U.S. patent application number 16/140270 was filed with the patent office on 2019-05-09 for live being optical analysis system and approach.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junio University. Invention is credited to Eric David Cocker, Benjamin A. Flusberg, Juergen Claus Jung, Mark J. Schnitzer.
Application Number | 20190133449 16/140270 |
Document ID | / |
Family ID | 51177963 |
Filed Date | 2019-05-09 |
United States Patent
Application |
20190133449 |
Kind Code |
A1 |
Flusberg; Benjamin A. ; et
al. |
May 9, 2019 |
LIVE BEING OPTICAL ANALYSIS SYSTEM AND APPROACH
Abstract
Analysis of live beings is facilitated. According to an example
embodiment of the present invention, a light-directing arrangement
such as an endoscope is mounted to a live being. Optics in the
light-directing arrangement are implemented to pass source light
(e.g., laser excitation light) into the live being, and to pass
light from the live being for detection thereof. The light from the
live being may include, for example, photons emitted in response to
the laser excitation light (i.e., fluoresced). The detected light
is then used to detect a characteristic of the live being.
Inventors: |
Flusberg; Benjamin A.; (Palo
Alto, CA) ; Cocker; Eric David; (Palo Alto, CA)
; Jung; Juergen Claus; (Palo Alto, CA) ;
Schnitzer; Mark J.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junio
University |
Stanford |
CA |
US |
|
|
Family ID: |
51177963 |
Appl. No.: |
16/140270 |
Filed: |
September 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15967148 |
Apr 30, 2018 |
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16140270 |
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15727317 |
Oct 6, 2017 |
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15967148 |
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15443999 |
Feb 27, 2017 |
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15727317 |
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14322517 |
Jul 2, 2014 |
9636020 |
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15443999 |
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11338598 |
Jan 24, 2006 |
8788021 |
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14322517 |
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60646858 |
Jan 24, 2005 |
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60646711 |
Jan 24, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61B 5/0075 20130101;
G01N 21/6458 20130101; A61B 2576/026 20130101; A61B 5/0059
20130101; G01N 21/6486 20130101; G01N 21/65 20130101; A61B 5/0062
20130101; G01J 3/4406 20130101; A61B 5/0079 20130101; A61B 5/4064
20130101; A61B 5/0064 20130101; A61B 2503/40 20130101; G01N 21/6456
20130101; A61B 5/6814 20130101; G01N 21/6428 20130101; A61B 5/0068
20130101; G01J 3/44 20130101; A61B 5/0071 20130101; G01N 21/6408
20130101; A61B 5/0013 20130101 |
International
Class: |
A61B 5/00 20060101
A61B005/00; G01N 21/64 20060101 G01N021/64 |
Goverment Interests
FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with Government Support under
contract 0352456 awarded by the National Science Foundation and
contract N00014-04-1-0826 awarded by the Department of the Navy.
The U.S. Government has certain rights in this invention.
Claims
1-4. (canceled)
5. An imaging system for imaging a cell in vivo, comprising: a base
plate configured to affix on a subject, wherein said subject
comprises said cell; a housing capable of removably attaching to
said base plate; and an optical arrangement at least partially
enclosed by said housing, wherein said optical arrangement allows
for optical communication between said cell, a light source, and a
light detector.
6. The imaging system of claim 5, further including the light
source and the light detector configured and arranged to (1)
generate a signal from light collected by the light source, and (2)
pass said signal to an image processor that is configured to
generate structural cellular resolution images.
7. The imaging system of claim 6, wherein the optical arrangement
further includes an objective lens that is configured to (i)
provide an optical path for the source light from the light source
to one or more cells and (ii) provide an optical path for the
response light from the one or more cells to the light
detector.
8. The imaging system of claim 5, wherein the optical arrangement
further includes optics which direct the source light to one or
more cells and the light source is configured to provide source
light to the one or more cells, wherein the source light induces a
response light from the one or more cells.
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. 15/967,148 filed on
Apr. 30, 2018, which is a continuation of U.S. patent application
Ser. No. 15/727,317 filed on Oct. 6, 2017 (abandoned), which is a
continuation of U.S. patent application Ser. No. 15/443,999 filed
on Feb. 27, 2017 (abandoned), which is a continuation of U.S.
patent application Ser. No. 14/322,517 filed on Jul. 2, 2014 (U.S.
Pat. No. 9,636,020), which is a continuation of U.S. patent
application Ser. No. 11/338,598 filed on Jan. 24, 2006 (U.S. Pat.
No. 8,788,021), which claims the benefit under U.S.C. .sctn. 119(e)
of U.S. Provisional Patent Application Ser. No. 60/646,711,
entitled "Live Being Optical Analysis System and Approach" and
filed on Jan. 24, 2005, and of U.S. Provisional Patent Application
Ser. No. 60/646,858, entitled "Optical Analysis Systems and
Approaches" and also filed on Jan. 24, 2005; each of these patent
documents is fully incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to optical analysis,
and more particularly to in vivo imaging approaches involving the
analysis of live beings.
BACKGROUND
[0004] A variety of approaches to optical imaging have been used
for many different applications, such as for research, testing and
treatment of diseases or other illnesses. For example, endoscopes
and other imaging devices have been used for imaging tissue in
anesthetized animals.
[0005] With the growing number of approaches to the analysis of
live beings, there has been growth in technologies benefiting from
such analysis as well as in the need to perform extensive analysis
of the impact of such technologies. For example, imaging cellular
and sub-cellular functions in live animals is desirable for many
areas of biological research. Fluorescent probes have been
implemented for expression in specific cell classes of
genetically-engineered animals. Fluorescence microendoscopy
involving one- or two-photon fluorescence excitation has been used
to image biological cells in tissue, and have been implemented with
relatively deep-tissue analysis. See, e.g., J. C. Jung and M. J.
Schnitzer, Opt. Lett. 28, 902 (2003); see also J. C. Jung, A. D.
Mehta, E. Aksay, R. Stepnoski, and M. J. Schnitzer, J.
Neurophysiol. 92, 3121 (2004). These and other approaches are
useful, for example, in the development of new drugs and
therapeutics.
[0006] In many applications, optical imaging requires anesthetized
or otherwise immobilized subjects. These requirements have
presented challenges not only to the ability to obtain optical
data, but to the analysis of subjects. For instance, an
anesthetized or immobile state may be available under limited
conditions of time and environment.
[0007] The above and other issues have presented challenges to
optical analysis approaches and, in particular, to optical imaging
in live beings.
SUMMARY
[0008] The present invention is directed to overcoming the
above-mentioned challenges and others related to the types of
devices and applications discussed above and in other applications.
These and other aspects of the present invention are exemplified in
a number of illustrated implementations and applications, some of
which are shown in the figures and characterized in the claims
section that follows.
[0009] According to an example embodiment, an optical analysis
approach involves connecting a light-directing arrangement to a
live being for stimulating and detecting a response from the live
being. A light source, such as a laser, provides light to the
light-directing arrangement, which couples the light into a portion
of the live being using a directing mechanism such as a
micro-mirror or micro-movable actuator. The light is impinged upon
a target region of the live being. An optical response from the
live being is collected and passed to a light detector for
analysis. The connected nature of the light-directing arrangement
facilitates the maintenance of a spatial relationship between the
directing mechanism with the live being. With this approach, a live
being can be analyzed using a fixed light-directing arrangement
without necessarily anesthetizing the being and, where appropriate,
facilitating the ability of the live being to move while under
analysis.
[0010] According to another example embodiment of the present
invention, a freely-moving live being is analyzed. A housing is
fixed to the live being and a light scanning arrangement is coupled
to the housing to hold the light scanning arrangement in a position
relative to the live being. Stimulation light is passed to the
light scanning arrangement and, using the light scanning
arrangement, the stimulation light is selectively scanned across a
target portion of the live being while the freely-moving being
moves about a controlled environment. Response light is passed from
the light director arrangement to a light detector, which receives
and detects the response light. The detector further generates a
signal corresponding to the detected light for use in analyzing the
live being.
[0011] In another example embodiment of the present invention,
brain tissue in a freely-moving live animal is imaged using an in
vivo fluorescence approach. An endoscope arrangement is fastened to
the skull of the freely-moving live animal using, for example,
screws or other fasteners. The endoscope arrangement is thus held
in a position relative to the live animal's brain tissue, such that
the animal can move freely over a period of time. In this context,
free movement of the animal involves, for example, movement of the
animal within a cage or to the extent that light and/or electrical
conduits coupled to the endoscope arrangement allow. Pulsed laser
stimulation light is passed to the live animal's brain tissue via
the endoscope arrangement. The wavelength of the pulsed laser
stimulation is selected to stimulate a fluorescent response that is
limited to a point of focus of the pulsed laser stimulation light
in the brain tissue. A point of focus, in this context, involves a
small area, such as that immediately adjacent a focal point in a
plane of focus of the laser light. Photons emitted via the
fluorescent response of the brain tissue over the period of time
are passed via the endoscope to a detector where the photons are
detected. These detected photons are then analyzed to detect a
condition of the live animal.
[0012] According to another example embodiment of the present
invention, an optical analysis system facilitates in vivo analysis
of a freely-moving live being. The system includes a light source,
a light scanning arrangement and a housing to which the light
scanning arrangement is coupled. A light stimulation conduit such
as a fiber optic cable passes stimulation light from the light
source to the light scanning arrangement. The housing is fastened
to the freely-moving live being (e.g., to its skull where brain
tissue is to be imaged) and, thereby, holds the light scanning
arrangement in a position relative to the live being while it moves
about a controlled environment such as in a cage. The light
scanning arrangement selectively scans stimulation light from the
light source across a target portion of the live being. A light
response conduit passes response light from the light director
arrangement to a light detector that receives and detects the
response light and generates a signal corresponding to the detected
light for use in analyzing the live being.
[0013] In various applications, the above and other approaches are
implemented, for example, in applications such as biological
research, the development of new drugs and therapeutics, with the
stimulation of a live being, and corresponding detection of a
response, facilitated via these approaches.
[0014] 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
[0015] 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:
[0016] FIG. 1 illustrates an optical analysis system for imaging
within a live being, according to an example embodiment of the
present invention;
[0017] FIG. 2 illustrates optical and electronic components of an
imaging device, according to another example embodiment of the
present invention;
[0018] FIG. 3 illustrates a microendoscopy device that includes a
liquid lens, according to another example embodiment of the present
invention;
[0019] FIGS. 4A and 4B show a microendoscope device, according to
other example embodiments of the present invention; and
[0020] FIG. 5 illustrates an optical analysis device in which a
laser source and light detector are embedded in a chip that is
mountable on the head of a live being, according to another example
embodiment of the present invention.
[0021] 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
[0022] The present invention is believed to be applicable to a
variety of different types of devices and approaches, and the
invention has been found to be particularly suited for the analysis
of live beings, and of live beings in a freely-moving state. While
the present invention is not necessarily limited to such
applications, various aspects of the invention may be appreciated
through a discussion of various examples using this context.
[0023] According to an example embodiment of the present invention,
an approach to optical analysis with a live being involves the
mounting (e.g., coupling or affixing) of a light directing
arrangement to the live being in a manner that facilitates the live
being's movement while optically analyzing the being. In this
context, the being is generally free to move about as would occur
under regular living conditions for that being, such as in a home
setting (for human beings) or in a controlled environment such as a
caged or fenced-in type of setting (e.g., for an animal).
[0024] The fixed nature of the light directing arrangement relative
to the subject being analyzed facilitates the stimulation of, and
corresponding detection of related responses to, target portions in
the live being. The light directing arrangement is adapted to
direct source light into the being at a selected target location.
For instance, in some applications, light is directed to target
locations at subcutaneous locations in a live being, such as into
brain tissue of a mouse, rat or similar animal. Further, the fixed
nature of the light directing arrangement facilitates analysis over
time, such as hours, days, weeks or even months; this analysis is
thus facilitated while allowing the subject being analyzed to move
freely over such time periods under what could be considered
otherwise normal life conditions.
[0025] The source light is generated in a variety of manners. In
some applications, the source light is generated using a light
source mounted to the live being; in other applications, the source
light is received from an external light source using, e.g., a
fiber optic link to present the light to the light directing
arrangement. The light is directed into the live being using one or
more of a variety of approaches, such as those implementing an
actuator, micro-electrical mechanical (MEMS) micro-mirror or other
direction approach. In the context of these applications, the
source light may be implemented to stimulate a response in the live
being, or for collection upon reflection from the live being for
approaches such as illumination and/or those involved with the
detection of reflective characteristics of the being undergoing
analysis.
[0026] Light from the being (e.g., reflected or emitted) is
directed and/or collected using the light director discussed above
and/or another arrangement. Directed light is passed to a detector
where the light is detected and used in analyzing the being. The
detector is selectively located on the live being (e.g., mounted
with the light directing arrangement) or remotely from the live
being, with a light conduit type of device used to pass the light
to the detector.
[0027] In the context of the approaches discussed herein, a variety
of live beings may be analyzed using one or more of these
approaches. In some applications, the live being is an animal such
as a rat, bird or mouse, allowed to generally move freely about a
controlled environment such as a cage while undergoing analysis. In
certain implementations, the live being is an animal configured
(e.g., via genetics, substance injection or viral vector
introduction) to generate a particular response, relative to light
stimulation. For example, fluorescent markers are selectively
injected or genetically engineered into tissue to selectively tag a
particular molecular species within specific classes of cells;
responses associated with these fluorescent markers are detected
and used to analyze the tissue. With these approaches, responses of
the live being to particular treatments can be monitored and, where
appropriate, monitored over time while facilitating the ability of
the live being to move under generally normal conditions. Further,
certain response-generating activities such as those related to
treatment with pharmaceuticals can be monitored over time, where
treatment can be effected to the live being while the being is
undergoing analysis.
[0028] In various embodiments, the approaches discussed in the
previous paragraph are implemented in connection with the analysis
of outwardly behavioral aspects of the live being undergoing
analysis. For example, responses of a live being to treatment can
be monitored using the above approaches while also monitoring
behavioral factors of the live being under relatively normal
movement conditions. Relative to the analysis of anesthetized
beings, these approaches facilitate a real-time analysis of a live
being as it experiences relatively normal living conditions. One
such approach may involve, for example, the monitoring of brain
tissue in a live being over a period of time (e.g., weeks, months
or longer) as the live being undergoes a particular treatment;
during this time, physical behavior of the live being can also be
monitored. Psychological responses and/or characteristics of the
live being can be similarly monitored. In this regard, a variety of
behaviors such as those relating to drug abuse, athletic
performance and learning can be correlated to detected
characteristics of underlying cells supporting the behavior over
the course of time. Changes in behavior relative to subcutaneous
tissue monitoring, at an instant and/or over a period of time, can
further be detected and used with corresponding conditions of
underlying cells in research and/or treatment of conditions. In
some applications, such approaches are used with drug research,
facilitating the discovery of new drugs and the testing of new
pharmaceutical agents.
[0029] Various embodiments are implemented with the observation of
both internal response and outward behavior as discussed above.
With these approaches, the behavior of cells in physiologic
contexts is used to reveal and to confirm native cellular
characteristics, where cellular properties mutually interact with
organ systems physiology. For example, anesthetized animals may not
necessarily experience stress, exercise, fear, hunger, arousal or
other conditions that can be implemented with a freely-moving being
with approaches discussed herein. In this regard, interplay between
cellular mechanisms and physiological factors in alert animals are
thus studied using detected responses to stimulation applied via a
light directing arrangement as discussed above. Furthermore,
certain applications are directed to the examination of cellular
underpinnings of phenomena such as problem solving, memory, or
social behavior.
[0030] In another example embodiment of the present invention, an
optical imaging system comprises a light director arrangement and a
housing structure. The light director arrangement is adapted to
selectively direct stimulation light from a light source to a
target area in the live being. The stimulation light generates
response light in the target area, and the light director
arrangement is adapted to direct the response light from the live
being to a light detector. The housing structure is adapted to be
fixed to the live being, such as by mounting via screws or glue to
tissue, bone or other portions of the live being. The housing
structure is further adapted to hold the light director arrangement
in relative proximity to the live being to facilitate the selective
direction of stimulation light to the target area and to facilitate
the direction of the response light. For instance, where the light
director arrangement includes a fixed device such as a mirror or
fiber optic actuator, the housing is adapted to hold the fixed
device in a position that is relative to the live being. In this
regard, when the live being moves, the housing holds the fixed
device in a consistent position, relative to the position of the
target area.
[0031] In various example embodiments, an optical imaging system
attached to a live being is formed in a relatively compact and
lightweight arrangement amenable to use with small beings, such as
an adult mouse. For instance, an adult mouse typically used with
testing and analysis approaches often can bear up to about 3.5-4
grams of weight on its head while still being able to behave
normally. In this regard, light direction and collection components
of optical analysis equipment are manufactured in a manner that
facilitates equipment exhibiting such light weight, such as by
using small-scale devices such as MEMS-type devices
(micro-electro-mechanical systems devices). In some applications,
the optical imaging system is flexibly implemented so that the live
being is free to move around during imaging, with system components
that are not mounted directly on the mouse being connected to the
mouse by wires, optical fibers, wireless links or other flexible
components.
[0032] In one example embodiment of the present invention, an
optical imaging system employs scanning imaging modality such as
two-photon fluorescence imaging, and includes components for
scanning a light beam that is delivered to the live being's body.
For instance, a laser source can be implemented to direct laser
light (e.g., pulsed at a femtosecond rate) via an optic conductor
such as a fiber optic cable. The optic conductor is coupled to
imaging components at the live being's body. Certain applications
are directed to the generation of the laser light at the live
being's body, with the light from the laser passed to a target
region of the live being directly from the laser or via a
relatively short on-body type of light conductor.
[0033] In various applications, fluorescence imaging approaches as
discussed herein involve multiphoton fluorescence excitation, with
multiple photons emitted (e.g., scattered) from a target location
in a live being and detected to facilitate three-dimensional
imaging. Such approaches facilitate imaging of thin optical
sections that can be obtained through hundreds of microns of tissue
(e.g., with the point of focus of laser or other light directed to
the target location).
[0034] Lasers and/or other light sources and arrangements
implemented with these approaches are adaptable for directing light
to target locations at various portions in a live being. For
instance, surface or near-surface structures, such as those at or
just below a cutaneous layer, are readily imaged using such
approaches. Similarly, deep tissue structures of live beings in a
conscious, behaving state can also be imaged, in connection with or
independently from near-surface imaging. As discussed at various
portions herein, certain deep tissue structures can be imaged using
a capillary-type approach, where a semi-permanent capillary is
selectively implanted into the live being, leaving an exposed end
of the capillary open to acceptance of optical imaging components
such as light source (or conduit) and light direction
components.
[0035] In various implementations, one or more imaging and
spectroscopic modalities are used to probe cellular or molecular
properties in a freely moving, genetically engineered or transgenic
animal such as a mouse, using an attached light direction type
arrangement that facilitates optical analysis thereof. Other
applications are directed to the analysis of an animal having
undergone delivery of viral vectors (e.g., that encode fluorescent
or non-fluorescent proteins), or having undergone RNAi (RNA
interference). Examples of applicable analysis approaches include,
but are not limited to, conventional fluorescence imaging, confocal
fluorescence imaging, multi-photon fluorescence imaging, second
harmonic generation (SHG), third harmonic generation (THG), Raman
spectroscopy, and coherent anti-stokes Raman scattering (CARS),
fluorescence lifetime imaging, fluorescence resonance energy
transfer (FRET), fluorescence recovery after photobleaching (FRAP),
and types of polarization sensitive imaging. Such an imaging system
could comprise miniaturized microscopes, endoscopes, or other types
of biosensors.
[0036] In one particular embodiment, two-photon fluorescence
microendoscopy is used to probe cellular and molecular properties
in freely moving, genetically engineered mice. Two-photon
fluorescence facilitates (e.g., relative to certain confocal
modalities for imaging within biological tissue) one or more of:
depth sectioning without use of a confocal pinhole, reduced
photobleaching and phototoxicity, and reduced scattering through
the use of longer wavelength excitation. Moreover, certain
applications are directed to the use of a longer wavelength,
relative to single-photon fluorescence imaging, to facilitate the
nonlinear excitation of a fluorochrome in the being and thereby
generally limit the fluorochrome excitation to the point of focus
of the photons. Limiting the fluorochrome excitation in this manner
facilitates the aforesaid reduced photobleaching and
phototoxicity.
[0037] In some applications, two-photon fluorescence microendoscope
probes are implemented with minimally invasive compound gradient
refractive index (GRIN) lenses that are about 350-1000 .mu.m in
diameter. The GRIN lens microendoscopes approach is implemented
with flexible fiber-optic technology and a stable, compact and
lightweight housing for attaching to live beings such as mice. With
this approach, the imaging system can be used for probing both
surface and deep tissue structures in freely moving beings such as
genetically engineered mice. Certain figures and corresponding
discussion below describe examples equipment that can be
implemented with such applications.
[0038] According to another example embodiment of the present
invention, an optical imaging system is fastened or coupled to a
live being for imaging the live being by selectively implementing
one or more approaches as described above. The system includes a
light source, light detector, a light director arrangement, a
housing, a light conduit that passes source light from the light
source to the light director arrangement, and another (common or
separate) light conduit that passes response light from the light
director arrangement to the light detector.
[0039] The light director arrangement is adapted to couple to the
live being via the housing and to selectively direct stimulation
light from the light source to a target portion in the live being
and to direct response light from the live being to the light
detector. The housing is fixed to the live being and holds the
light director arrangement in a position relative to the live being
while the light director arrangement selectively directs
stimulation and response light.
[0040] The light detector is adapted to receive the light directed
from the live being, via the light conduit that passes response
light. The light conduits (separably or in combination) may
include, for example, one or more of a fiber optic cable, a mirror
and an air medium. Furthermore, the light director arrangement may
include and/or make up part of one or both of the light
conduits.
[0041] The light director arrangement is implemented using one or
more of a variety of components and approaches, depending upon the
application. In one implementation, the light director arrangement
includes a fiber optic cable coupled to an actuator, with the
actuator adapted to move the fiber optic cable to selectively
direct light into the live being (e.g., to scan light across tissue
in the live being). In another implementation, the light director
arrangement includes a mirror arrangement adapted to move to
selectively direct light into the live being, such as via
translation and/or rotation.
[0042] The light source includes one or more of a variety of types
and arrangements of light sources, such as those discussed herein.
In one instance, the light source is an external source such as a
laser, with light from the source being coupled to the light
director arrangement using a fiber optic cable or other means for
passing source light. In another instance, the light source is a
local source coupled to the light director arrangement and
includes, for example, a laser mounted to the housing. Where the
laser is local, the light conduit is selectively integrated with
the light director arrangement, for example where a MEMS mirror is
implemented to receive light from a local laser source and scan
that light into the live being.
[0043] The light detector arrangement includes one or more of a
variety of detectors, such as those discussed herein. The light
detector may be located remote to the light director arrangement,
with light coupled to the remote detector via a fiber optic cable,
a mirror or other device for passing response light. In some
instances, a light detector is implemented locally with the light
director arrangement and includes, for example, a light responsive
component and a communications arrangement for communicating a
signal representing the detected light for use in analyzing the
live being.
[0044] FIG. 1 shows an arrangement 100 that can be implemented with
a variety of approaches to two-photon imaging of brain material,
such as in a freely moving mouse 102 as shown, according to another
example embodiment of the present invention. The arrangement 100
readily facilitates analysis of brain tissue while allowing the
being undergoing analysis to move relatively freely (e.g., about a
cage), further facilitating the observation of behavioral
characteristics of the being. Such an approach is applicable, for
example, to those approaches discussed above in connection with the
combined analysis of internal and external portions of a being over
time to facilitate real-time analysis of responses to drugs or
other conditions of treatment. That is, the mouse 102 can be
observed for physical or psychological behavior from an outward
perspective while correspondingly observing the mouse's brain
tissue, with observations being correlated to one another and,
where appropriate, to treatment applied to the mouse.
[0045] The arrangement 100 includes an endoscope arrangement 110
coupled to a live being (here shown coupled to the mouse 102), and
which selectively includes GRIN optics. The endoscope arrangement
110 is implemented using one or more of a variety of devices, such
as that shown in and discussed in connection with FIG. 2. In some
applications, the endoscope arrangement 110 includes a base 112
adapted to mount to a live being and, in some applications, to
actuate, translate or otherwise control endoscope components. For
purposes of this discussion, the remaining portions of FIG. 1 are
discussed in the context of analyzing the mouse 102; however, as
described herein a multitude of different types of live (or
non-live) beings are selectively analyzed using a similar
approach).
[0046] The endoscope arrangement 110 passes light from a light
source to the mouse 102, using one or more of a variety of
approaches such as scanning, pulsing, or otherwise directing the
light. The endoscope arrangement 110 also passes light from the
mouse to an external detector. This light from the mouse may
include, for example, reflected light and/or emitted light, and in
the latter case may include light emitted in response to light
directed to the mouse, such as that stimulated via single- or
two-photon excitation.
[0047] A variety of light sources and accompanying light-directing
arrangements can be implemented with the endoscope 110 for
analyzing the mouse 102. By way of example, a laser 120 is shown as
a light source and may include one or more of a variety of lasers.
In some applications, the laser 120 is a Ti:sapphire laser that
generates short pulses of laser light at intervals on about a
picosecond or femtosecond frequency (e.g., 100-150 fs intervals at
790-810 nm).
[0048] Light from the laser 120 is selectively pre-chirped using
optics including one or more of a combination of mirrors and
gratings or prisms to compensate for possible chromatic dispersion
incurred when passing the light to the mouse 102. When such a
pre-chirping approach is used, light from the laser 120 is passed
via light-directing devices including a (beamsplitting) mirror 130,
grating 132 and 134, mirror 136 and lens 138, all of which are
selectively included in a common arrangement as suggested by the
dashed lines around the same. In this regard, light from the laser
120 is selectively passed through the mirror 130 to the grating
132, on to grating 134 and reflects off of the mirror 136 back
through the grating 134 and 132, respectively, and from mirror 130
to lens 138.
[0049] The laser light is focused into a light conduit 140, which
can be implemented using a variety of light-conducting devices. In
this instance, the light conduit 140 is shown by way of example as
implemented with a hollow-core photonic crystal fiber that
generally mitigates unwanted nonlinear optical effects, such as
self-phase modulation, via mode propagation mainly in the air core.
When implemented, for example, with a Ti:sapphire laser with short
pulses as discussed above, the laser light passes in the
lowest-order mode of the light hollow-core photonic crystal
fiber.
[0050] The light exits the light conduit 140 and enters the
endoscope device 110, which focuses the light into the brain of the
mouse 102 for exciting tissue therein. The endoscope device 110 is
selectively controlled by a computer 150, via a wired link 152 or
wireless link for passing control signals (e.g., for scanning or
otherwise directing light to the mouse 102). In some applications,
the light facilitates two-photon endoscopy via two-photon
excitation of tissue in the mouse 102 (or other subject).
Fluorescence photons emitted in response to the laser excitation
are collected by the endoscope 110 and then delivered using another
light conduit 142, shown by way of example as a large-core
multimode fiber, to a light detector 160, such as a photomultiplier
tube.
[0051] The light detector 160 generates an output signal 162 that
is passed to the computer 150, which is programmed with image data
processing code. The computer 150 uses the output signal 162 to
construct an image or other information characterizing the tissue
in the mouse 102, and selectively displays the image or information
on a monitor. This output is selectively used, as discussed above,
to correlate conditions of the tissue in the mouse 102 with
behavior of the mouse that is outwardly observable (e.g., by a
caretaker, either directly or as monitored electronically).
[0052] FIG. 2 shows a microendoscope arrangement 200 adapted for
mounting to a live being, according to another example embodiment
of the present invention. In some applications, the microendoscope
arrangement 200 is implemented with an approach such as that shown
in FIG. 1, in connection with the endoscope arrangement 110. A base
205 is used to couple the arrangement 200 to a live being, such as
by inserting a fastener such as a screw through the base 205 and
into the skull of a mouse for neural observation.
[0053] The microendoscope arrangement 200 includes an optics
arrangement 210 having a micro-prism 212 (e.g., beam splitter) and
a probe arrangement (e.g., a doublet GRIN lens probe) including a
relay lens 214 and an objective lens 216 (e.g., implemented with
one or more lenses, or a lens array, of glass, plastic or other
material). In one application, the objective lens 216 has a 0.48 NA
and 0.22 pitch, and the relay lens 214 has a 0.2 NA and 0.14 pitch
or a 0.11 NA and 0.18 pitch.
[0054] Light received via the coated microprism 212 is passed
through the relay lens 214 and the objective lens 216 to a target
location of a live being. Light from the live being is passed
through the objective lens 216, the relay lens 214 and the
micro-prism 212 to a light conduit 220 such as a multimode fiber
(e.g., similar to light conduit 142 in FIG. 1, such as a polymer
fiber having a 980 micrometer-diameter core and 0.51 NA).
[0055] A micromotor 250 and piezo scanner 240 are coupled to a
source light conduit 222, such as a photonic bandgap fiber, that
supplies light from a source such as a laser. The light conduit 222
may, for example, be implemented in a manner similar to the light
conduit 140 in FIG. 1. A stiffening piece 230 is optionally
implemented to stiffen or otherwise facilitate a connection between
the light conduit 222 and the piezo scanner 240.
[0056] The micromotor 250 is optionally coupled to the base 205 via
a translation arrangement 255, such as a track, gear or other
device that allows controlled movement of the micrometer relative
to the base in a manner that moves the light conduit 222 laterally
to focus light to the micro-prism 212. In certain applications, the
micromotor 250 is fixed and includes a screw, track or gear coupled
to the piezo scanner 240 for moving the scanner laterally. For
example, the piezo scanner 240 can be clamped to a shuttle that
runs along a threaded output shaft of the micromotor 250. An
electrical signal applied to the micromotor 250 translates the
piezo scanner 240 and light conduit 222 axially with respect to the
micro-prism 212. This motion adjusts the location of the light
focus (e.g., excitation laser light) within the subject by tens to
hundreds of microns and gives the user fine control over the
focus.
[0057] The piezo scanner 240 (e.g., about 5-10 mm long) is coupled
to the micromotor 250 and moves therewith, and is further coupled
to the light conduit 222 directly and, in some applications, the
stiffening piece 230. In this regard, when the micromotor 250 moves
laterally, the piezo scanner 240 and correspondingly, the light
conduit 222, move with it. The piezo scanner 240 moves the light
conduit 222 in a vertical direction as shown by the double arrow
241, to effect the scanning of the resultant light source as shown
by the double-arrow 242. The piezo scanner 240 bends from microns
to hundreds of microns in response to an applied voltage, thus
actuating the light conduit 222 to scan a target area. A length of
the light conduit 222 (e.g., less than one millimeter to tens of
millimeters) extends from the piezo scanner 240. The stiffening
piece 230 couples the orthogonal axes of the piezo scanner 240, and
thus the one-dimensional motion of the piezo scanner causes the tip
of the light conduit 222 to move.
[0058] Movement of the light conduit 222 is effected in different
ways to achieve resultant scanning of a live being. For example,
the piezo scanner 240 is selectively actuated to facilitate
resonant vibration of the tip of the light conduit 222. In some
applications, the stiffening piece 230 or another piece of fiber is
implemented between the piezo scanner 240 and the light conduit 222
to facilitate a split in resonant frequencies of lateral vibration.
The piezo scanner is correspondingly driven with a voltage signal
including both resonant frequencies to drive the tip of the light
conduit in a Lissajous pattern, with the ratio of the driving
frequencies selected to set the sampling density. In some
applications, the Lissajous pattern is demagnified 3.2 to
5.2.times. (times) at the sample (live being) and is selectively
adjusted in size by setting the amplitude of the aforesaid drive
voltages. With these approaches, scan fields of up to about 145 to
215 micrometers are selectively achieved, depending on the
endoscope probe used. For general information regarding Lissajous
patterns and for specific information regarding Lissajous-type
patterns that may be implemented in connection with example
embodiments discussed herein, reference may be made to F. Helmchen,
M. S. Fee, D. W. Tank, and W. Denk, Neuron 31, 903 (2001), which is
fully incorporated herein by reference.
[0059] In some applications, the translation arrangement 255
provides a third degree of movement in a direction generally
perpendicular to the shown movement with double arrows 241 and 242
(i.e., in a Cartesian "Z" direction, where lateral movement of the
micromotor 250 is in the "X" direction and the vertical movement
with the piezo scanner 240 is in the "Y" direction). With this
approach, light from the light conduit 222 can be scanned in a
plane.
[0060] In one implementation, the arrangement 200 is used to
facilitate two-photon excitation fluorescence with a live being,
wherein the light conduit 222 is scanned vertically to facilitate
the lateral scanning across a sample to which the arrangement 200
is coupled. The piezo actuator scans the light conduit 222 in a
two-dimensional Lissajous pattern.
[0061] In various applications, the tip of the optics arrangement
210 at the objective lens 216 is placed above or inserted into
tissue of a live being and positioned over a target region to be
imaged. Light from the live being is passed via the light conduit
220 to a remotely placed optical detector. In some applications,
the light conduit 220 is removed and a microscope objective is
positioned over the micro-prism 212 to facilitate one-photon
fluorescence imaging in an epi-fluorescence configuration.
[0062] In one embodiment, the working distance of the system
microendoscope arrangement 200 (the distance from the exit face of
the relay lens 214 to the focal plane) is selectively set via the
optical properties of the particular relay lens chosen and by the
distance between the light conduit 222 and the micro-prism 212.
This working distance is selected to effect a change in the working
distance of the imaging system, and is facilitated further via the
adjustment of focus achieved with lateral movement effected via the
micromotor 250 as discussed above.
[0063] In some applications, a set screw and spring loaded, movable
wedge included with the base 205 facilitates the adjustment of the
height of the light source optics (micromotor 250, piezo scanner
240 and light conduit 222) with respect to the base. This
adjustment may, for example, involve a vertical adjustment of a few
millimeters to a few centimeters, and is selectively used for
choosing a depth and region within the tissue for imaging. In
certain implementations, such a screw and movable wedge are
implemented to facilitate the use of different length relay lenses
(214) with different optical properties for tailoring the
microendoscope arrangement 200 for resolution, field of view and/or
working distance conditions in delivering light to a live being.
Once the region to be imaged is located, the optics arrangement 210
is left in place, and all subsequent focal adjustments are
performed by the micromotor.
[0064] In another embodiment, a clip or other fastener holds the
piezo scanner 240 and is adapted to swivel about two orthogonal
axes, facilitating the alignment of the light conduit 222 to direct
light (e.g., excitation laser light) to the center of the
micro-prism 212. In some applications, this approach is implemented
to compensate for any misalignment of the light conduit 222.
[0065] As discussed above, the components described with FIG. 2 can
be attached to a compact, protective housing (i.e., base 205) that
is mounted to one or more of a variety of live beings, such as a
mouse's skull. The weight of the entire device, including the
housing and all of the optical and electronic components, is
selected to facilitate implementation with the particular live
being undergoing imaging. For instance, where a mouse's brain is
imaged, the weight is small enough to allow the mouse to carry the
device freely on its head (e.g., less than about 4 g for most adult
mice) and go about relatively normal activity. The connections
between the device mounted on the live being and remotely located
components may include, for example, flexible optical fibers for
delivering the excitation light and for collecting a response such
as light emitted via fluorescence, and any control wires for
controlling the piezo scanner 240 and micromotor 250.
[0066] In some applications, the endoscope arrangement 200 is
controlled via wireless communications to control, for example, the
micromotor 250 and piezo scanner 240. A wireless receiver is
included with one or both of the micromotor 250 and piezo scanner
240, or otherwise with the arrangement 200 and coupled to control
the micromotor and piezo scanner. By way of example, a wireless
receiver 207 is shown implemented with the base 205 and
communicates using, for example, wireless Ethernet signals,
Bluetooth signals, infrared signals or other signals. Control
signals received via the wireless receiver are processed and used
in the actuation of one or both of the micromotor 250 and piezo
scanner 240.
[0067] In another example embodiment and referring again to FIG. 2,
a light source 260 is implemented at the endoscope arrangement 200
and at the live being. This light source 260 may include, for
example, a chip-mounted laser or other small-scale light source.
The light conduit 222 is arranged to receive light from the light
source 260 and to pass light to the micro-prism 212 as discussed
above. In some applications, the light source 260 is mounted to
move with the light conduit 222 in response to movement from one or
both of the micromotor 250 and piezo scanner 240.
[0068] Another example embodiment is directed to the use of a light
collector or detector at the endoscope arrangement 200. By way of
example, a light detector 270 is shown arranged to receive light
via the light conduit 220. The light collector 270 may include, for
example, a photomultiplier tube as discussed in connection with
FIG. 1 (item 160), or another arrangement adapted to detect light
and to generate a signal characterizing detected light. Any such
signal generated is passed to an external device, either via a
wired connection or via a wireless connection as discussed above
(e.g., selectively using wireless receiver 207 or another wireless
receiver, separate and/or in connection with the light detector
270.
[0069] In another implementation, a tunable lens is implemented in
the optical path within the microendoscope device 200 to provide
fine focal control. In some applications, the tunable lens is
implemented in lieu of the micromotor 250. By way of example, a
tunable lens 280 is shown arranged between an end of the light
conduit 222 and before the micro-prism 212. In various
applications, the tunable lens 280 includes one or more of a liquid
lens or a liquid crystal lens (see, e.g., FIG. 3 and discussion
below for an example liquid lens that can be implemented in
connection with the tunable lens 280). An electrical signal applied
to the tunable lens 280 changes the focal length of the lens and
thus changes the working distance of the device 200. In this way,
focal control is obtained without necessarily implementing moving
parts, facilitating relatively small size and complexity.
[0070] A multitude of optical analysis approaches are selectively
implemented in connection with one or both of FIGS. 1 and 2, in
addition to or separately from the approaches discussed above such
as two-photon fluorescence imaging. The following approaches are
thus selectively implemented with the above-discussed approaches:
conventional fluorescence imaging, confocal fluorescence imaging,
multi-photon fluorescence imaging, second harmonic generation
(SHG), third harmonic generation (THG), Raman spectroscopy,
coherent anti-stokes Raman scattering (CARS), fluorescence lifetime
imaging, fluorescence resonance energy transfer (FRET),
fluorescence recovery after photobleaching (FRAP), and types of
polarization sensitive imaging. For these embodiments, appropriate
filters and optical elements are selectively implemented in
connection with or in alternative to those shown in FIGS. 1 and 2,
with the optics and arrangements shown in those figures selectively
modified to suit each particular application. Furthermore, one or
more embodiments discussed herein are implemented in a manner not
inconsistent with that described in Benjamin A. Flusberg, Juergen
C. Jung, Eric D. Cocker, Erik P. Anderson, and Mark J. Schnitzer,
"In vivo brain imaging using a portable 3.9 gram two-photon
fluorescence microendoscope," OPTICS LETTERS/Vol. 30, No. 17/Sep.
1, 2005, which is fully incorporated herein by reference.
[0071] Referring again to FIG. 2, a variety of other approaches to
directing light from the light conduit 222 are implemented in
various example embodiments. For example, different configurations
of piezo actuators could be used with or in an alternative to the
piezo scanner 240. In another example, the light conduit 222 is
fixed and small-scale mirrors such as MEMS mirrors are used to
deflect light from the light conduit 222. A general MEMS mirror
approach that may be implemented in connection with such an example
embodiment is described in U.S. patent application Ser. No.
11/338,592 (U.S. Pat. No. 7,307,774) entitled "Micro-optical
Analysis System and Approach Therefor" and filed on Jan. 24, 2006
(now U.S. Pat. No. 7,307,774), which is fully incorporated herein
by reference. In addition, the light conduit 222 includes a
coherent bundle of optical fibers, with scanning implemented at a
distal end of the fiber bundle (i.e., at an end of the light
conduit 222 that is opposite the micro-prism 212).
[0072] In another example embodiment, a coating on the micro-prism
212 separates source (excitation) and response (fluorescence)
light. That excitation light is transmitted through the coated
micro-prism while the fluorescence light is reflected off the
hypotenuse face thereof.
[0073] The micro-prism 212 is replaced with a dichroic mirror in
another example embodiment. The dichroic mirror is used to separate
source light from the light conduit 222, and response light from
the live being undergoing analysis. For example, when the source
light passed via the light conduit 222 is laser excitation light
used to stimulate a fluorescence response in a live being, the
dichroic mirror separates the laser light from fluorescence
photons.
[0074] FIG. 3 shows a liquid lens arrangement 300 having a liquid
lens 310 placed in an optical path between an excitation photonic
bandgap fiber 320 and a GRIN endoscope/prism cube 330, according to
another example embodiment of the present invention. Excitation
light from a laser light source 322 is passed by the photonic
bandgap fiber 320 to the liquid lens 310, which passes the light to
the GRIN endoscope/prism cube 330 and into a microendoscope probe
340, which directs the excitation laser light to tissue under
investigation 305. The approach shown in FIG. 3 may be implemented,
for example, in connection with the approaches discussed above with
and shown in FIGS. 1 and 2, such as with the tunable lens 280.
[0075] A variable voltage is selectively applied to the liquid lens
310 to change the shape of a liquid meniscus inside the liquid
lens, and thus changing the liquid lens' focal length. This change
in the liquid lens' focal length in turn changes the working
distance of the liquid lens arrangement 300.
[0076] In some applications, an AC voltage is applied to the liquid
lens 310 at a specified frequency to facilitate axial scanning of
the tissue 305 for volumetric imaging approaches. In various
implementations, other optical elements (e.g., lenses, filters) are
placed in the optical path, either before or after the liquid lens
310, with optical elements 312 and 314 shown by way of example.
[0077] FIGS. 4A and 4B show a microendoscope device 400, according
to another example embodiment of the present invention. FIG. 4A
shows the device 400 without a cover and FIG. 4B shows the device
with a cover in place. These approaches may be implemented, for
example, in connection with the example embodiments shown in and
described with FIG. 2.
[0078] FIG. 4A shows a base plate 405 for mounting a scanning
device on a live being's head, such as a mouse's head. A
microendoscope probe 410 is arranged to supply excitation light to
the live being, the excitation light being supplied via a bandgap
optical fiber 415. A piezoelectric actuator 420 held with a piezo
clip 422, and micromotor 425 with a shuttle 430, are mounted
together on an optical alignment arm. The bandgap optical fiber 415
is glued to the piezoelectric actuator 420 and the fiber is aligned
with a micro-prism 440 and the microendoscope probe 410. The
arrangement shown in FIG. 4A may, for example, be implemented in a
manner similar to that shown in and discussed in connection with
FIG. 2, with similar items implemented in a similar manner as
appropriate.
[0079] In FIG. 4B, a protective housing 460 is shown coupled to the
base plate 405. Optical and electrical inputs 465 respectively pass
excitation light into the microendoscope device 400 and pass
electrical signals into (and, where appropriate, from) the
microendoscope device. A light conduit 470 passes light detected
from the live being to an external analysis arrangement such as a
light detector. The housing 460 and/or base plate 405 and/or
various components shown therewith include materials such as
Delrin, nylon, titanium, and stainless steel. In one
implementation, the dimensions of the housing are approximately
35.times.15.times.8 mm, with the microendoscope device 400 weighing
less than about 4 grams. The protective housing 460 is further
selectively removed and/or modified to facilitate epifluorescence
imaging using a microscope objective; this approach is used, for
example, to determine whether fluorescent objects are in the field
of view prior to laser-scanning imaging as discussed above.
[0080] FIG. 5 shows an arrangement 500 for scanning light to tissue
in a freely-moving, live being 502, according to another example
embodiment of the present invention. The approach shown in FIG. 5
may, for example, be implemented using an approach similar to that
discussed above in connection with FIG. 1.
[0081] A chip 512 includes a laser source and light detector and is
mounted on a live being's head, here shown as a mouse by way of
example. The laser source passes light into the mouse's brain and
the light detector detects light emitted, reflected or otherwise
passed via the mouse's brain. Electronic control lines 510 couple
the chip 512 to a computer 550 and pass information from the light
detector that can be used to characterize the light from the
mouse's brain. In some implementations, the electronic control
lines are omitted and a wireless signal is passed between the
computer and the chip to facilitate communications
therebetween.
[0082] In some applications, the arrangement 500 includes an optics
and/or mechanical arrangement that effects scanning of laser light.
For instance, one such application involves the generation of laser
light on-chip with the chip 512, and directing the laser light via
one or more micromirrors as discussed hereinabove. The one or more
micromirrors are controlled to facilitate the scanning.
[0083] The various embodiments described above and shown in the
figures 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. For example, a
variety of types of light sources, light conduits, actuators and
optics can be implemented in connection with and/or as an
alternative to those shown in the figures. These approaches are
implemented in connection with various example embodiments of the
present invention. Such modifications and changes do not depart
from the spirit and scope of the present invention, including that
set forth in the following claims
* * * * *