily
apparent using either intrinsic signal, especially after band-pass
filtering the images to remove spatial frequencies representing
distance-scales outside the plausible range of sarcomere lengths
(1-5 .mu.m). Overlaid images of autofluorescence and
second-harmonic signals revealed that the two arise spatially out
of phase within sarcomeres, as expected if autofluorescence were to
come mainly from Z-disc mitochondria and SHG from myosin rods.
[0093] For use in live subjects, we imaged based on epi-detected
SHG and autofluorescence signals from the lateral gastrocnemius
muscle of anesthetized adult mice. Although SHG primarily arises in
the forward-propagating direction, we hypothesized that in thick
tissue there would be sufficient backward-propagation to allow in
vivo microendoscopy, due to multiple scattering of photons that
were originally forward-propagating. We discovered that in vivo SHG
imaging of sarcomeres was feasible by microendoscopy using
illumination wavelengths of .about.820-980 nm and generally led to
better sarcomere visibility than autofluorescence imaging (see
Methods, supra). SHG is an effective, endogenous contrast parameter
that can be used to visualize sarcomeres in living subjects, and
for subsequent imaging we used SHG and 920 nm illumination.
[0094] We further explored capabilities for imaging sarcomeres in
anesthetized mice. After inserting a microendoscope into the
gastrocnemius, we regularly imaged large assemblies of individual
muscle sarcomeres (n=23 mice). Cardiac and respiratory movements
often caused significant motion artifacts at image frame
acquisition rates of <4 Hz, but at 4-15 Hz sarcomeres were
readily identifiable within raw images. Insertion of the
microendoscope helped stabilize underlying tissue, reducing tissue
motion and enhancing image quality. To test the utility of our
data, we performed several illustrative analyses of muscle fiber
structure in live mammals.
[0095] First, we determined average sarcomere lengths and their
variability within individual muscle fibers and between adjacent
fibers. Uncertainties in measurements of average sarcomere length
within individual fibers can be reduced to limits set by the
inherent biological variability, rather than by instrumentation,
since the distance spanned by a large, countable number of
sarcomeres can be determined at a diffraction-limited resolution.
Thus, with 20-50 nm sarcomeres often visible concurrently, our
measurements of average sarcomere length have .about.20-50 nm
accuracy. In connection with the invention, we discovered that
individual sarcomere lengths can be variable, with up to .about.20%
variations within a .about.25-.mu.m-diameter vicinity. The degree
of local variability is likely influenced by passive mechanical
inhomogeneities and could not be examined previously without a
technique such as ours for visualizing individual sarcomeres.
[0096] We created three-dimensional models of muscle fiber
structure from stacks of SHG images acquired at 0.5 .mu.m depth
increments within tissue. Construction of these models used the
optical sectioning provided by SHG imaging which, like two-photon
imaging, generates signals from a spatially restricted laser focal
volume (see Campognola, P. J. et al., "Three-dimensional
High-resolution Second-harmonic Generation Imaging of Endogenous
Structural Proteins in Biological Tissues." Biophy J 82, 493-508
(2002)). We thereby verified that the muscle fibers we imaged were
almost exactly parallel to the face of the endoscope, thus
permitting us to make accurate sarcomere length determinations by
imaging in the two lateral spatial dimensions (Methods).
[0097] We next measured sarcomere lengths at different body
positions. In the gastrocnemius of anesthetized mice (n=7),
sarcomere lengths depended on the angle of the ankle, as shown in
FIG. 6A, due to changes in total muscle length. Across mouse
subjects, sarcomere lengths shortened from 3.15.+-.0.06 (s.e.m)
.mu.m to 2.55.+-.0.14 .mu.m during changes in ankle angle from
70-170 degrees. This matches the operating range of 3.18-2.58 .mu.m
that we estimated based on a biomechanical analysis (Delp, S. L. et
al., "An Interactive Graphics-based Model of the Lower Extremity to
Study Orthopedic Surgical Procedures." IEEE Trans Biomed Eng 37,
757-767 (1990)) using measurements of muscle length, pennation
angle, moment arm length, and an assumed optimal sarcomere length
of 2.8 .mu.m for a 120.degree. ankle angle.
[0098] We further used such microendoscopy to capture the dynamics
of sarcomere contractions. Because these dynamics elapse over
milliseconds, we performed laser line-scans at 200-1000 Hz
perpendicularly across rows of sarcomeres undergoing changes in
length. To induce muscle contraction in anesthetized mice, we
electrically stimulated the gastrocnemius proximal to the site of
microendoscopy (see Methods, supra). This triggered a contraction,
which we visualized with .about.1-3 ms time resolution (FIG. 6B).
Across multiple mice (n=5) in which the microendoscope was inserted
a similar distance from the ankle, mean sarcomere length was
3.05.+-.0.02 (s.e.m) .mu.m prior to stimulation and 2.55.+-.0.03
.mu.m afterwards (FIG. 6C). Mean contraction speed peaked at
8.00.+-.0.05 (s.e.m) .mu.m s.sup.-1 during electrical stimulation
(FIG. 6D), which is within the range of maximum fibers responding
in vitro to a chemical stimulus.
[0099] To demonstrate the applicability of microendoscopy to
studies and diagnostics in humans, we visualized individual
sarcomeres within the extensor digitorum muscle of healthy human
subjects (n=3). After placing a 20-gauge hypodermic tube into the
extensor digitorum, we inserted a 350-.mu.m-diameter microendoscope
through the tube and into the muscle. The hypodermic was removed
and the microendoscope held in place. The subject's arm was placed
in a brace, immobilizing the forearm and wrist but leaving the
fingers mobile. After commencing SHG imaging we are able to
visualize sarcomeres and their length fluctuations. Motion
artifacts were often substantial but were reduced by bracing the
limb. This tactic does not eliminate artifacts due to involuntary
muscle twitching, which could only be overcome by increasing the
laser-scanning speed to 400-1000 Hz. Subjects were asked to move
their fingers into fully flexed and extended positions. Systematic
variations in sarcomere length between these two positions were
evident from images across all subjects, but each person exhibited
slightly different ranges of sarcomere operation. With fingers
flexed, mean sarcomere lengths from three subjects were
3.15.+-.0.03 .mu.m, 3.30.+-.0.01 .mu.m and 3.25.+-.0.05 .mu.m
(n=12, 17 and 11 trials, respectively); with fingers extended these
values were 2.97.+-.0.03 .mu.m, 3.24.+-.0.02 .mu.m, 3.12.+-.0.02
.mu.m (n=10, 10, and 7 trials), illustrating our ability to
determine how human sarcomere lengths depend on body posture.
Subjects reported feeling only mild discomfort during imaging
sessions due to insertion of the microendoscope, indicating a
potential suitability for eventual use during routine diagnostics
of human sarcomere function.
[0100] Growing evidence from tissue biopsies indicates sarcomere
structure and lengths are altered in numerous neuromuscular
disorders that result from mutations in sarcomeric proteins.
Visualization of sarcomeres by microendoscopy can facilitate
efforts to diagnose the severity of these conditions, monitor
progression, and assess potential treatments. Other syndromes in
which monitoring sarcomere lengths might inform treatment choices
include geriatric muscle loss and contractures due to cerebral
palsy or stroke (see Plotnikov, S. V. et al., "Measurement of
Muscle Disease by Quantitative Second-harmonic Generation Imaging,"
Journal of Biomedical Optics in press (2008), and Ponten, E.,
Gantelius, S. & Lieber, R. L., "Intraoperative Muscle
Measurements Reveal a Relationship Between Contracture Formation
and Muscle Remodeling," Muscle Nerve 36, 47-54 (2007). Combined SHG
and two-photon microendoscopy of sarcomere lengths and fluorescent
sensors or proteins in mouse models of diseases is a scientific
tool to aid in the understanding of muscle biology and
pathophysiology. Intraoperative sarcomere imaging during orthopedic
reconstructions or tendon transfer facilitates efforts by surgeons
to identify and set optimal sarcomere operating ranges. By reducing
reliance on unproven assumptions, such as regarding the
distribution of sarcomere lengths, in vivo sarcomere measurements
improve biomechanical models that inform the understanding of human
motor performance and development of rehabilitation technology,
robotics, and prosthetic devices. For further discussion in this
regard, reference may be made to Lieber, R. L., Murray, W. M.,
Clark, D. L., Hentz, V. R. & Friden, J., "Biomechanical
Properties of the Brachioradialis Muscle: Implications for Surgical
Tendon Transfer." J Hand Surg [Am] 30, 273-282 (2005), Delp, S. L.
et al., "OpenSim: Open-source Software to Create and Analyze
Dynamic Simulations of Movement," IEEE Trans Biomed Eng 54,
1940-1950 (2007), and Manal, K., Gonzalez, R. V., Lloyd, D. G.
& Buchanan, T. S., "A Real-time EMG-driven Virtual Arm," Comput
Biol Med 32, 25-36 (2002).
[0101] Optical degradation can occur when changing focal planes
using a GRIN lens. In an example embodiment, a telecentricaly
oriented GRIN endoscope is designed in a manner that allows a user
of the endoscope to change the focal plane while maintaining
constant power, maintaining and maximizing the resolution, and
maintaining a constant magnification. The GRIN endoscope, which is
a doublet consisting of a relay and objective lens, operates at
infinite conjugates. In other words, collimated light, light whose
rays are in parallel, in the endoscope is pivoted at the back
aperture rather than focusing a point at the back of the GRIN lens.
The focal plane of the GRIN endoscope of the instant embodiment can
be changed by the user via an afocal focusing mechanism. The afocal
focusing mechanism adds convergence or divergence to the normally
collimated light and shifts the focal plane at the sample. This is
opposed to translating a focusing lens to change the image planes.
Further, in another example embodiment, the afocal mechanism is
configured and arranged to keep the beam waist constant at the back
aperture of the GRIN endoscope. Therefore, the power of the light
delivered to the sample does not fluctuate during focusing.
Further, the collimated light beam supplied to the GRIN endoscope
can be provided such that the beam overfills the back aperture of
the GRIN endoscope. In this manner, the endoscope delivers the
maximum available resolution, irrespective of the instant focal
plane of the endoscope.
[0102] In the embodiments described herein, the focal plane of a
GRIN endoscope accepting collimated light, and that delivers a
focused spot at the sample's plane, can be altered by changing the
shape of the incoming light beam. Turning now to FIG. 7, which
shows a GRIN endoscope in accordance with an example embodiment of
the instant disclosure. In an example embodiment, a pair of lenses
700/710 is arranged in an afocal arrangement. The pair of lenses
700/710 is used to change the shape of light beam provided to the
GRIN endoscope 760. The example embodiment of the GRIN endoscope
shown in FIG. 7 also includes a scan mirror 720, a scan lens 730, a
tube lens 740, and a dichroic mirror 750. The lens closest to the
scanning mirror 720 is fixed, the fixed focus lens 710, while the
lens nearest the light source, the mobile focus lens 700, moves on
an actuated stage. When the mobile focus lens 700 translates, the
light beam diverges or converges depending on the direction the
lens moved. The divergence and convergence of the light beam is
shown in FIG. 8. FIG. 8 displays the focus lens arrangement for
three different desired image planes. The left lens of the pair is
the mobile focus lens 800, whereas the right lens is a fixed lens
810. The position of the lens shown at the top of FIG. 8, also
labeled as position 1, is designed for shallow imaging (less than
100 .mu.m). The position of the lens shown in the middle of FIG. 8,
position 2, shows the neutral location where the outgoing beam is
unaltered and yields a focal depth of 100 .mu.m. The position shown
in the bottom of FIG. 8, position 3, shows the lens arrangement
designed for deeper imaging (greater than 100 .mu.m). The mobile
lens 800 can be placed at any location within its range, allowing
the user to change the focal depth at the sample continuously
between 0 and 150 .mu.m. In each of the lens positions shown in
FIG. 8, the exiting beam waist is constant within the focal plane
of the fixed lens 810, regardless of the position of the mobile
lens.
[0103] Turning again to FIG. 7 and also to FIG. 8, placing the scan
mirror 720 in the focal plane of the fixed lens 710/810 provides a
constant beam waist at the back aperture of the GRIN endoscope 760
because the scan lens 730 and tube lens 740 image the mirror to the
same plane. Therefore, regardless of the position of the mobile
lens 700/800, the intensity profile at the back aperture of the
GRIN remains the same, and therefore power delivered to the sample
remains constant. Further, in certain embodiments, the beam waist
is sized so that it is slightly larger than the back aperture of
the GRIN endoscope. As a result, the beam diameter stays fixed, and
light overfills the GRIN endoscope, which delivers a maximum
available resolution for all focal planes. Moreover, in certain
embodiments, when the focal lengths of the two focusing lenses, the
mobile focusing lens 700/800 and the fixed focusing lens 710/810,
are different, this mechanism doubles as a beam
expander/de-expander.
[0104] In FIGS. 9A-C, the resulting beam waist at the back aperture
of the GRIN endoscope is shown for the same lens positioning in
FIG. 8. FIG. 9A shows the beam waist of position 1 shown in FIG. 8.
FIG. 9B shows the beam waist of position 2 shown in FIG. 8. FIG. 9C
shows the beam waist of position 3 shown in FIG. 8. Also shown in
FIGS. 9A-C is the tube lens 910 and the dichroic mirror 900, shown
in the example embodiment of the GRIN arrangement in FIG. 7. In
each of the arrangements, the diameter of the beam waist at the
back of the GRIN endoscope 920 is constant. This keeps the power at
the sample constant and the resolution at an optimal level for all
focusing positions. Although the above embodiment is detailed with
reference to a GRIN lens arrangement, the afocal focusing method
described could be employed in any scanning microscope (e.g., laser
scanning microscope) to change focus of the microscope without
physically translating the objective.
[0105] The magnification of the GRIN endoscope can also change when
shifting the focus to different planes. In certain instances,
precise measurements require the user of an endoscope to record and
track the instantaneous magnification, and correct for that factor
in the measurement. This type of repeated scale corrections is an
unnecessary hindrance, which can be eliminated using the
telecentric lens arrangement describe in the instant
embodiment.
[0106] "Telecentric" describes a lens arrangement whose chief rays
exiting the GRIN endoscope objective are parallel to the optical
axis. The parallel nature of the chief rays is independent of focus
position, therefore, the size of the image plane remains constant
apart from the depth that the probe is imaging.
[0107] The optical path through the GRIN endoscope of the instant
embodiment is shown in FIG. 10A-B. A cutaway view of the back end
of the GRIN endoscope is shown in FIG. 10A, where collimated light
1040 pivots near the back aperture of the relay lens 1000. A
cutaway view of the front end of the GRIN endoscope is shown in
FIG. 10B, highlighting the objective lens 1010, a 90.degree.
reflecting prism 1020 (modeled as a solid square of glass for
simplicity), and a 100 .mu.m thick layer of tissue 1030. In FIG.
10A-B the double dashed lines indicate the location of the cutaway.
The relay lens 1000 consists of a half pitch multiple and therefore
acts as a one-to-one imaging system, transmitting the pivoted
collimated rays from its back aperture to its front aperture. The
angle of the pivoted rays exiting the relay is identical to the
incoming angle for even multiples of the half pitch length, and is
reversed for odd multiples. The objective takes this light and
focuses it to a point in the imaging plane. When the light entering
the endoscope is pivoted, the focal point generated by the
objective lens translates within the sample, making it possible to
generate a scanned image. When the scanned light pivots at the
front focal plane of the objective, the chief rays exit parallel to
the optical axis, and there is no magnification effect that results
from changing the focus position. In order to achieve the lack of
magnification effect (interference), rather than pivot the light
directly at the back aperture of the relay, the scan plane is
shifted by a small amount (approximately 400 .mu.m) relative to the
back aperture of the relay lens. This shift makes the apparent scan
plane reside at the front focal plane of the objective, and
eliminates the magnification effect. Alternatively, the length of
the relay lens itself can be altered to eliminate the magnification
effect.
[0108] Also shown in FIG. 10 is a close up of the back of a GRIN
endoscope in accordance with an example embodiment of the instant
disclosure. Further, FIG. 10 shows where the pivoting of the beam
takes place (approximately 400 .mu.m above the surface of the relay
lens 1000). This shifts the plane of scanning downstream so that
the beam pivots at the front focal plane of the GRIN objective,
shown in detail in FIG. 10B. The central "chief rays" of the
different scanned angles are parallel to the axis of the endoscope,
and therefore no magnification changes will result when shifting
the focal plane.
[0109] The telecentric effect is further demonstrated in FIG. 11,
which shows only the chief rays of the same scan angles from FIG.
10 to highlight the fact that they are parallel 1100 to the optical
axis. Shown in FIG. 11 is the front focal plane 1110 of the
objective 1150. FIG. 11 also shows the ray trace of the chief rays
scanning at 0 degrees (1140), 1.5 degrees (1130) and 3 degrees
(1120) at the scan mirror. All three are parallel to the center
axis of the endoscope. Further, the pivot point of the chief rays
occurs at the front focal plane 1110 of the objective 1150 which
lies within the relay lens 1160.
[0110] The GRIN optics with the telecentric arrangement, the afocal
focusing mechanism, and the scan mirror and accompanying optics,
can be incorporated into a single imaging system. An imaging
system, consistent with the instant disclosure, is further
described below. The description of the system is separated into
two parts: description of a handheld device (HD), and description
of an integrated endoscope (IE). When an HD is combined with an IE,
scanning and signal collection necessary to generate images of deep
tissue can be performed. An example embodiment of an IE connected
with the HD is shown in FIG. 12.
[0111] An example embodiment of a handheld device (HD) can be seen
in FIG. 13A. Description of the HD will often include reference to
an IE, which will be discussed subsequently in further detail. In
the example embodiment shown in FIG. 13A, an HD 1300 is shown
connected to an attached integrated endoscope (IE) 1310 via a
precision clamping mechanism (PCU) 1305. Also included in this
example HD 1300 is a collimating unit 1345, for collimating light
1330 provided to the HD. The light 1330 is delivered by a flexible,
low dispersion fiber optic, most likely an air-core photonic
crystal fiber. Further, a focus stage 1325 is included to change
the focal plane at the sample. The collimated and focused light
then is provided to a MEMS scanning mirror 1320, as detailed above,
which reflects the light towards a dichroic mirror 1315. The light
is reflected by the dichroic mirror 1315 though an integrated
endoscope (IE) 1310. The precision clamping mechanism (PCU) 1305,
with translational adjustment, aligns the IE 1310 to the optical
axis within the HD 1300 and to the scanning mirror plane.
[0112] Also included in the arrangement shown in FIG. 13A is an
integrated power sensor 1350, which is aligned with the optical
axis within the HD 1300. The integrated power sensor 1350 provides
information regarding the alignment and functional status of the
proceeding optical elements. The integrated power sensor 1350 can
be calibrated to indicate the power delivered to the sample while
imaging.
[0113] These components can also be seen in FIG. 13B, which shows
the HD prior to integration of the components. The collimating unit
1345 of the HD is shown at the top of FIG. 13B. The collimating
unit 1345 collimates light from a light source connected to the
exterior of the unit. The collimated light would then pass through,
assuming all the components of the HD are connected, the focusing
unit 1325. A scanning unit 1355 provides a base for the MEMS mirror
that reflects the collimated light towards the dichroic mirror,
through the scan and tube lens (as described above with reference
to telecentric focusing) and then towards the sample (as described
above in FIG. 13A and further with reference to FIG. 14 below).
Also shown in FIG. 13B are example embodiments of an integrated
endoscope 1310, and a probe-clamping mechanism 1305, both discussed
in further detail below. The collection unit 1360 is the component
of the HD that both houses the dichroic mirror, directing light
towards the sample, and additionally collects the light from the
sample, and directs that light towards a signal fiber alignment
unit 1350.
[0114] Turning now to FIG. 14, which shows a light path through the
HD, an integrated power sensor 1480 utilized in an HD 1410 can
constantly measure the amount of optical power transmitted through
the optical path 1400 of the HD 1410, as reflected off a MEMS
mirror 1490 and provided to a dichroic mirror 1450, prior to
entering the optics of the IE 1420. The integrated power sensor
1480 can be situated behind a dichroic mirror 1450 in and HD
arrangement 1410. The integrated power sensor 1480 is configured to
measure the approximately 1% of the excitation light, termed
leakage light 1440, that leaks through the dichroic mirror 1450,
and the remaining 99% of the light 1430 reaches the sample. The
leakage light 1440 is directly proportional to the amount of light
that is incident on the IE optics.
[0115] The integrated power sensor 1480 can consist of a photodiode
1470 in series with a fixed value resistor. When light falls on the
sensor, the photons are converted by the photodiode 1470 into
electric current, and by passing through the resistor, a voltage is
generated that is proportional to the power of the incident light.
The voltage measurement is provided via electrical leads 1460, and
is monitored by, a highly sensitive multi-meter.
[0116] The integrated power sensor, as consistent with the instant
disclosure, can provide valuable information during sample imaging.
For example, the power sensor can indicate the functional status of
all the proceeding components. The sensor reading is recorded at a
benchmark power level after achieving optical alignment. When the
reading at that same power level decreases either gradually or
rapidly, the power change indicates that an optical element has
been damaged, or has fallen out of alignment. Further, this
indicator can be used to optimize the laser coupling into the
delivery fiber because the indicator gives a direct reading of how
much light is getting from the laser, through the fiber, and
through the optics of the device. Moreover, the integrated power
sample can be used as a real time indicator of the power delivered
to the sample while imaging. After an IE is aligned to the HD,
laser power is varied, and the power exiting the HD is correlated
to the reading on the power sensor. Using these readings, the
operator will have knowledge of how much power is present at the
tip of the endoscope, and therefore prevent tissue damage from
excessive laser power.
[0117] An integrated endoscope (IE) has been developed that allows
for puncturing of thick tissue (e.g., muscle), and delivery of
imaging optics in the same step. The IE has additional features
that make it possible to collect both high quality static and
dynamic images. The basic construction of the IE is seen in FIGS.
15A and 15B. The IE is composed of a GRIN based microendoscope and
a rigid needle with a cutting point 1510; an outer insulating
jacket, and a solid base that connects to a handheld device (as
described in detail above). The IE also contains additional
adjustment points to fine tune the alignment with the optical
pathway (shown in further detail in FIGS. 23A-B and FIG. 24) within
the HD. FIG. 15A also shows a wire for stimulation or sensing 1500,
a part of the IE, that is connected to the needle with cutting tip
and internal GRIN endoscope 1510. Further, the IE includes a
suction port connection 1520 for blood removal. FIG. 15B shows an
embodiment of the IE containing two ports: a blood removal suction
port 1520, and a second port 1540 for providing a fluid to the
sample. FIG. 15B additionally shows the separate elements of the
needle 1510, which is described in further detail with reference to
FIG. 16 and FIGS. 18-21.
[0118] The microendoscope, described in detail above and shown
again in FIG. 16, consists of a relay lens 1640, a more powerful
objective lens 1630, and a 45-45-90 prism 1620. The prism allows
for "side viewing" with the endoscope. The "side viewing"
orientation provides multiple benefits. The tissue can be punctured
directly with the IE itself. Additionally, there is no need for a
separate delivery cannula. Furthermore, by translating the IE
axially, it is possible to image different fibers within the same
injection.
[0119] The endoscope cannot directly image along its axis due to
the IE incorporating a needle with a solid point 1650. The needle
1650 is displayed transparently in FIG. 16 to facilitate
visualization of the internal components and their spatial
relationship to the needle. The prism 1620 bends the focused light
from the objective 1630 out to the side of the IE. A laser light
1610 is provided through the endoscope, and passes through the GRIN
relay 1640, and the GRIN objective 1630, where it is reflected by
the prism 1620 and creates a focused spot 1600 outside of the IE.
In this way, a sample positioned at the side of the IE can be
visualized.
[0120] Turning now to FIGS. 17A and 17B, which show muscle fibers
1700 positioned along the side of the IE. The muscle fibers make
contact with the outer surface of the IE 1720, putting them in
direct contact with the imaging optics. In FIG. 17A, the striped
pattern represents the sarcomere pattern 1700. The focus 1730
exiting the prism, or excitation cone, is perpendicular to the axis
of the muscle fibers 1700. From the side, the focus 1730 is in the
fibers near the periphery. Translating (indicated by the arrows in
FIG. 17B) the needle 1710 within the hole allows direct imaging of
separate fibers without the need for multiple injections.
[0121] Turning to FIG. 18, which shows an example needle 1810 that
is used with the IE. The needle shown in FIG. 18 is machined out of
a straight wire. A channel 1800 is cut along the axis of the wire
that allows the GRIN optics to fit inside with the prism at the end
of the channel, and flush to the perimeter. The needle 1810 has a
solid tip (shown in detail in FIG. 19) which can be machined into a
tri-facet trocar geometry, which is highly efficient at penetrating
tissue. The three faces intersect at three edges which act like
blades to slice through tissue. This arrangement helps ensure that
the needle punctures the fascia that envelops the muscle, and also
minimizes patient discomfort since there is less force needed to
deliver the needle. The tri-facet trocar geometry can be replaced
with an additional arrangement that minimizes damage or distortion
of the sample at the injection site. In another example embodiment
of the needle design, a hollow steel tube is used, as opposed to a
manufactured wire, and a solid tip is attached. The solid tip can
be manufactured into a cutting tip.
[0122] Turning now to FIG. 19, this figure shows a detailed
arrangement of the three faces of the needle 1900. The three faces
of the needle 1900 intersect in a symmetric 120 degree pattern. The
facets are aligned such that the cutting edge formed by the bottom
two facets lies within the plane 1920 that bisects the optics
channel. This orientation minimizes the likelihood that one of the
cutting edges would slice the imaged muscle fibers. Instead, the
muscle fibers will run along the flat face, and reach the prism
intact. Images taken of punctured muscle verify that the fibers at
the imaging site are not damaged. Other tip geometries could also
be used for the solid tip. Also shown in FIG. 19 is the focused
spot 1910 relative to the three faces of the needle 1900.
[0123] FIG. 20 shows suction line and GRIN optics placement in a
needle in an example embodiment of the instant disclosure. The
channel 2040 can be machined with a standard endmill, which
produces an extruded rectangular pocket. The channel 2040 is large
enough for the prism to fit inside, which means that the round
cross sections of the GRIN lenses 2010 do not fully fill it. This
provides an area where suction lines 2000 can be placed to remove
blood. Additionally, the suction lines 2000 can be used for
delivery of a liquid. For example, formulations can be injected to
the imaging site to either aid with data collection, or to produce
some other observable effect. The formulations can be a drug to
reduce pain, clotting, to produce sustained contractions, or to
inhibit contractions. In an example embodiment, the suction lines
2000 are cast into place using a medical device grade epoxy and
Teflon.RTM. coated wires. The coated wires are placed in the gap
between the GRIN endoscope 2010, and the side wall of the needle
channel 2040. The coated wires run the length of the endoscope, and
rest above the prism 2030 so that they exit the needle channel near
the end 2020. When the endoscope is secured in place, epoxy fills
the voids between the channel 2040 and the GRIN lenses 2010, and
encapsulates the coated wires. Epoxy does not stick to the Teflon.
Therefore, after the epoxy sets, the coated wires are removed,
leaving a clean tube that runs the length of the IE, thus creating
the suction lines 2000.
[0124] In certain embodiments, the suction lines are merged at a
suction connector, therefore, both lines either provide suction or
both deliver fluid. In other embodiments, the suction lines are
independent of one another, therefore, one line could be used to
inject saline, for example, while the other provides suction to
clean the image site.
[0125] In an example embodiment, wires that are 100 .mu.m in
diameter are used. The holes at the top end of the IE can be
plugged later, and a connector is secured to allow a small tube to
connect to the IE, which draws blood out.
[0126] Turning now to FIG. 21, a wire segment 2120 is attached at
the end of the suction lines in order to avoid the suction lines
coring out muscle tissue when the IE is injected. The smooth
suction inlets 2100 are shown relative to the focused spot 2110.
The smooth suction lines 2100 can be achieved, for example, by
scraping by the epoxy until the wire 2120 is exposed, and polishing
the arrangement clean. Tissue that contacts the inlets 2100 while
the needle is being injected will run into the wire 2120, and slide
over without coring because the wire 2120 is round and smooth. In
another example embodiment, the wire segment is not used, and
instead a geometry of the suction site that prevents coring is
utilized.
[0127] The needle-optics package is secured inside a polymer jacket
that electrically insulates the endoscope. A wire is attached to
the needle at the top of the IE that enables the user to monitor
electric signals at the image site, or to apply a voltage to the
muscle and illicit a local contraction. In FIG. 22, the outer
jacket 2210 and the wire 2200 are shown near the base of the IE.
The wire attaches to the base of the needle, so the tip of the IE
is in electrical communication with this wire. In another example
embodiment, the needle can have multiple, isolated conducting
pathways that extend to the tip. Therefore, the needle can
simultaneously excite contraction, and record electrical signals,
or a differential method of measuring signals can be utilized.
[0128] Incorporating a separate integrated endoscope (IE) into a
small, all-in-one device, such as the handheld device (HD), is
difficult due to the need for precise alignment of the optics. The
alignment procedure of an IE and HD is described in detail with
reference to the figures. Turning now to FIGS. 23A and 23B, the
base of the IE is a large "button" 2350 that has a trapezoidal
cross section. The button 2350 can be made out of brass or any
acceptable metal substitute. The faces of the IE button 2350 are
coupled to the clamping surfaces 2300 on the HD, and rigidly fix
the IE to the Probe Clamp Unit body 2370. The "plane-line-point"
clamping mechanism, described in further detail below, enables the
IE to be taken on and off the HD, and return to the same clamped
position with very high precision allowing for sterilizing the IE.
The IE should first be aligned, and then autoclaved, as the IE
should be autoclaved between uses. The clamps 2300 on the HD can be
moved to provide coarse alignment of the IE to the optical path of
the HD. In another example embodiment, the optics of the needle
could be changed to have a selection of different IEs with
different optical properties. Therefore, the "plane-line-point"
clamping mechanism would allow the user to swap the different IEs
with different optical properties in and out at will without the
need for individual alignment.
[0129] This clamping mechanism is referred to as the Probe Clamp
Unit (PCU). Proper position of the IE with the HD is important for
maintaining optical alignment, which is directly related to the
optical performance of the HD. The clamping strategy aims to locate
the IE relative to the HD with particularity. A body in space has
six degrees of freedom, and therefore can be fixed in a space by
utilizing six points of constraint. A plane is defined by three
points, a line by two, and a point by one yielding six points in
total. FIGS. 23A and 23B show the PCU 2370 and an IE that is
clamped in place.
[0130] The bottom of the button 2350 rests against the base plane
2320 of the PCU 2370. The base of the PCU acts as a planar
constraint since it is machined flat. When clamped, the IE is
constrained to translations within this plane. The line constraint
exists in the form of a long pin 2340 that contacts one of the
angled surfaces 2330 of the IE base. The long pin 2340 is held in
one of the clamp jaws 2300, and contacts the 45 degree face 2330 on
the button. This constrains the IE to translations along this line
within the original plane. The flat side of the button 2350
contacts a vertical pin 2310 which constrains the IE to a point on
the previously fixed line 2340, and therefore fully constrains the
IE.
[0131] The function of the PCU is precision (not accuracy). In
other words, the PCU mechanism positions an IE in a particular
location, with a low level of variation, but the location is not
unique. The clamped location of the IE can be changed by
translating either of the constraint clamps 2300. Translating the
constraint clamps alters the actual location of the clamped IE, but
will not change the repeatability of the positioning in the new
location. The translating constraint clamps can be utilized to
adjust the optical alignment between the HD and the IE prior to
imaging. The location of the laser beam within the HD can certainly
deviate slightly from its ideal location. Therefore, when the laser
beam reaches the IE, the optics within the IE may be slightly out
of alignment. The moveable clamps 2300 on the PCU 2370 allow the IE
to translate within the constraint plane, and maximize optical
alignment.
[0132] To insure each individual probe is optimally aligned, the
instant embodiment also allows for additional alignment freedom in
the IE, because there is also variation within the IE itself. The
base of the IE 2350 allows additional alignment of the optics
within the needle to the optical path of the HD once the clamp
positions are set. As shown in FIG. 24, the additional alignment
can be accomplished by loosening and tightening the opposing pairs
of set screws 2400/2410 to move the needle-endoscope portion of the
IE within the pocket of the IE base. Once optimal alignment is
achieved, the needle-endoscope portion of the IE can be fixed
rigidly to the base. Different IEs can be swapped in and out of the
HD in any given order without the need for additional alignment.
This functionality allows for imaging multiple subjects
quickly.
[0133] In order to ensure accurate alignment of the IE and the HD,
an example clamping procedure of the IE and the PCU is provided.
Turning to FIGS. 25A-D as a reference, a first clamping force is
generated by the spring loaded block 2500. When engaged, the
springs within this mechanism push an angled block 2505 against the
IE base 2510, and generate a lateral and downward force that
secures the IE relative to the HD.
[0134] The solid arrows of FIGS. 25A-D represent an applied forced
in proportion to arrow size, and a dotted line indicates the
translation of the components on which the dotted lines rest.
[0135] In order to secure the IE to the HD, turning to FIG. 25A,
the spring loaded block 2500 is retracted to make space for the IE
base 2510, and position the HD over the IE base 2510. As shown in
FIG. 25B, gentle pressure should be applied on the spring loaded
block 2500, which will squeeze the IE base 2510 between the line
constraint pin 2515, and the angled portion 2505 of the spring
loaded block 2500.
[0136] While maintaining pressure on the spring loaded block 2500,
slight pressure is applied to a push rod 2520 to translate the IE
base along the pin line until it contacts the point constraint 2530
on the opposing clamp 2535, as shown in FIGS. 25B-C. Turning to
FIG. 25D by way of example, while continuing to maintain pressure
on both the spring loaded block 2500 and the push rod 2520, the
pressure on the spring loaded block 2500 should be increased. The
increased pressure applied to the spring loaded block 2500 will
compress the springs until a latch engages, and fixes the spring
loaded block 2500 in place. The springs provide a consistent
lateral clamping load that also pushes the IE base 2510 down into
the PCU base 2540 due to the angled surfaces 2545 of the IE base
2510.
[0137] In certain embodiments of the instant disclosure, a rapid
injector is provided to deliver the integrated endoscope (IE) to a
sample (i.e., muscle) of interest beneath the skin. The use of an
injector yields repeatable, clean, and less painful injections of
the IE. As can be seen in FIG. 26, the injector consists of a
spring 2620 loaded plunger that connects to the base of the IE
prior to injection. A locking mechanism 2630 prevents the IE from
accidentally shooting out of the injector. The locking mechanism
2630 can be a dual movement locking mechanism. Further, the
injector includes a threaded portion on the end of the plunger with
a stroke limiting block 2600 which makes it possible to adjust the
depth of needle injection. A trigger 2610 is provided to release
the spring 2620, and inject the needle.
[0138] FIG. 27 shows the dual locking mechanism in more detail. In
the normal position, as shown in FIG. 27A, the base of the knob
2700 prevents the angled clamp 2710 from pivoting so the IE 2720
cannot be removed. FIG. 27B shows that the knob 2700 must be
rotated clockwise 2730, and pushed down 2740, to pivot the angled
clamp 2710 and release the IE 2720.
[0139] An example procedure for imaging a human muscle with a
handheld device (HD), including an integrated endoscope (IE), can
be seen with reference to FIG. 28A-C. An appendage of a human is
first stabilized, and an area of interest is identified, FIG. 28A.
A needle is inserted into the subject, FIG. 28B, and the handheld
device, FIG. 28C, is attached and aligned (as described in detail
above).
[0140] Clear muscle sarcomere images can be achieved through use of
the attachment procedure. For example, a tibialis anterior muscle
image is shown in FIG. 29.
[0141] In an example embodiment, consistent with the instance
disclosure, a method is described for imaging an aspect of
biological tissue, including muscle, via a light-delivering optical
probe (in the biological tissue). The method is performed by using
an objective, during focal plane changes, to focus the objective in
the probe on the biological tissue while lessening adverse
image-quality degradation. In certain specific embodiments,
magnification effects are eliminated during focal plane changes.
The method continues by using the optical probe to send light
pulses toward structure in the biological thick tissue at a
sufficiently fast line-resolution rate to mitigate motion artifacts
due to physiological motion. In certain embodiments, the light
pulses overfill the optical probe to deliver maximum resolution
regardless of the focal plane changes, and in other embodiments,
the light pulses provided to the optical probe are collimated and
pivoted at a back aperture of the optical probe. Using the optical
probe, as part of the method described, causes, in response to the
light pulses, signals to be generated from and across a sufficient
portion of the structure to span a sarcomere length. In using the
probe select ones of the generated signals are collected, the
signals are predominantly present due to properties intrinsic to
the structure. As part of the method, data is provided in response
to the collected signals. The data is used for high-resolution
imaging of said portion of the tissue structure.
[0142] In certain specific embodiments of the method for imaging,
the objective of the optical probe, used during focal plane
changes, is characterized as telecentric. Further, magnification of
the optical probe remains constant during focal plane changes.
Moreover, in other specific embodiments of the method of imaging,
constant power and maximum resolution of the light pulses are
maintained during the focal plane changes. Constant power is
maintained by providing the light-pulses with a constant beam
waist. Maximum resolution is maintained by provided light-pulses to
overfill a back aperture of the optical probe.
[0143] Another embodiment of the method uses an afocal lens
arrangement to add convergence or divergence to the light pulses
thereby shifting the focal plane at the biological thick tissue. In
those embodiments utilizing an afocal lens arrangement, the afocal
lens arrangement includes a mobile lens and a fixed lens. In other
embodiments of this method, the light pulses provided to the
optical probe are collimated and pivoted at a back aperture of the
optical probe.
[0144] The instant disclosure is also directed towards an apparatus
for imaging an aspect of biological tissue, which includes muscle.
In certain embodiments, this apparatus can be used in a method of
imaging an aspect of biological tissue (including muscle). The
apparatus includes a light-delivering optical probe. The optical
probe is configured and arranged with one end to be placed in the
biological tissue. The light-delivering optical probe is further
configured and arranged to send light pulses toward structure in
the biological thick tissue at a sufficiently fast line-resolution
rate to mitigate motion artifacts due to physiological motion.
Moreover, the light-delivering optical probe is designed to cause,
in response to the light pulses, signals to be generated from and
across a sufficient portion of the structure to span a sarcomere
length; and collect select ones of the generated signals that are
predominantly present due to properties intrinsic to the structure.
The apparatus additionally includes a telecentric objective and a
collector. The telecentric objective in the light-delivering
optical probe is designed to lessen adverse image-quality
degradation during focal plane changes on the biological tissue by
maintaining constant magnification during the focal plane changes.
The collector is configured and arranged to provide data in
response to the collected signals for high-resolution imaging of
said portion of the tissue structure.
[0145] In certain specific embodiments of the apparatus, the
optical probe further includes an afocal lens arrangement that is
designed to maintain maximum resolution of the optical probe. In
other specific embodiments, the optical probe can include an afocal
lens arrangement configured and arranged to maintain constant power
of the optical probe. The telecentric objective, in other
embodiments of the apparatus for imaging an aspect of biological
tissue, is designed to maintain constant magnification of the
optical probe. In other embodiments, the apparatus also includes an
afocal lens arrangement configured and arranged to provide
convergence or divergence to the light pulses.
[0146] The instant disclosure also details a method for imaging an
aspect of biological tissue, including muscle, via a
light-delivering optical probe in the biological tissue. The method
of the instant example embodiment is utilized by providing the
optical probe with a needle, the optical probe and needle being
integrated with an electro-mechanical end portion that is
configured and arranged to puncture the tissue and while in the
tissue, electro-optically access the biological tissue. In certain
specific embodiments, the signals communicated between the
electro-mechanical end portion and the biological tissue are those
optically sensed for muscular contraction and/or for measuring the
resulting sarcomere changes in response to electrically stimulating
as another inventive aspect. Moreover, in other embodiments, the
signals communicated between the electro-mechanical end portion and
the biological tissue are to electrically stimulate. The method
continues by using the optical probe to send light pulses toward
structure in the biological thick tissue, and cause, in response to
the light pulses, signals to be generated from and across a
sufficient portion of the structure in the biological thick tissue
to span a sarcomere length. Further, in response to signals
communicated between the electro-mechanical end portion and the
biological tissue, selected ones of the generated signals are
collected, the generated signals are predominantly present due to
properties in the structure. The method then operates by providing
data in response to the collected signals for high-resolution
imaging of said portion of the tissue structure.
[0147] In certain specific embodiments, the light pulses are sent
toward structure in the biological thick tissue at a sufficiently
fast line-resolution rate to mitigate motion artifacts due to
physiological motion. Another embodiment of the method of imaging
an aspect of biological tissue is further characterized in that the
signals communicated between the electro-mechanical end portion and
the biological tissue include signals which are electrically
stimulating signals as well as the responsive optically sensible
signals. Moreover, the responsive optically sensible signals are
useful for detecting muscular contraction and/or for measuring the
resulting sarcomere changes.
[0148] The needle of the optical probe used in the method can be,
in certain embodiments, translated axially relative to tissue
enabling multiple independent measurements from a single injection.
Further, in other embodiments, the needle has differing optical
properties for wide field or high resolution imaging.
[0149] The instant disclosure is also directed towards an optical
imaging apparatus for imaging an aspect of biological tissue
including muscle via an imaging-processing microscope and a
light-delivering optical probe in the biological tissue. The
optical imaging apparatus, in the example embodiment now described,
includes an optical probe with a needle. The optical probe and
needle are integrated with an end portion that is designed to
puncture the tissue, and while in the tissue, electro-optically
access the biological tissue. The optical imaging apparatus further
includes an engageable clamp-mechanism interface. The engageable
clamp-mechanism interface is configured and arranged to attach the
optical probe with the imaging-processing microscope while
maintaining optical alignment for imaging processing of the
biological tissue.
[0150] Another embodiment of the instant disclosure is directed
towards a method for imaging an aspect of biological tissue
(including muscle) using a light-delivering optical probe having an
objective. The method, of an example embodiment consistent with the
instant disclosure, operates by providing a portable device for
processing optical-signal data from the optical probe. Further, the
method works by puncturing the tissue with the probe, and while in
the tissue, collecting signals for high-resolution imaging of the
tissue while lessening adverse image-quality degradation by
controlling and maintaining power level and light-beam resolution
for light pulsed through the light-delivering optical probe and
objective. In certain specific embodiments, the portable device
utilized in the method is hand-held.
[0151] The instant disclosure is also directed towards a method for
imaging an aspect of biological tissue including muscle via a
light-delivering optical probe having an objective. The method is
characterized by puncturing the tissue with the probe, and while in
the tissue, collecting signals for high-resolution imaging of the
tissue while lessening adverse image-quality degradation by moving
fluid near a biological-tissue image site for improving optical
clarity via the objective. In certain specific embodiments of this
method, moving fluid involves removing blood from the
biological-tissue imaging site, and in other embodiments, moving
fluid involves providing saline to the biological-tissue imaging
site.
[0152] In certain specific embodiments, the method of imaging is
further characterized in that the optical probe is used to send
light pulses toward structure in the biological thick tissue at a
sufficiently fast line-resolution rate to mitigate motion artifacts
due to physiological motion; cause, in response to the light
pulses, signals to be generated from and across a sufficient
portion of the structure to span a sarcomere length; and collect
selected ones of the generated signals that are predominantly
present due to properties intrinsic to the structure. In this
specific embodiment, the method provides data, in response to the
collected signals, for high-resolution imaging of said portion of
the tissue structure.
[0153] Methods Summary
[0154] Instrumentation.
[0155] In vivo imaging was performed on a laser-scanning microscope
(Prairie) equipped with a wavelength-tunable Ti:Sapphire laser (Mai
Tai, Spectra-Physics) and adapted to accommodate a microendoscope
(see Jung, J. C. & Schnitzer, M. J., "Multiphoton Endoscopy,"
Opt Lett 28, 902-904 (2003), and Jung, J. C. Mehta, A. D., Aksay,
E., Stepnoski, R. & Schnitzer, M. J., "In Vivo Mammalian Brain
Imaging Using One- and Two-photon Fluorescence Microendoscopy," J
Neurophysiol 92, 3121-3133 (2004). In most SHG studies, we used
920-nm-illumination. Epi-detected emission was band-pass filtered
(ET460/50m, Chroma). A 10.times.0.25 NA objective (Olympus, PlanN)
focused illumination onto the microendoscope. Static images were
acquired at 512.times.512 pixels with 8 .mu.s pixel dwell time.
Line-scans were 256-512 pixels long with 4 .mu.s dwell time.
[0156] Animal Imaging.
[0157] After anesthetizing adult C57bl/6 mice, we placed a
microendoscope inside or atop the muscle via a small skin incision.
We used 1-mm- and 350-.mu.m-diameter doublet microendoscopes
(Grintech), respectively exhibiting 0.48 and 0.4 NA and 250-.mu.m-
and 300-.mu.m-diameter working distances in water.
[0158] Human Imaging.
[0159] Under sterile conditions, a stainless steel clad
350-.mu.m-diameter microendoscope was inserted into the proximal
region of extensor digitorum via a 20-gauge hypodermic. We used a
350-.mu.m-diameter microendoscope (Grintech) with a 1.75 pitch
relay and a 0.15 pitch objective of 0.40 NA and 300-.mu.m-working
distance. In one situation, line-scan images were acquired at 488
Hz in the exterior digitorum muscle of a human subject with digits
of the hand flexed and/or extended.
[0160] Data Analysis.
[0161] Mean sarcomere lengths in static and dynamic images were
computed in Matlab (Mathworks) by calculating the autocorrelation
across an image region that was one pixel wide and parallel to the
muscle fiber's long axis. An 11.sup.th-order Butterworth band-pass
filter selective for 1-5 .mu.m periods was applied to the
autocorrelation. Fitting a sine to the resultant yielded the
dominant periodicity and mean sarcomere length. Analysis of length
variations relied on measurement of individual sarcomere lengths
performed at each pixel by finding distances between successive
intensity peaks along a line parallel to the fiber's long axis.
Locations of these peaks were found by fitting a one-dimensional
Gaussian to each high-intensity region. A 2.times.5 pixel median
filter, with its long axis aligned to the muscle fiber, smoothed
the resultant image of sarcomere lengths.
[0162] Methods
[0163] In Vitro Imaging.
[0164] Single muscle fibers were prepared by enzymatic dissociation
of tibialis anterior from C57bl/6 mice using a method modified from
Carroll et al. Tibialis anterior from a freshly sacrificed adult
C57lb/6 mouse was incubated in 0.2% collagenase (Sigma, type IV)
solution for 3-4 hours. After incubation, single fibers were
obtained by trituration with a wide-mouth pipette, transferred to
90% Ringer's solution (in mM, 2.7 KCl, 1.2 KH.sub.2PO.sub.4, 0.5
MgCl.sub.2, 138 NaCl, 8.1 NaHPO.sub.4, 1.0 CaCl.sub.2); pH 7.4) and
10% fetal bovine serum, and incubated for <1 day. The imaging
system comprised a custom laser-scanning microscope equipped with a
wavelength-tunable, ultrashort-pulsed Ti:Sapphire laser
(Spectra-Physics, Mai Tai) and a 40.times. water 0.80 NA objective
(Olympus, LUMPLFL). 720-nm-illumination was used to generate
autofluorescence that was collected in the epi-direction and
filtered with BG40 colored glass (Schott). 920-nm-illumination was
used to generate SHG that was collected in the trans-direction by
an identical 40.times. water microscope objective and filtered by
an ET460/50m filter (Chroma). In some experiments using SHG, the
polarization of the laser light was varied with a half-wave plate
to verify polarization dependence or to optimize signal intensity.
Acquired images were four frame averages of 512.times.512 pixels
using an 8 .mu.s pixel dwell time.
[0165] Animal imaging. All animal procedures were approved by the
Stanford Institutional Animal Care and Use Committee. Adult C57bl/6
mice were anesthetized by injection of ketamine (0.13 mg/g) and
xylazine (0.01 mg/g i.p.). The hindlimb was shaved and fixed to a
frame such that joint angles could be controlled. The imaging site
was periodically irrigated with Ringer's solution. In experiments
on sarcomere dynamics, we stimulated the muscle supra-maximally
using a muscle stimulator (Medtronic, model 3128) with tungsten
wires surrounding the proximal tibial nerve, which innervates the
lateral gastrocnemius. We generally used either a 1-mm-diameter
doublet microendoscope (Grintech, GmbH), composed of a 0.75 pitch
Li-doped gradient refractive index (GRIN) relay lens of 0.2 NA
coupled to a 0.22 pitch Ag-doped GRIN objective lens of 0.48 NA and
250-.mu.m-working distance in water, or a stainless steel clad
350-.mu.m-diameter doublet microendoscope (Grintech, GmbH),
composed of a 1.75 pitch Li-doped GRIN relay lens of 0.2 NA coupled
to a 0.15 pitch Ag-doped GRIN objective lens of 0.40 NA with a
300-.mu.m-working distance in water. We performed laser
line-scanning by first acquiring a reference image in two spatial
dimensions and then choosing a linear path parallel to the long
axis of the fiber for subsequent line-scanning.
[0166] Model of sarcomere length versus joint angle. The change in
muscle-tendon length (dl.sup.int) with change in ankle joint
rotation angle (d.theta.) was determined using:
dl mt d .theta. = ma , ##EQU00001##
where ma is the moment arm of the muscle. The moment arm and its
variation with joint angle were determined by calculating the
distance to the joint's center of rotation along the direction
normal to the muscle's line of action. We calculated the change in
muscle fiber length (dl.sup.m) with change in ankle angle during
passive motion by assuming that tendon stretch was negligible and
thus:
dl m d .theta. = ma ( cos .alpha. ) , ##EQU00002##
where a is the pennation angle of the muscle fibers. Once the
change in muscle fiber length with ankle angle was computed, the
change in sarcomere length (dl.sup.s) with joint angle (FIG. 3) was
estimated using:
dl s d .theta. = ma cos .alpha. ( l o s / l o m ) ,
##EQU00003##
where the optimal muscle fiber length (l.sup.m.sub.o) was
determined by measuring the fiber length at the resting joint
angle. The sarcomere length at the optimal fiber length
(l.sup.s.sub.o) was assumed to be 2.8 .mu.m.
[0167] Human Imaging.
[0168] All human imaging procedures were performed in accordance
with FDA guidelines for the protection of human subjects (21 CFR
50) and approved by the Stanford Institutional Review Board.
Subjects' forearms were restrained in a brace and fixed to the
microscope's vibration-isolation table. All optical components were
identical to those used during animal studies. However, all
components, including microendoscopes and mounting components,
contacting or potentially contacting human subjects at the imaging
site were sterilized by autoclaving. After insertion of the
microendoscope, subjects were asked to flex and extend their
fingers and changes in sarcomere length were monitored. Duration of
testing was <60 minutes in all cases.
[0169] Data Analysis. Band-pass filtered images of sarcomeres were
computed from raw images by applying an 11.sup.th-order Butterworth
filter that acts as a band-pass for spatial periods between 1-5
.mu.m. All analyzed images contained between 20 and 50 sarcomeres.
For each muscle fiber, average sarcomere length was determined
along each of a series of parallel lines aligned with the axis of
the fiber. We report the mean and s.e.m. of this collection of
measurements. Determinations of accuracy in average sarcomere
length measured along a single line used the 95% confidence
interval generated by a nonlinear least-squares curve fitting
algorithm (Trust-Region algorithm, nonlinear least-squares method).
All data analysis was done in Matlab (Mathworks).
[0170] Assessment of Sarcomere Visibility.
[0171] The intensities of epi-detected SHG and autofluorescence
signals are influenced by several wavelength-dependent processes,
including attenuation of illumination in thick tissue, generation
of signal photons at the focal plane, scattering of signal photons,
and attenuation of signal photons within the detection pathway.
Both the spatial arrangement and the contrast ratio between the
maximum and minimum signal intensities observed within individual
sarcomeres also influence sarcomere visibility. After exploring the
illumination wavelength range of 720-980 nm using our tunable
Ti:Sapphire laser, we found that given this light source and the
transmittance characteristics of our microscope, SHG imaging with
illumination of .about.920 nm was most effective at revealing
sarcomeres in vitro. We do not claim that 920 nm is the optimum
excitation wavelength for imaging sarcomeres in thick muscle
tissue, but rather that SHG imaging with 920-nm-illumination
permits characterization of sarcomere lengths and dynamics in live
subjects.
[0172] Potential Measurement Errors.
[0173] To minimize chances of photo-damage during imaging we
maintained incident laser illumination below 30 mW, a reported
approximate threshold for tissue damage. We also monitored for any
physical signs of damage in the tissue. If a component of a muscle
fiber or its lateral inter-fiber connections were substantially
damaged, one might expect to see punctate, local differences in
sarcomere structure distinct from surrounding tissue. We did not
observe such effects, but rather observed sarcomeres with
relatively uniform and smoothly varying lengths. We also performed
control studies in which we tested quantitatively for any
differences in sarcomere lengths between paired measurements
obtained just prior to and then immediately after insertion of the
microendoscope into the muscle. Prior to insertion we measured
sarcomere lengths in the unperturbed muscle using an air objective
(Olympus, 20.times., 0.4 NA, LMPlanFL). We then inserted a
microendoscope into the same tissue site and measured sarcomere
lengths again. Comparison of the paired data sets revealed that
sarcomere length determinations were virtually identical under the
two conditions, differing by only 3.8.+-.2.4% (mean.+-.s.d.; n=45
measurement sites) and thereby precluding any substantial errors
due to microendoscope insertion.
[0174] Another potential source of measurement error is parallax
due to misalignment of the microendoscope's optical axis relative
to the muscle fibers' transverse planes. However, a measurement
error of just 1% would require a misalignment of over 8 deg, which
was not observed in our three-dimensional data sets acquired with
the microendoscope placed atop the muscle. In the mouse lateral
gastrocnemius we found that muscle fibers were nearly parallel to
the face of the microendoscope. From three-dimensional image sticks
we measured an average misalignment of 3.3.+-.1.8 deg
(mean.+-.s.d.; n=37 measurements from 4 stacks acquired in 4 mice).
Such consistent mechanical alignment probably results in part due
to pressure from the microendoscope on the muscle fibers. We
conclude that in the lateral gastrocnemius measurement errors due
to orientational misalignment are usually negligible. Similarly,
misalignment errors seem likely to be minor in muscles in which the
fibers lie parallel to the surface of the muscle, but perhaps less
so in muscles in which the fibers vary significantly from this
orientation.
[0175] For discussion relating to the above embodiments, reference
may be made to the appendix document (Appendix A) filed in the
underlying provisional patent application and entitled, "Direct
Observation Of Mammalian Sarcomere Extension In Skeletal Muscle
Using Minimally Invasive Optical Microendoscopy." This appendix
document and all other patent and non-patent documents cited herein
are incorporated by reference, each in its entirety.
[0176] The various embodiments described above are provided by way
of illustration only and should not be construed to limit the
invention. Based on the above discussion and illustrations, those
skilled in the art will readily recognize that various
modifications and changes may be made to the present invention
without strictly following the exemplary embodiments and
applications illustrated and described herein. As an example,
technology other than GRIN-lens technology may be used in
implementing the microendoscopes discussed above. As another
example, the above-described methods and arrangements for using
lead channels having multiple optic probes are applicable to both
skeletal sarcomere and cardiac sarcomere. Such modifications and
changes do not depart from the true spirit and scope of the present
invention, which is set forth in the following claims.
* * * * *