U.S. patent application number 17/626266 was filed with the patent office on 2022-09-08 for microscopy with ultraviolet surface excitation (muse) imaging implemented on a mobile device.
The applicant listed for this patent is Case Western Reserve University. Invention is credited to Michael JENKINS, Yehe LIU.
Application Number | 20220283421 17/626266 |
Document ID | / |
Family ID | 1000006419062 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220283421 |
Kind Code |
A1 |
LIU; Yehe ; et al. |
September 8, 2022 |
MICROSCOPY WITH ULTRAVIOLET SURFACE EXCITATION (MUSE) IMAGING
IMPLEMENTED ON A MOBILE DEVICE
Abstract
A external accessory can allow a mobile device to perform
microscopy imaging with Type-C ultraviolet (UVC) light excitation.
The external accessory includes a compound lens placed in front of
a camera lens of the mobile device. A light transparent optical
window configured to be placed in front of the compound lens and
positioned such that a front surface of the optical window overlaps
the front focal plane of the compound lens. One or more light
emitting diode (LED) that emits UVC light positioned at one or more
side-edges of the optical window. The one or more LED emits UVC
light through the optical window so that the UVC light undergoes
total internal reflection. An externally triggered LED driver
configured to power and control the LED(s).
Inventors: |
LIU; Yehe; (Cleveland,
OH) ; JENKINS; Michael; (Cleveland, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Case Western Reserve University |
Cleveland |
OH |
US |
|
|
Family ID: |
1000006419062 |
Appl. No.: |
17/626266 |
Filed: |
July 16, 2020 |
PCT Filed: |
July 16, 2020 |
PCT NO: |
PCT/US2020/042214 |
371 Date: |
January 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62874688 |
Jul 16, 2019 |
|
|
|
62936757 |
Nov 18, 2019 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2201/0221 20130101;
G01N 2201/0621 20130101; G02B 21/367 20130101; G02B 21/16 20130101;
G01N 2021/6439 20130101; G01N 21/6458 20130101 |
International
Class: |
G02B 21/16 20060101
G02B021/16; G01N 21/64 20060101 G01N021/64; G02B 21/36 20060101
G02B021/36 |
Claims
1. An external accessory that allows a mobile device to perform
microscopy imaging with Type-C ultraviolet (UVC) light excitation,
the external accessory comprising: a compound lens configured to be
placed in front of a camera lens of the mobile device, wherein the
compound lens forms an imaging relay with the camera lens so that
an image of a front focal plane of the compound lens is generated
at an image sensor of the camera lens of the mobile device; a UVC
light transparent optical window configured to be placed in front
of the compound lens and to hold a sample, wherein the optical
window is positioned such that a front surface of the optical
window overlaps the front focal plane of the compound lens; one or
more light emitting diode (LED) at one or more side-edges of the
optical window, wherein the one or more LED is configured to emit
UVC light through the optical window to facilitate the UVC light
excitation of at least a portion of a sample via frustrated total
internal reflection; and an externally triggered LED driver
configured to power and control the one or more LED.
2. The external accessory of claim 1, wherein a transmittance of
the optical window is greater than 50% at the center wavelength of
the LEDs.
3. The external accessory of claim 1, wherein a center wavelength
of the UVC light is between 240 nm and 290 nm.
4. The external accessory of claim 1, wherein the compound lens is
configured to center align with the camera lens of the mobile
device.
5. The external accessory of claim 1, wherein the compound lens
comprises a reversed camera lens or an infinite corrected
microscope objective.
6. The external sensor of claim 1, wherein the optical window
comprises quartz, fused silica, and/or UV transparent sapphire.
7. The external accessory of claim 1, wherein the optical window
has a thickness that is greater than 0.5 times either a length or a
width of the one or more LED.
8. The external accessory of claim 1, wherein the LED driver is
triggered by a mechanical button, a digital signal from the mobile
device, and/or a flash light signal from the mobile device.
9. The external accessory of claim 1, wherein the LED driver is
powered by an external battery or an integrated battery of the
mobile device.
10. A method comprising: placing a sample within an imaging field
of an external accessory to a mobile device, wherein the external
accessory comprises: a compound lens configured to be placed in
front of a camera lens of the mobile device, wherein the compound
lens forms an imaging relay with the camera lens of the mobile
device so that an image of a front focal plane of the compound lens
is generated at an image sensor of the camera lens of the mobile
device; a Type-C ultraviolet (UVC) light transparent optical window
configured to be placed in front of the compound lens and to act as
a sample holder, wherein the optical window is positioned such that
a front surface of the optical window overlaps the front focal
plane of the compound lens; one or more UVC light emitting diode
(LED) positioned at one or more side-edge of the optical window,
wherein the one or more LED is configured to emit UVC light through
the optical window and wherein the UVC light undergoes total
internal reflection in the optical window until frustrated by the
sample; and an externally triggered LED driver configured to power
and control the one or more LED; and taking a photograph of the
imaging field to provide a microscopy image of the sample.
11. The method of claim 10, wherein the mobile device comprises one
or more integrated photographic camera.
12. The method of claim 10, wherein the mobile device is a
smartphone, a laptop computer, a tablet computer, or a digital
camera.
13. The method of claim 10, further comprising center aligning the
compound lens with the camera lens.
14. The method of claim 10, further comprising staining the sample
with a fluorescent histology dye.
15. The method of claim 10, further comprising dissolving a
fluorescent histology dye with a first solvent; absorbing the
solvent with a first piece of absorbent material; and drying the
first piece of absorbent material in a container.
16. The method of claim 15, further comprising wetting the first
piece of absorbent material and a second piece of absorbent
material with a second solvent; and contacting the sample with the
first piece of absorbent material and then the second absorbent
material to stain the biological sample.
17. The method of claim 16, wherein a surface tension from residual
liquid on the sample holds the sample in place within the imaging
field.
18. The method of claim 17, wherein the first piece of absorbent
material and/or the second piece of absorbent material is made of
at paper, cotton, sponge, and/or a synthetic polymer.
19. The method of claim 16, wherein the first solvent and/or the
second solvent comprises one or more of water, methanol, ethanol,
isopropanol, acetone, and xylene.
Description
CLAIM OF PRIORITY
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 62/874,688, filed Jul. 16, 2019, entitled
"Improved Microscopy Devices, Systems, and Methods" and to U.S.
Provisional Application Ser. No. 62/936,757, filed Nov. 18, 2019,
entitled "Systems and Methods for Improved Light Sheet Microscopy".
The entirety of these provisional applications is hereby
incorporated by reference for all purposes.
TECHNICAL FIELD
[0002] The present disclosure relates generally to mobile device
microscopy and, more specifically, to systems and methods that can
implement Microscopy with Ultraviolet Surface Excitation (MUSE)
imaging on a mobile device microscope.
BACKGROUND
[0003] With only small modifications (e.g., an external aspheric
compound lens), mobile devices can to be used to perform microscopy
as a research and diagnostic tool. Most mobile devices are already
equipped for photography and include at least one integrated
photographic camera (including at least a camera lens and a high
quality digital image sensor). More importantly, these mobile
devices also have sufficient computational power for image
processing and provide an internet connection for data transfer.
Such mobile devices can become microscopes in a cost effective way
by mounting a reversed camera lens (obtained for a low cost and/or
repurposed from retired technology) as the external compound lens
(or external objective lens) in front of the mobile device's
camera. Using the reversed camera lens in this manner can optimize
both the resolution (dependent on pixel size) and the field of view
(dependent on sensor size) without requiring a complex and costly
lens assembly. The resolution can be increased even more by using a
reversed camera lens designed for image sensors that are smaller
than the high quality digital image sensor of the integrated
photographic camera in the mobile device.
[0004] The focal length of these mobile device microscopes is
necessarily limited in order to achieve higher resolution. Limited
focal length is not a problem for microscopy imaging of thin
samples with transmitted light. However, limited focal lengths are
a problem when using techniques like Microscopy with Ultraviolet
Surface Excitation (MUSE) imaging, which requires type-C
ultraviolet light (UVC) to be delivered to a sample surface. UVC
cannot transmit through most glass and polymer materials, so the
original MUSE microscope configuration cast the UVC light onto the
sample surface with a light emitting diode (LED) placed between the
microscope objective and the sample. However, the original MUSE
microscope configuration would not work in a mobile device
microscope due to very limited spacing between the reversed lens
and the sample, so the MUSE microscope requires a significantly
more complicated and costly setup.
SUMMARY
[0005] Provided herein is a solution that allows imaging techniques
that can image thick samples, like Microscopy with Ultraviolet
Surface Excitation (MUSE), to be used with a mobile device
microscope. The systems and method described herein employ a
compact and low cost attachment based on frustrated total internal
reflection (FTIR) that can be used to perform high resolution MUSE
imaging (while still having the functionality of a basic
bright-field microscope).
[0006] In one aspect, the present disclosure can include an
external accessory that allows a mobile device to perform
microscopy imaging with Type-C ultraviolet (UVC) light excitation.
The external accessory includes a compound lens (also referred to
as an objective lens) that can be placed in front of a camera lens
of the mobile device. The compound lens forms an imaging relay with
the camera lens so that an image of a front focal plane of the
compound lens is generated at an image sensor of the camera lens of
the mobile device. A UVC light transparent optical window can be
placed in front of the compound lens, positioned such that a front
surface of the optical window overlaps the front focal plane of the
compound lens. One or more light emitting diode (LED) can be placed
at one or more side-edges of the optical window and can be
configured to emit UVC light through the optical window, where the
UVC light undergoes total internal reflection within the optical
window. An externally triggered LED driver can power and control
the one or more LED.
[0007] In a further aspect, the present disclosure can include a
system that can be used to perform high resolution MUSE imaging.
The system can include a mobile device and an external accessory.
The mobile device can have a camera. The external accessory can
allow the mobile device to perform microscopy imaging with UVC
light excitation. The external accessory includes a compound lens
that can be placed in front of a camera lens of the mobile device.
The compound lens forms an imaging relay with the camera lens so
that an image of a front focal plane of the compound lens is
generated at an image sensor of the camera lens of the mobile
device. A UVC light transparent optical window can be placed in
front of the compound lens, positioned such that a front surface of
the optical window overlaps the front focal plane of the compound
lens. One or more UVC-generating LED can be placed at one or more
side-edges of the optical window and can emit UVC light through the
optical window, where the UVC light undergoes total internal
reflection within the optical window. An externally triggered LED
driver can power and control the one or more LED.
[0008] In another aspect, the present disclosure can include a
method for performing high resolution MUSE imaging. A biological
sample can be placed within an imaging field of an external
accessory to a mobile device. The external accessory includes a
compound lens configured to be placed in front of a camera lens of
the mobile device to form an imaging relay with the camera lens of
the mobile device so that an image of a front focal plane of the
compound lens is generated at an image sensor of the camera lens of
the mobile device. A UVC light transparent optical window can be
placed in front of the compound lens and positioned such that a
front surface of the optical window overlaps the front focal plane
of the compound lens. One or more UVC-generating LED positioned at
one or more side-edge of the optical window and can emit UVC light
through the optical window, where the UVC light undergoes total
internal reflection within the optical window that is frustrated by
the biological sample. An externally triggered LED driver can power
and control the one or more LED. A photograph can be taken of the
imaging field of the compound lens to provide a microscopy image of
the biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The foregoing and other features of the present disclosure
will become apparent to those skilled in the art to which the
present disclosure relates upon reading the following description
with reference to the accompanying drawings, in which:
[0010] FIG. 1 is a diagram showing an example of an external
accessory that allows a mobile device to perform microscopy imaging
with Type-C ultraviolet (UVC) light excitation;
[0011] FIG. 2 is a diagram showing an example of a system that
performs microscopy imaging using the external accessory of FIG.
1;
[0012] FIG. 3 is a diagram showing an example operation of the LEDs
and optical window of the external accessory of FIG. 1 exciting a
sample particle, and the excitation being detected by the mobile
device of FIG. 2;
[0013] FIGS. 4 and 5 include photographs showing example uses of
the external accessory of FIG. 1;
[0014] FIG. 6 is a process flow diagram showing an example method
for performing mobile device microscopy with an external
accessory;
[0015] FIG. 7 is a process flow diagram showing an example method
for staining a sample for mobile device microscopy;
[0016] FIG. 8 is a process flow diagram of an example method for
performing mobile device microscopy with an external accessory of a
stained sample;
[0017] FIG. 9 is an exploded schematic showing the major components
of Pocket MUSE;
[0018] FIG. 10 shows histology images acquired with Pocket MUSE;
and
[0019] FIG. 11 shows images of a 500 .mu.m thick Thy2-GFP brain
slice acquired with Pocket MUSE.
DETAILED DESCRIPTION
I. Definitions
[0020] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which the present disclosure pertains.
[0021] As used herein, the singular forms "a," "an" and "the" can
also include the plural forms, unless the context clearly indicates
otherwise.
[0022] As used herein, the terms "comprises" and/or "comprising,"
can specify the presence of stated features, steps, operations,
elements, and/or components, but do not preclude the presence or
addition of one or more other features, steps, operations,
elements, components, and/or groups.
[0023] As used herein, the term "and/or" can include any and all
combinations of one or more of the associated listed items.
[0024] As used herein, the terms "first," "second," etc. should not
limit the elements being described by these terms. These terms are
only used to distinguish one element from another. Thus, a "first"
element discussed below could also be termed a "second" element
without departing from the teachings of the present disclosure. The
sequence of operations (or acts/steps) is not limited to the order
presented in the claims or figures unless specifically indicated
otherwise.
[0025] As used herein, the term "Microscopy with Ultraviolet
Surface Excitation (MUSE) imaging" can refer to a microscopy method
utilizing ultraviolet (UV) surface excitation to generate
high-quality images of one or more samples.
[0026] As used herein, the term "imaging" can refer to methods and
technologies for visualizing and examining structures not
observable with the naked eye. One example type of imaging is
medical imaging, in which visual representations of anatomical
structures and biological samples are created for diagnostic,
treatment, or research purposes.
[0027] As used herein, the term "microscopy" can refer to a type of
imaging that examines a sample using a microscope. Types of
microscopy can include, for example, bright field microscopy, MUSE,
a hybrid between bright field microscopy and MUSE, or the like.
[0028] As used herein, the term "microscope", also referred to as
an optical microscope or a light microscope, can refer to an
instrument that uses light and a system of lenses to generate
magnified images of a sample.
[0029] As used herein, the term "ultraviolet (UV) light" can refer
to a type of electromagnetic radiation with wavelength shorter than
that of visible light, but longer than X-rays. For example, UV
light can have a wavelength from 10 nm to 400 nm. One specific type
of UV light is Type-C ultraviolet (UVC) light, having a center
wavelength from 240 nm to 290 nm.
[0030] As used herein, the term "mobile device microscope" can
refer to a mobile device (also referred to as a mobile electronic
device) with the capability of photography, such as a smartphone, a
tablet computer, a digital camera, a laptop computer, or the like,
being used as a microscope (either alone or with one or more
accessories).
[0031] As used herein, the terms "accessory", "external accessory",
"attachment", "external attachment", or the like, can refer to a
device that can be attached or added to a mobile device to increase
the utility, efficiency, or versatility of the mobile device. For
example, an accessory can be attached to the mobile device to
increase the ability of the mobile device to operate as a
microscope.
[0032] As used herein, the term "resolution" can refer to the level
of detail contained within an image (represented by the number of
pixels, the size of the pixels, or the like). A high resolution
image can include more detail than an image with a lesser
resolution and, for example, can have pixels sized on the order of
microns/micrometers.
[0033] As used herein, the term "focal length" can refer to the
distance between the optical center of the lens to its focal
point.
[0034] As used herein, the term "imaging relay" between at least
two lenses can refer to the transmission of an image of the front
focal plane of one lens to be generated at an image sensor of
another lens. The imaging relay can be used to magnify the
specimen.
[0035] As used herein, the term "photographic camera" can refer to
an optical instrument that includes at least one camera lens and at
least one digital image sensor. The photographic camera may be
integrated within a mobile device.
[0036] As used herein, the term "camera lens" (also referred to as
a "photographic lens" or "photographic objective") can refer to an
optical lens or assembly of lenses of a photographic camera that
can be paired with an image sensor.
[0037] As used herein, the term "image sensor" can refer to a
device that converts light striking a camera lens into an
electronic signal (the electronic signal can be transformed into a
digital image by a processor, for example).
[0038] As used herein, the term "objective lens" can refer to a
lens within an optical system that is located closest to the
sample. The objective lens can be a compound lens and/or be part of
a compound lens.
[0039] As used herein, the term "compound lens" can refer to
multiple lenses that are arranged on a common axis. The compound
lens can be used to increase magnification of a sample. At least
one of the multiple lenses within the compound lens can be an
aspheric lens (in these instances, the compound lens is referred to
as an "aspheric compound lens").
[0040] As used herein, the term "sample" can refer to a small part
used for testing or examination to show what the whole is like. For
example, the sample can be a biological sample, in which the whole
is an organic material, such as blood, interstitial fluid, tissue,
bone, etc.
[0041] As used herein, the term "total internal reflection" can
refer to a phenomenon in which light traveling through one
transparent medium reaches an interface with a second, less dense
transparent medium and fully reflects back into the denser medium
at the angle of incidence (the angle at which the light hits the
interface between the two media). Frustrated total internal
reflection can refer to a phenomenon in which some of the light
hitting the interface between the two transparent media is not
reflected. This can occur when a third medium with a higher
refractive index abuts the interface between the first two
media.
[0042] As used herein, the term "exogenous" can refer to a
substance originating outside of a body.
[0043] As used herein, the term "excite", "excitation" or the like,
can refer to raising the energy of a particle, atom, nucleus, or
molecule above its ground or baseline state.
[0044] As used herein, the term "histology" can refer to the study
of microscopic biological structures.
[0045] As used herein, the term "stain", "staining" or the like,
can refer to the use of one or more selected dyes on specimens to
increase the visibility of certain structures. Examples of stains
can include a fluorescent probe, a fluorophore, or the like.
[0046] As used herein, the terms "fluorescent probe" and
"fluorophore" can refer to a fluorescent material, molecule,
substance, or the like that can change its fluorescence emission
(re-emit light) upon light excitation.
II. Overview
[0047] Mobile devices can to be used to perform microscopy using an
external aspheric compound lens (e.g., a reversed camera lens
designed for a smaller image sensor) mounted in front of the mobile
device's camera. Higher resolution can be achieved with this
microscopy set up while sacrificing the focal length, which is not
a problem for microscopy imaging of thin samples with transmitted
light, but becomes a problem when imaging thick samples. Microscopy
with Ultraviolet Surface Excitation (MUSE) imaging is a type of
imaging that can be used to image thick samples, but requires
type-C ultraviolet light (UVC) to be delivered to a sample surface
to excite the sample. UVC cannot be transmitted through most glass
and polymer materials, so the UVC must be delivered between the
microscope objective and the sample. Delivery between the
microscope objective and the sample is impossible using a mobile
device microscope due to very limited spacing between the compound
lens and the sample. The systems and methods described herein
provide a solution that allows imaging techniques like MUSE imaging
to be used with a mobile device microscope.
[0048] The systems and methods described herein employ a compact
and low cost external accessory for a mobile device that can excite
a sample based on frustrated total internal reflection (FTIR) and
this excitement can be used to perform high resolution MUSE
imaging. (It should also be noted that the external accessory
leaves the mobile device with the capabilities of a basic
bright-field microscope.) The external accessory requires only
minor structural modifications based on the original design
concepts of MUSE imaging and mobile device microscopes. The
external attachment uses a UVC-transparent optical window/sample
holder as the total internal reflection waveguide, allowing for
uniform delivery of UVC light to the sample (held onto the
microscope by surface tension) through the space between the
objective lens and the sample. Traditionally, a thin optical window
(<0.25 mm--quartz or fused silica) has been used as the sample
holder, but the external attachment uses a thick optical window
(>0.5 mm, similar to or thicker than the width of a conventional
UVC LED emitter) as both the sample holder and the total internal
reflection (TIR) waveguide. When the sample is in contact with the
optical window, TIR is disrupted and the UVC light leaks out to the
sample. Using a UVC LED with a few milliwatt optical power is
sufficient to generate detectable fluorescence signals and create
MUSE contrast with a mobile device microscope.
III. System
[0049] With only minor modifications, many mobile devices can be
used as high performance microscopes for research and diagnostic
applications. One of the easiest and most cost effective ways to
create a mobile device microscope involves mounting a reversed
camera lens in front of the mobile device camera. Such mobile
device microscopes are very useful to take bright field microscopy
images of thin samples, but suffer when imaging thick samples
(using methods like Microscopy with Ultraviolet Surface Excitation
(MUSE) imaging). MUSE imaging requires type-C ultraviolet light
(UVC), having a center wavelength from 240 nm to 290 nm, which
cannot transmit through most glass and polymer materials, to be
delivered to a sample surface to excite the sample. So, the UVC
must be delivered between the reversed camera lens and the sample,
which would not work in a mobile device microscope due to very
limited spacing between the reversed camera lens and the sample.
The external accessory 10 as shown in FIG. 1 provides a solution
that allows a mobile device microscope to perform MUSE imaging to
image thick samples.
[0050] The external accessory 10 uses the original design concepts
of MUSE microscopy and mobile device microscopes with only minor
modifications. As such, the external accessory 10 is a compact and
low cost attachment to a mobile device. The external accessory 10
includes an objective lens 12 (or compound lens), an optical window
14, one or more light emitting diodes (LEDs) 16 and/or 17, and an
LED driver 18. At least a portion of one or more of the objective
lens 12, the optical window 14, the one or more LEDs 16 and/or 17,
and the LED driver can be encased in one or more housings
(illustrated as housing 19). The housing 19, in some instances, can
be configured to attach to a mobile device to position the external
accessary 10 relative to the mobile device. In other instances, the
housing 19 can simplify focusing by ensuring that a focal spot is
pre-aligned, making the configuration very convenient to work with
in the field when a stable work bench is not available. Moreover,
it will also be understood that the LED driver 18 need not be
physically connected to the LED 17. Instead, the LED driver 18 can
be coupled to any part of the external accessory 10 to power and/or
control the LEDs 16, 17. The components of the external accessory
10 can be constructed at a low cost--for example, the whole
assembly can be produced easily for under $30 in material cost with
widely-available tools.
[0051] The external accessory 10 can be attached to a mobile device
22 to form a system 20 as shown in FIG. 2. For example, the housing
19 can include an attachment mechanism to facilitate the attachment
of the external accessory 10 to the mobile device. As an example,
the attachment mechanism can include an adhesive material. As
another example, although not illustrated, the attachment mechanism
can include the LED driver 18 being connected to the mobile device
22.
[0052] The objective lens 12 (or compound lens) can be configured
to be placed in front of a camera lens 24 of the mobile device 22
(so that the objective lens 12 center aligns with the camera lens
24, which may include an automatic process). For example, the
housing 19 can be specifically configured for the mobile device 22
to ensure that the objective lens 12 aligns with the camera lens
24. The objective lens 12 (or compound lens) can be an inexpensive
lens and/or an aspherical lens, such as a reverse camera lens from
an older-model smartphone, an infinite corrected microscope
objective, or the like. While the inexpensive lens and/or the
aspheric lens generally has good microscopy performance, the
inexpensive lens can generally have a very limited focal length; in
order to achieve higher resolution, the inexpensive lens is
designed for an image sensor with an even shorter focal length
(<1 mm, for example). The purpose of the objective lens 12 (or
compound lens) is to form an imaging relay with the camera lens 24
so that an image of a front focal plane of the objective lens 12
(or compound lens) is generated at an image sensor 26 of the camera
lens 24 of the mobile device 22.
[0053] The optical window 14 can be configured to be located/placed
in front of (or above) the objective lens 12 (or compound lens)--in
other words, between the objective lens 12 (or compound lens) and
the sample 28 (which can, in some instances, be a biological sample
and/or may be stained. The optical window 14 can be positioned such
that a front surface of the optical window overlaps the front focal
plane of the objective lens 12 (or compound lens). In some
instances, at least a portion of the optical window 14 can include
a sample holder. However, the optical window 14 may include
additional hardware to facilitate positioning or holding the sample
28. In some instances, the sample 28 can be held onto/within the
sample holder by surface tension (e.g., provided by liquid 29). The
optical window 14 can be transparent to UVC light. For example, the
optical window 14 can be at least partially made of quartz, fused
silica, and/or UV transparent sapphire.
[0054] One or more light emitting diode (LED) (e.g., LED 16 and/or
LED 17) can be positioned at one or more side-edges of the optical
window 14. The one or more LED (e.g., LED 16 and/or LED 17) can be
configured to emit UVC light through the optical window 14. As the
UVC light transmits through the optical window 14, the UVC light
can excite at least a portion of the sample 28 (e.g., the stain
associated with the sample 28). As an example, the optical window
14 can exhibit a transmittance greater than 50% at the center
wavelength of the one or more LEDs (e.g., LED 16 and/or LED
17).
[0055] The one or more LEDs (e.g., LED 16 and/or LED 17) are
powered and controlled by an externally triggered LED driver 18 (in
some instances, the LED driver 18 can power and control the one or
more LEDs, but in other instances, the LED driver 18 can be linked
to/paired with a separate LED controller that can, for example,
regulate current to the LEDs). The LED driver 18 can be triggered
by a mechanical button (which is pressed electronically, by a
person, or the like), a digital signal from the mobile device 22, a
flash light signal from the mobile device 22, or the like. The LED
driver 18 can be powered by an external battery or other external
power source, in some instances. In other instances, the LED driver
18 can be powered by an integrated battery of the mobile device 22
(in instances when the LED driver 18 is connected to the mobile
device 22). It should be understood that in instances where the LED
driver 18 and LED controller are separate, each may be powered by
an integrated battery of the mobile device 22 and/or powered by an
external battery or other external power source.
[0056] In order to acquire good microscopy images, many modalities
require thin samples, which are difficult to prepare in many
scenarios. The external accessory 10 allows for imaging using thick
samples by sampling the surface directly, enabling a broad range of
applications that could not be done with mobile device microscopes
previously. Since MUSE imaging requires illumination with UVC light
and UVC light cannot transmit through most glass and polymer
materials, it is impossible to use conventional microscope lenses
or mobile device camera lenses to deliver the light. In traditional
MUSE microscope configurations, UVC light is cast onto the sample
surface by an LED placed between the microscope objective and the
sample. However, with limited spacing between the objective lens 12
(or compound lens) and the sample 28, the traditional approach
would require a significantly more complicated and costly
setup.
[0057] The external accessory 10 is designed based on the optical
concept of frustrated total internal reflection (FTIR). FTIR is
easy to implement and has been used in many consumer applications,
such as touch sensing and transparent drawing boards. The optical
window 14 of the external accessory 10 facilitates the FTIR, acting
as the optical window, the sample holder, and a total internal
reflection waveguide, as shown in FIG. 3 (FIG. 2 will also be used
in the description of FIG. 3). Acting as the TIR waveguide, the
optical window 14 allow UVC to be uniformly delivered to the sample
in the space between the objective lens 12 (or compound lens) and
the sample 28.
[0058] The external accessory 10 only requires minor structural
modifications compared to the original design concepts of MUSE
imaging and traditional mobile device microscopes. Instead of using
a thin optical window (<0.25 mm--quartz or fused silica) as the
sample holder (like with traditional MUSE imaging), the external
accessory 10 uses a thick optical window 14 (>0.5 mm, similar to
or thicker than the width or length of a conventional UVC LED
emitter) as both the sample holder and the TIR waveguide. The UVC
LED emitter (e.g., used as elements 16 and/or 17) can have an
optical power of a few milliwatts.
[0059] The external accessory 10 employs the one or more LEDs 16
and/or 17 on one or more lateral side-edges of the optical window
14 to emit and transmit UVC light 32 through the optical window.
The UVC light 32 emitted from one or more of the LEDs 16, 17 is
guided through the optical window 14 of the attachment 10 using
TIR. When the UVC light contacts at least a portion of the sample
28 (in contact with the optical window 14 through the liquid 29),
the TIR is disrupted and the UVC light 32 can leak out into the
sample 28. The leaked UVC light 32 can excite the sample to create
an excited emission 34. The emitted light 36 is transmitted through
the objective lens 12 (or compound lens), through the camera lens
24, to the image sensor 26. The UVC LED emitter (e.g., used as
elements 16 and/or 17) can have an optical power of a few
milliwatts and be sufficient to generate detectable fluorescence
signals and create MUSE imaging contrast with a mobile device
camera.
[0060] The external attachment 10 can excite a sample based on
frustrated total internal reflection (FTIR) and this excitement can
be used to perform high resolution MUSE imaging. (It should also be
noted that the external accessory leaves the mobile device with the
capabilities of a basic bright-field microscope.) The external
attachment uses a UVC-transparent optical window/sample holder as
the total internal reflection waveguide, allowing for uniform
delivery of UVC light to the sample (held onto the microscope by
surface tension) through the space between the objective lens and
the sample. Traditionally, a thin optical window (<0.25
mm--quartz or fused silica) has been used as the sample holder, but
the external attachment uses a thick optical window (>0.5 mm,
similar to or thicker than the width of a conventional UVC LED
emitter) as both the sample holder and the total internal
reflection (TIR) waveguide. When the sample is in contact with the
optical window, TIR is disrupted and the UVC light leaks out to the
sample. Using a UVC LED with a few milliwatts of optical power is
sufficient to generate detectable fluorescence signals and create
MUSE contrast with a mobile device microscope.
[0061] The external attachment device 10 can be small to allow for
portable imaging with the mobile device 22. For example, FIG. 4 is
a photograph of an example attachment device A (with the components
of external attachment device 10) mounted on a Xiaomi Mi 6 android
smartphone B. FIG. 5 is a photograph of an example attachment
device A (with the components of external attachment device 10)
mounted on an Apple iPhone 6s+ A and the device being used for an
imaging operation B. The external attachment device 10 does not
remove abilities of the mobile device to act as a bright field
microscope. This opens the possibility to create hybrid images
showing more features of the sample (e.g., overlaying MUSE images
over bright field images).
IV. Methods
[0062] Another aspect of the present disclosure can include methods
60-80, as shown in FIGS. 6-8, for employing a compact and low cost
external accessory for a mobile device that can excite a sample
based on frustrated total internal reflection (FTIR) and this
excitement can be used to perform high resolution MUSE imaging. The
methods 60-80 can be performed by the system of FIG. 2, as shown
and described further in FIGS. 1 and 4-5.
[0063] The methods 60-80 are illustrated as a process flow diagram
with flow chart illustrations. For purposes of simplicity, the
methods are shown and described as being executed serially;
however, it is to be understood and appreciated that the present
disclosure is not limited by the illustrated order, as some steps
could occur in different orders and/or concurrently with other
steps shown and described herein. Moreover, not all illustrated
aspects may be required to implement the methods.
[0064] Referring now to FIG. 6, illustrated is method 60 for
performing mobile device microscopy with an external accessory
(e.g., external accessory 10 attached to mobile device 22). At 62,
a sample (e.g. sample 28, which may be stained and held in place by
liquid 29) can be placed within an imaging field of an external
accessory (e.g., external accessory 10) to a mobile device (e.g.,
mobile device 22). At 64, a photograph of the imaging field (of the
external accessory 10) can be taken (e.g., by the mobile device
with an integrated photographic camera) to provide a microscopy
image of the sample (e.g., a MUSE microscopy image of the sample
28).
[0065] FIG. 7 shows a method 70 for staining a sample for mobile
device microscopy (as described with respect to FIG. 6). At 72, the
sample (e.g., sample 28, which can be a biological sample) can be
stained with a dye (e.g., that is excitable by UVC light). For
example, the dye can be a fluorescent histology dye. The
fluorescent histology dye can be dissolved in a first solvent
(e.g., one or more of water, methanol, ethanol, isopropanol,
acetone, xylene, etc.). The first solvent can be absorbed with a
first piece of absorbent material, which can be dried in a
container. The first piece of absorbent material and a second piece
of absorbent material can be wetted with a second solvent (e.g.,
one or more of water, methanol, ethanol, isopropanol, acetone,
xylene, etc.). The second solvent may be the same as the first
solvent, but need not be the same as the first solvent. The sample
can be contacted with the first piece of absorbent material and
then the second piece of absorbent material to stain the sample.
The surface tension from residual liquid on the sample holds the
sample in place within the imaging field. The first piece of
absorbent material and/or the second piece of absorbent material
can be made using at least one of paper, cotton, sponge, a
synthetic polymer, or the like. At 74, the stained sample can be
held in place in the imaging field (e.g., of the external accessory
10).
[0066] Referring now to FIG. 8, illustrated is a method 80 for
performing mobile device microscopy with an external accessory
(e.g., external accessory 10 attached to mobile device 22) of a
stained sample (e.g., stained according to the method 70). At 82, a
sample can be exposed to one or more stains that accumulate in a
structure of interest. At 84, the one or more stains within the
sample can be excited with type-C ultraviolet (UVC) light. At 86, a
signal emitted from the one or more stains can be collected (as
shown in FIGS. 2 and 3). At 88, an image of the sample can be
generated (e.g., by a processor of mobile device 22) based on the
signal.
V. Experimental
[0067] The following example shows the use of an example of
Microscopy with Ultraviolet Surface Excitation (MUSE) imaging
implemented on a mobile device. The following example is for the
purpose of illustration only and is not intended to limit the
appended claims.
Materials and Methods
Fabrication
[0068] Aspheric compound lenses, 285-nm LEDs, fused silica optical
windows and other general supplies were purchased from various
online vendors and modified as follows: 1. aspheric compound lenses
were gently removed from the aftermarket replacement cameras using
plastic tweezers; 2. quartz windows of the LEDs were removed using
a razor blade and the height of the LED packaging was further
reduced to -1 mm (from -1.25 mm) by manual sanding with a file (180
Grit); 3. fused silica optical windows were cut into
.about.10.times.10 mm.sup.2 squares using a diamond scribe, with
two opposite edges polished sequentially using 40/30/12/9/3/1/0.3
.mu.m grade lapping films. The base plate and the sample holder
retainer were designed with Solidworks, and 3D printed with
polylactide using an FDM printer (Snapmaker). The modified LEDs
were soldered on customized printed circuit board (PCB) adaptors
(designed with Autodesk EAGLE, fabricated by OSHPark.com). The LEDs
were wired to a DC up-regulator with a push button switch in
between. The components were assembled as shown in FIG. 9.
Alignment
[0069] An easy and robust alignment procedure was developed to
tolerate the limited accuracy of inexpensive components (e.g., 3D
printing and optical window thickness) and allow nonprofessionals
to align the system. It is critical to align the sample holder to
the focal plane of the reversed aspheric compound lens (RACL). To
tolerate variations from the manufacturing process, the base plate
is designed to be slightly thicker, so the focal plane of the RACL
offsets .about.150 .mu.m below the sample surface. Alignment of
Pocket MUSE is an iterative process where the baseplate surface
facing the smartphone is sanded with 1000-3000 grit sandpaper until
the sample surface is in focus. Taking advantage of the focus
adjustment function of smartphone cameras, the focal plane of the
microscope can swing by tens of microns, reducing the accuracy
needed from the sanding step. The thickness of the base plate
(measured with a caliper) and alignment of the system (evaluated
qualitatively by image sharpness) is verified regularly (e.g.,
every .about.30 .mu.m) until good alignment is achieved.
Imaging
[0070] The Pocket MUSE component is mounted onto the smartphone
with double sided tape. The DC up-regulator is either connected to
the smartphone USB outlet (for Android phones), Lightning outlet
(for iPhones, with an On-The-Go (OTG) converter) or an external
battery. After samples are loaded on the sample holder by surface
tension, microscopy images can be taken directly with the default
smartphone camera apps. For advanced controls of imaging parameters
(e.g., ISO (gain), exposure time, focus, output format, etc.), it
is helpful to use third-party or customized camera apps (e.g.,
Halide). For MUSE imaging, UV illumination should be enabled with
the push button switch before the focus and exposure adjustments.
Exposure time varies between 10 ms and 1 s depending on the sample
type and dye concentration. Smaller ISO (gain) is desired for
better signal to noise ratio. To prevent background light, external
lights can be dimmed or aluminum foil can be used to cover the
microscope. Bright-field transillumination is achieved by facing
the smartphone towards a white scattering surface (e.g., white
wall, printing paper, etc.). Instability by hand is usually well
tolerated because relative sample motion with respect to the
smartphone is extremely small, especially for exposure times
shorter than 250 ms.
Data Processing
[0071] Unlike scientific cameras, smartphone camera apps usually
automatically process raw image data and save the data as 24-bit
RGB color images. Therefore, data processing (e.g., white balance,
digital filters, etc.) can take place even before (e.g., in preview
mode) an image is acquired. Although it is difficult to determine
the actual data processing algorithm performed by different
smartphones, such information is not required for most Pocket MUSE
applications. Still, it is possible to use third-party camera apps
(e.g., Halide on iOS and ProCam on Android) to save raw
(unprocessed) image data, which is especially beneficial when
extended dynamic range, lossless data, and advanced processing are
needed. To visualize camera raw data, it is necessary to first
convert the data (e.g., DNG file) into 24-bit RGB formats (e.g.,
TIFF). Data conversion can be performed with software such as Adobe
Camera Raw (in Photoshop) and RawTherapee (in GIMP). These programs
are commonly used for non-scientific photo editing, so they could
be easily adapted by non-professional users.
Whole Mount Samples
[0072] Excised mouse tissue was obtained from unrelated studies
with IACUC approval. The tissue was either used fresh directly
following dissection, or fixed in 4% paraformaldehyde overnight and
stored in 1X phosphate buffered saline (PBS) at 4.degree. C. Other
animal and plant samples were collected from the subject's kitchen
(e.g., vegetables, meat, etc.), university campus (e.g., algae,
pine needles, etc.) and backyard (e.g., garden plants, roundworms,
etc.). All samples were manually cut or torn with tweezers into
smaller pieces (<3.times.6.times.3 mm.sup.3). For each sample,
at least one relatively flat imaging surface is created. Staining
solutions were prepared by dissolving dyes in 30-70% v/v alcohol.
One commonly used staining solution in this study is 0.05% w/v
rhodamine B and 0.01% w/v DAPI in 50% v/v methanol, which was used
for most histology samples and some plant samples. The sample is
immersed in the staining solution for 5-20 s, rinsed with tap water
and briefly dried with an absorbent material (e.g., tissue paper).
Pseudo H&E color mapping was performed using the method
described previously.
[0073] For the IHC staining demonstration, a piece of fixed
Thy1-GFP (Jackson Laboratory, CAT #011070) mouse brain slice
(500-.mu.m thick) was obtained from unrelated studies with IACUC
approval. A universal buffer (e.g., for blocking, staining and
washing) containing 3% v/w bovine serum albumin, 1% v/w Triton
X-100, 0.05% v/w sodium azide and 1.times.PBS was prepared ahead of
time. For blocking, the brain slice was first incubated in an
excess amount of the universal buffer for .about.2 h at 37.degree.
C. For whole-mount staining, the blocking buffer was then replaced
with 500 .mu.L fresh universal buffer containing 1% v/v GFP
Polyclonal Antibody (Alexa Fluor 488 conjugate, Thermo Fisher
Scientific, CAT #A-21311) and 0.05% w/v propidium iodide. The
sample was shaken at 37.degree. C. for 16 hours. After staining,
the sample was washed again in an excess amount of the universal
buffer for .about.2 h at 37.degree. C., followed by a 30 min wash
in PBS. Channel unmixing was performed using ImageJ.
Cytology Samples
[0074] Blood samples were collected from a subject with a consumer
lancing device (for blood glucose monitoring). The experiment was
determined as a non-human subject research project by Case Western
Reserve University's Internal Review Board (IRB) and was conducted
under the consent of the subject who provided the sample. 100 .mu.L
of blood was mixed in 100 .mu.L PBS containing 4 mM
ethylenediaminetetraacetic acid and 0.01% w/v sodium azide. For
nuclei staining, 10 .mu.L of the blood sample was mixed with 1
.mu.L of 0.1% w/v acridine orange in 50% v/v methanol. For dense
blood smear imaging, 1 .mu.L of the stained sample was dropped on
the sample holder and air dried prior to imaging. For thin blood
smear imaging, the stained sample was further diluted 10 times with
PBS prior to imaging. Similarly, cheek swab samples were collected
from one subject using consumer cotton swabs. The experiment was
also determined as a non-human subject research project by the
university's IRB and was conducted under the consent of the subject
who provided the sample. After swabbing the inner surface of the
cheek, the cotton swab was dipped in a staining solution containing
10% v/v CytoStain (Richard-Allan Scientific) and 0.01% w/v
propidium iodide for 5 s. The cotton swab was then briefly rinsed
with tap water and dried with an absorbent material. The stained
cell lesions were either imaged after being smeared on the sample
holder surface, or directly on the cotton swab.
Bacteria Samples
[0075] To test non-specific bacterial labeling, a random mixture of
bacteria was collected from the supernatant of a mouse tissue
specimen that was improperly stored in non-sterile PBS at 4.degree.
C. for 6 months. The sample was diluted 10 times with PBS, and 100
.mu.L of the sample was mixed with 10 .mu.L of 0.1% w/v acridine
orange in 50% v/v methanol. 2.5 .mu.L of the mixture was dispensed
on the Pocket MUSE sample holder and the aliquot was imaged
directly with Pocket MUSE. To test Gram-specific bacterial
labeling, Escherichia coli (E. coli) was generously provided from
an unrelated study. Bacillus subtilis (Ehrenberg) Cohn (B.
subtilis) was ordered from American Type Culture Collection (ATCC,
CAT #23857). Both bacteria were cultured in lysogeny broth
overnight at room temperature. For the experiment, 4 samples were
prepared as follows: 1. 500 .mu.L PBS as a control; 2. 100 .mu.L E.
coli culture in 400 .mu.L PBS; 3. 100 .mu.L B. subtilis culture in
400 .mu.L PBS; 4. 50 .mu.L E. coli culture and 50 .mu.L B. subtilis
culture in 400 .mu.L PBS. Each sample was mixed with a 100 .mu.L
staining solution, containing 0.05% w/v DAPI and 0.1% w/v WGA-AF594
in 50% methanol. 2.5 .mu.L of each mixture was imaged with Pocket
MUSE with the same camera configuration. Distribution of pixel
values was plotted using Matlab.
Results
Overview of Pocket MUSE Design and Operation
[0076] To ensure low cost and ease of production, Pocket MUSE
features a simple design while maintaining the ability to obtain
high-quality images. It consists of only 4 major components: an
objective lens, a sample holder, UV LED light sources and a base
plate (FIG. 9). A reversed aspheric compound lens (RACL) serves as
the proximal optical element, centered immediately in front of the
smartphone camera, and provides a relatively wide field of view
(FOW) of .about.1.5.times.1.5 .mu.m.sup.2. The sample holder, a 0.5
mm-thick quartz optical window, has its top surface pre-aligned
with the focal plane of the objective lens. This eliminated the
need for fine coarse focusing mechanics that are essential for
traditional microscope designs. The required fine focusing is
performed via smartphone camera focus adjustment. The sample holder
also serves as a waveguide for the frustrated TIR illumination. The
light sources, two miniature 275-285 nm UV LEDs, are powered
directly with the smartphone battery via the USB port through a
step-up regulator. All the components are integrated in an
ultra-compact base plate, designed to be 3D printable using simple
fused-deposition modeling (FDM), utilizing <2 g of material.
[0077] By eliminating all adjustment mechanisms, even first-time
users can easily operate Pocket MUSE. To image, samples (tissue or
fluid) are attached to the sample holder by surface tension. As the
sample holder is pre-aligned to the focal plane of the objective,
the sample is always in focus during normal operation. In addition,
similar to conventional smartphone photography, Pocket MUSE is
designed to take quality images while holding the phone with one
hand. This provides extra convenience for applications in the
field, where a stable working bench is not always available. After
imaging, the sample holder can be easily cleaned using cotton swabs
and common solvents (e.g., 70% isopropanol). For heavy duty
cleaning or sterilization, the sample holder can also be detached
from the device.
Objective Lens Selection
[0078] The microscope compartment of Pocket MUSE is improved over
previous RACL smartphone microscope designs. In an early-phase
implementation, the RACL design delivered good resolution and a
large FOV while maintaining relatively low cost (lens cost
<$10). The principle behind this design is simple and robust.
Because smartphone camera lenses are capable of telecentric
imaging, stacking a pair of such lenses face-to-face creates 1:1
finite image conjugation (object size:image size) between their
back focal planes, corresponding to the object plane (sample
surface) and the image plane (sensor surface) of the microscope.
However, this original design had a critical limitation. While a
common smartphone camera lens often has f-numbers around 1.5-2.4
(corresponding to a numerical aperture of .about.0.2-0.3), and
provides .about.1-2-.mu.m optical resolution, the actual resolution
is pixel-limited because a typical smartphone camera sensor often
has a pixel size of .about.1.5-2 .mu.m. The pixels are grouped in
2.times.2 as part of RGB Bayer filter configuration, further
reducing the effective pixel size to .about.3-4 .mu.m.
[0079] Improving the resolution of previous RACL designs would
further expand the capabilities and potential applications for
smartphone microscopes. The original RACL manuscript suggested that
a smartphone with a large sensor and small pixel size (e.g., Nokia
808) can help improve the effective resolution 10. However, it is
not a universal solution for most smartphones. Here, the effective
resolution of the RACL design can be further improved using a
smaller RACL with a shorter focal length (e.g., <1.5 mm).
Through Zemax optical simulation, it was confirmed that a smaller
RACL can effectively reduce the image conjugation ratio (e.g., from
1:1 to 1:2) while maintaining good optical performance over a >1
mm FOV. While preserving optical resolution, the smaller RACL
increases the magnification of the system, and in turn, boosts the
effective resolution through denser spatial sampling when projected
onto the cellphone camera sensor. As the simulation did not take
into account the specific optics and sensor size for each
smartphone, a wide range of lens samples were obtained from
aftermarket consumer products and tested them with different
smartphones. Among the lenses tested, 1 .mu.m effective resolution
and 1.5.times.1.5 mm.sup.2 FOV can be achieved using a smaller RACL
designed for 1/7'' image sensors. By comparison, this optical
design greatly outperforms a conventional benchtop microscope with
a high quality 10.times. objective. Therefore, these lenses were
chosen for the Pocket MUSE design.
Frustrated TIR Illumination
[0080] A smaller RACL often has a large entrance aperture (>3 mm
diameter) and a short working distance (<1 mm). Within this
narrow working distance, it is necessary to fit a sample holder
(optical window). Because conventional sub 285 nm UV LEDs often
have package sizes (3.5.times.3.5.times.1 mm.sup.3) that are even
larger than the RACL, implementing the original MUSE illumination
configuration becomes nearly impossible due to limited spatial
clearance. To overcome this problem, frustrated TIR was identified
as an effective approach to deliver light to the sample surface. In
the configuration, LED-based sub 285 nm UV illumination is coupled
into the sample holder (a 0.5 mm thick quartz optical window) from
the side faces of the optical window. Above the glass-air critical
angle, the coupled light bounces between the two glass surfaces
through TIR. When a sample is present, the glass-air interface
turns into a glass-sample (glass-water) interface. It changes the
TIR critical angle and allows some light to refract out of the
glass, facilitating sample illumination. In addition, the TIR
illumination was further optimized by implementing two LEDs.
Because a significant amount of light is absorbed by sample regions
closer to the LED, a single LED could not effectively illuminate
the entire FOV. Through optics-based simulation, a >50% energy
drop across 2 millimeters of sample was noted, causing
significantly non-uniform illumination. To compensate for this
drop, another LED was added on the opposite edge of the optical
window. Through both modeling and experiments, it was shown that
relatively uniform illumination (<.+-.10% variation across 3 mm)
can be achieved with the dual-LED setup.
Histology Imaging
[0081] Slide-free histology is one of the most well-established
MUSE applications. Therefore, as the first demonstration, Pocket
MUSE is shown to be fully capable of producing high quality
histology images similar to those acquired from benchtop MUSE
systems. With a single-dip staining process followed by brief
tap-water washing, high image contrast was achieved on a large
variety of tissue samples (e.g., kidney, muscle, etc.) within
minutes (FIG. 10, elements a-d; all samples were stained with 0.05%
w/v Rhodamine B and 0.01% w/v DAPI unless otherwise specified. a)
Image of a thick section of mouse kidney sliced with a razor blade.
A close-up view of the region in the white box (box size:
320.times.320 .mu.m.sup.2) is shown on the right. b) Image of a
piece of mouse skeletal muscle torn with tweezers. c) Image of the
serosal surface of a piece of mouse small intestine. d) Image of a
piece of salmon steak sliced with a kitchen knife. e) Image of a
thick section of mouse liver sliced with a razor blade.). In
addition, Pocket MUSE provides a similar FOV compared to a
conventional 10.times. objective (e.g., .about.1.5.times.1.5
mm.sup.2). With sufficient resolution to resolve individual cell
nuclei, it is readily useful for a number of histology-centered
applications. Using images captured from Pocket MUSE, it was also
possible to implement the color remapping technique described in to
generate histology images mimicking the color contrast of
conventional H&E staining. Conventional fluorescence
immunohistochemistry (IHC) stained tissue can also be imaged with
Pocket MUSE (FIG. 11, element a (RGB image acquired with Pocket
MUSE)). The sample was stained with anti-GFP antibody (Alexa Fluor
488 conjugate and propidium iodide. Scale bar: 300 .mu.m.). With
overnight staining at a slightly higher concentration, fluorophore
conjugated antibodies can provide sufficient contrast between the
structures of interest and the background fluorescence. Common cell
nuclei dyes (e.g., DAPI, propidium iodide, etc.) could also be
easily incorporated in the IHC staining process. Different labels
can be readily separated by unmixing the RGB channels (FIG. 11,
elements b (Alexa Fluor 488 signal (from thy1-GFP) unmixed from the
green channel, showing Thy1 positive neurons) and c (Propidium
iodide signal unmixed from the red channel of the RGB image,
showing cell nuclei.)). The sample was stained with anti-GFP
antibody (Alexa Fluor 488 conjugate) and propidium iodide. Scale
bar: 300 .mu.m.).
Plants and Environmental Sample Imaging
[0082] Pocket MUSE is also a promising tool for imaging various
plants (e.g., vegetables, algae, etc.) and environmental samples
(e.g., micro-animals, synthetic pollutants, etc.). Many samples
(e.g., cilantro, micro-plastic particles, etc.) are intrinsically
fluorescent when excited around 265-285 nm. These samples are
capable of generating structural contrast without any staining.
Compared to conventional bright-field imaging, Pocket MUSE reveals
more of the cellular morphology in bulk plant structures. As with
animal tissues, plant tissue could also be stained to produce
additional micro-structural contrast with a single dip staining
process. For instance, DAPI effectively labels polysaccharides
moieties (found in, e.g., cell walls, root saps and starch) in
addition to cell nuclei, while rhodamine demonstrates accumulation
in the xylem. It was also observed that some absorptive staining
(e.g., iodine-stained starch) could be effectively incorporated
with fluorescent stains to create additional color contrast between
different plant structures.
Bright-Field and Hybrid Imaging
[0083] Pocket MUSE can also easily acquire bright-field images when
UV illumination is not enabled. This provides a simple and
effective method for visualizing naturally colored thin samples
(e.g., blood smears). A conventional fluorescence microscope
requires switching the filter cube to an open setting for
bright-field microscopy which is difficult in a compact smartphone
microscope. Because Pocket MUSE does not rely on filters, no
mechanical switching is required to change between fluorescence and
bright-field imaging. Trans-illumination bright-field microscopy
can be realized simply by directing the sample holder towards a
bright diffusive surface (e.g., white wall, printing paper, etc.)
in the far field. Regular room light and/or natural light (>100
lumen/m2) provide sufficient illumination.
[0084] Overlaying the fluorescence and bright-field images is a
common and useful technique to highlight the structures of interest
in biological samples. With Pocket MUSE, fluorescence and
bright-field contrasts can be combined through a single capture,
simply by enabling the UV illumination during bright-field imaging
(hybrid mode). As an example, with a thin blood smear, white blood
cells (WBC, fluorescence) can be highlighted in a crowd of red
blood cells (RBC, bright-field) by simply mixing in a small amount
of fluorescent nuclei dyes (e.g., 0.01% w/v acridine orange) in the
specimen. It is also possible to apply similar approaches to dense
blood samples for cytological quantification and infection
evaluation.
Mucosal Smear Imaging
[0085] Mucosal smears are used in many medical diagnostic
applications such as Pap smears. Mucosal smear preparation for
Pocket MUSE is extremely simple and can be performed within 30
seconds. The specimen is collected with a cotton swab that is then
dipped in a dye (e.g., propidium iodide with CytoStain.TM.),
briefly washed in tap water and smeared onto the sample holder.
Compared to bright-field cytology staining, MUSE fluorescence
results in a significantly higher contrast between the cell bodies,
nuclei and the background. Cell morphology can be clearly
visualized over the majority of the FOV due to low aberrations at
the edges. Although only a single FOV could be imaged at a time, a
larger population of cells could be rapidly reviewed by repeated
repositioning of the same swab. In addition, as conventional
mucosal smear cytology imaging requires cells to be attached to a
flat glass surface, Pocket MUSE allows cells to be imaged directly
on the cotton fiber matrices. Finally, because MUSE captures the
surface, some volumetric aspects of cell morphology can be
visualized.
Selective Bacteria Imaging
[0086] Fluorescent staining has been widely used to examine
bacteria in liquid samples. As a preliminary demonstration, a
bacterial suspension was labeled with fluorescent dyes (e.g.,
acridine orange) in a simple mixing step. Individual bacteria are
smaller than the resolution limit of Pocket MUSE, but their
presence can be effectively visualized when if sparsely dispersed
in a fluid sample. Suspended bacteria show a distinct twinkling in
preview mode due to their movement in and out of the focal plane.
In addition, with bacteria-specific fluorescent probes, Pocket MUSE
could also differentiate different populations of microorganisms.
As a preliminary demonstration, nucleic acid stain (DAPI, which
labels all bacteria) combined with peptidoglycan staining (wheat
germ agglutinin Alexa Fluor 594 conjugate (WGA-AF594), which labels
gram-positive bacteria) can differentiate Bacillus subtilis
(Gram-positive) and Escherichia coli (Gram-negative) bacteria
populations based on the color of microbe particles, which could be
further quantitatively assessed using signals found in the
different color channels of the RGB image.
[0087] From the above description, those skilled in the art will
perceive improvements, changes and modifications. Such
improvements, changes and modifications are within the skill of one
in the art and are intended to be covered by the appended claims.
All patents, patent applications, and publications cited herein are
incorporated by reference in their entirety.
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