U.S. patent application number 11/970189 was filed with the patent office on 2008-05-01 for system and method for increasing the contrast of an image produced by an epifluorescence microscope.
This patent application is currently assigned to IKONISYS, INC.. Invention is credited to Triantafyllos Tafas, Petros Tsipouras.
Application Number | 20080100911 11/970189 |
Document ID | / |
Family ID | 23058579 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080100911 |
Kind Code |
A1 |
Tafas; Triantafyllos ; et
al. |
May 1, 2008 |
SYSTEM AND METHOD FOR INCREASING THE CONTRAST OF AN IMAGE PRODUCED
BY AN EPIFLUORESCENCE MICROSCOPE
Abstract
The contrast of an image produced by epifluorescence microscopy
may be increased by placing a dichroic reflector behind the sample.
The dichroic reflector reflects the emission light emitted by the
fluorescent tags in the sample back through the objective lens
while allowing the shorter wavelength excitation light to pass
through the sample holder.
Inventors: |
Tafas; Triantafyllos; (Rocky
Hill, CT) ; Tsipouras; Petros; (Madison, CT) |
Correspondence
Address: |
KELLEY DRYE & WARREN LLP
400 ALTLANTIC STREET , 13TH FLOOR
STAMFORD
CT
06901
US
|
Assignee: |
IKONISYS, INC.
NEW HAVEN
CT
06511
|
Family ID: |
23058579 |
Appl. No.: |
11/970189 |
Filed: |
January 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11225460 |
Sep 13, 2005 |
7330309 |
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11970189 |
Jan 7, 2008 |
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10102500 |
Mar 19, 2002 |
6956695 |
|
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11970189 |
Jan 7, 2008 |
|
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60276906 |
Mar 19, 2001 |
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Current U.S.
Class: |
359/385 |
Current CPC
Class: |
G02B 21/16 20130101;
G01N 21/6458 20130101; G02B 21/34 20130101; G02B 21/082
20130101 |
Class at
Publication: |
359/385 |
International
Class: |
G02B 21/06 20060101
G02B021/06 |
Claims
1. An epifluorescence microscope for imaging a biological sample
having fluorescent tag molecules, the tag molecules emitting an
emission light at an emission frequency when illuminated by an
excitation light having an excitation frequency, the microscope
comprising: an excitation light source generating an excitation
light; a first dichroic mirror reflecting the excitation light; an
objective lens disposed to receive the excitation light reflected
by the dichroic mirror and to illuminate the sample with the
excitation light; an imaging lens disposed to receive emission
light from the sample through the objective lens and first dichroic
mirror; and a dichroic sample reflector disposed behind the sample
reflecting the emission light back through the sample, objective
lens, first dichroic mirror and imaging lens, while transmitting
the excitation light through the reflector.
2. The epifluorescence microscope of claim 1 wherein the dichroic
sample reflector is concave having a focal point disposed in the
sample.
3. The epifluorescence microscope of claim 1 wherein said
biological sample is placed directly on the dichroic sample
reflector.
4. A method for increasing the contrast of an image produced by an
epifluorescence microscope of a sample emitting an emission light
when illuminated by an excitation light comprising the steps of:
illuminating the sample with the excitation light; collecting a
first portion of the emission light; reflecting a second portion of
the emission light; collecting the reflected portion of the
emission light; and producing an image using the collected first
portion of the emission light and the collected reflected portion
of the emission light.
5. The method in claim 4 wherein the reflected portion of the
emission light is collected from a focal point disposed in said
sample.
6. The method of claim 4 wherein said sample is placed directly on
a dichroic sample reflector.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of continuation application
U.S. patent application Ser. No. 11/225,460, filed Sep. 13, 2005,
and U.S. patent application Ser. No. 10/102,500, filed Mar. 19,
2002, which claims priority of U.S. Provisional Application No.
60/276,906, filed Mar. 19, 2001; the entire contents of which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to microscope slides and the
like for use in epifluorescence microscopy of biological
specimens.
BACKGROUND OF THE INVENTION
[0003] Citation or identification of any reference in this section
or any section of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
[0004] An epifluorescence microscope is similar to a conventional
reflecting optical microscope in that both microscopes illuminate
the sample and produce a magnified image of the sample. An
epifluorescence microscope, however, uses the emitted fluorescent
light to form an image whereas a conventional reflecting optical
microscope uses the scattered illumination light to form an image.
The epifluorescent microscope uses a higher intensity illumination,
or excitation, light than a conventional microscope. The higher
intensity excitation light is needed to excite a fluorescent
molecule in the sample thereby causing the fluorescent molecule to
emit fluorescent light. The excitation light has a higher energy,
or shorter wavelength, than the emitted light. The epifluorescence
microscope uses the emitted light to produce a magnified image of
the sample. The advantage of a epifluorescence microscope is that
the sample may be prepared such that the fluorescent molecules are
preferentially attached to the biological structures of interest
thereby producing an image of the biological structures of
interest.
[0005] A common problem in epifluorescence microscopy is the low
contrast, or low signal-to-noise (S/N) ratio, of the fluorescent
image. This is due to the low intensity of the emitted light
compared to the high intensity of the excitation light. A dichroic
mirror is usually used to reduce the scattered excitation light
before the image is viewed or recorded.
[0006] The dichroic mirror is only partially effective in removing
the excitation light from the emitted light so other measures must
be taken to increase the S/N ratio of the fluorescent image. In
order to assist in the discussion of the other approaches to
increasing the S/N ratio of the fluorescent image, reference to
FIG. 1 is helpful.
[0007] FIG. I illustrates the optical path and components of a
typical epifluorescence microscope. A sample 100 is placed on a
sample holder 105, which is normally a microscope slide. The sample
is prepared prior to being placed on the holder 105 with
fluorescent tags that bind to the biological structures of
interest. The fluorescent tags may be a single type of fluorescent
tag that binds to a particular biological structure or may be a
mixture of several fluorescent tag types with each tag type binding
to a different biological structure. The sample 100 is illuminated
by a light source 110 that produces the excitation light with
sufficient intensity to cause the tags to emit fluorescent light.
The excitation light generated by the light source 110 follows a
path 115 through an excitation filter 120 that acts as a band-pass
filter allowing only a narrow range of frequencies to pass through
the excitation filter 120. The excitation filter 120 is chosen to
allow only the light of a frequency that will cause the tags to
fluoresce. The excitation light is reflected by a dichroic mirror
130 into the objective lens 140 of the microscope following path
125. A dichroic mirror separates the excitation light from the
emission light, in this example, by reflecting the excitation light
while transmitting the emission light. The excitation light
propagates through the objective lens 140 and illuminates the
sample 100 and excites the tags in the sample to emit fluorescent
light, also referred to as emission light. The emission light
propagates along path 125 in the opposite direction as the
excitation light. The emission light passes through the objective
lens 140 and through the dichroic mirror 130 and continues along
path 135 through an emission filter 150. The emission filter 150 is
selected to allow only light matching the frequency of the emission
light to pass through the filter. The emission filter 150 may be a
band-pass fitter, or a long-pass filter that allows the longer
wavelength emission light to pass through while stopping the
shorter wavelength excitation light. After filtering by the
emission filter 150, the emission light is formed into an image by
an imaging lens 160. If the emission filter 150 is perfectly
efficient in removing all but the emission light, the magnified
fluorescent image would have a very high contrast and S/N ratio.
Unfortunately, emission filters are not perfectly efficient so a
small amount of excitation light is transmitted though the emission
filters. Because the intensity of the excitation light is very
high, the small fraction of excitation light that passes through
the emission filter is sufficient to severely degrade the contrast
of the fluorescent image. In addition, the excitation frequency is
usually very close to the emission frequency of the fluorescent tag
molecule. The closeness of the two frequencies adds a further
requirement on the emission filter that the filter have a very
steep adsorption edge between the emission frequency and excitation
frequency.
[0008] U.S. Pat. No. 6,094,274 issued on Jul. 25, 2000 to Yokoi
teaches the use of two interference films as an emission filter.
The two interference films act to sharpen the adsorption edge
between the emission frequency and excitation frequency. The sharp
adsorption edge blocks more of the excitation light while
transmitting more of the emission light to the imaging lens.
[0009] Another approach to increasing the S/N ratio of a
fluorescent image is disclosed in Japanese Application Publication
No. 9-292572 by Sudo, et al. published on Nov. 11, 1997
(hereinafter referred to as "Sudo"). Sudo discloses the use of a
mirror behind the sample that reflects the excitation light back
through the sample. The reflected excitation light approximately
doubles the excitation light seen by the sample and therefore
approximately doubles the amount of emission light given off by the
sample. A portion of the reflected excitation light will, however,
also pass through the dichroic mirror and emission filter adding to
the "noise" of the higher emission signal. In addition, the
increased illumination of the sample from the reflected excitation
light increases the bleaching effect on the tagged sample.
Bleaching occurs when the fluorescent tag molecules emit decreasing
amounts of fluorescent light as the molecules are illuminated by
the excitation light. For example, a fluorescent tag molecule will
emit less than 10% of its emission intensity after only a minute of
being illuminated by the excitation light. As the intensity of the
excitation light increases the bleaching rate increases thereby
decreasing the emission light and reducing the contrast of the
fluorescent image.
[0010] Therefore, there still remains a need to provide a
microscope system capable of producing a high contrast fluorescent
image while reducing unnecessary bleaching of the sample.
SUMMARY OF THE INVENTION
[0011] One aspect of the present invention is directed to an
epifluorescence microscope for imaging a biological sample having
fluorescent tag molecules, the tag molecules emitting an emission
light at an emission frequency when illuminated by an excitation
light having an excitation frequency, the microscope comprising: an
excitation light source generating an excitation light; a first
dichroic mirror reflecting the excitation light; an objective lens
disposed to receive the excitation light reflected by the dichroic
mirror and to illuminate the sample with the excitation light; an
imaging lens disposed to receive emission light from the sample
through the objective lens and first dichroic mirror; and a
dichroic sample reflector disposed behind the sample reflecting the
emission light back through the sample, objective lens, first
dichroic mirror and imaging lens, while transmitting the excitation
light through the reflector.
[0012] Another aspect of the present invention is directed to a
sample holder for supporting a sample for epifluorescence
microscopy, the sample emitting an emission light when illuminated
by an excitation light, the sample holder comprising a base and a
dichroic reflector disposed on the base, wherein the dichroic
reflector reflects the emission light emitted by the sample while
transmitting the excitation light illuminating the sample.
[0013] Another aspect of the present invention is directed to a
microscope slide for supporting a sample, the slide comprising a
top surface and an infra-red reflecting film deposited on the top
surface, the film directly supporting the sample.
[0014] Another aspect of the present invention is directed to a
sample bolder holding a sample for an epifluorescence microscope,
the sample emitting an emission light when illuminated by an
excitation light, the sample holder comprising: a base supporting
the sample; and a sample reflector disposed on the base between the
sample and base, wherein the reflector reflects the emission light
emitted by the sample while transmitting the excitation light
illuminating the sample, wherein the sample reflector is concave
having a focal point disposed in the sample. Another aspect of the
present invention is directed to a sample holder for supporting a
sample emitting an emission light when illuminated by an excitation
light, the sample holder comprising: a top surface for directly
supporting a sample, the top surface having an infra-red reflecting
film deposited on the top surface; and a bottom surface having a
dichroic film deposited on the bottom surface, the dichroic film
reflecting emission light and transmitting excitation light.
[0015] Another aspect of the present invention is directed to a
sample holder for supporting a sample emitting an emission light
when illuminated by an excitation light, the sample holder
comprising: a top surface for directly supporting a sample; and a
dichroic film deposited on the top surface, the dichroic film
transmitting excitation light and reflecting emission light.
[0016] Another aspect of the present invention is directed to a
method for increasing the contrast of an image produced by an
epifluorescence microscope of a sample emitting an emission light
when illuminated by an excitation light comprising the steps of:
illuminating the sample with the excitation light; collecting a
first portion of the emission light; reflecting a second portion of
the emission light; collecting the reflected portion of the
emission light; and producing an image using the collected first
portion of the emission light and the collected reflected portion
of the emission light.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention may be understood more fully by
reference to the following detailed description of the preferred
embodiments of the present invention, illustrative examples of
specific embodiments of the invention and the appended figures in
which:
[0018] FIG. 1 is a view of a conventional epifluorescence
microscope.
[0019] FIG. 2 is a view of an embodiment of the present
invention.
[0020] FIG. 3 is a detail view of the sample holder of the
embodiment shown in FIG. 2.
[0021] FIG. 4 is a view of another embodiment of the present
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] FIG. 2 is a view of an embodiment of the present invention.
Excitation light generated by a light source 210 is filtered by an
excitation filter 220. The excitation filter 220 is preferably a
band-pass filter allowing excitation frequencies matched to the
fluorescent tags in the sample to pass through while absorbing the
rest. The excitation light is redirected by reflection from a
dichroic mirror 230 through an objective lens 240 to illuminate a
sample 200 having fluorescent tag molecules. The excitation light
causes the fluorescent tag molecules to emit fluorescent light. The
fluorescent light emitted by the tag molecules is collected by the
objective lens 240 and is transmitted through the dichroic mirror
230. The dichroic mirror 230 is selected to reflect the excitation
light emitted by the light source 210 toward the sample 200 while
transmitting the emission light emitted by the sample through the
dichroic mirror. The emission light is filtered by an emission
filter 250 to remove extraneous light such as scattered excitation
light. The emission light is formed into an image by an imaging
lens 260. The details of mounting and aligning the optical elements
described above are known to one of ordinary skill in the optical
microscopy art and are therefore not discussed.
[0023] The sample 200 is supported by a sample holder 205. The
sample holder 205 includes a sample reflector 207 positioned
directly behind the sample 200. It is understood that the term
"behind" is relative to the direction of the incident excitation
light. The sample reflector 207, in a preferred embodiment, is a
dichroic mirror selected to reflect the emission light while
transmitting the excitation light.
[0024] FIG. 3 is a detail view of the sample holder 205 and sample
reflector 207. A sample 300 such as a blood or cell smear is placed
on a sample support 305 such as a glass slide. The sample is
treated with a fluorescent tag that preferentially adsorbs to the
biological structures of interest. The sample 300 and sample
support 305 are supported by a sample holder 310. The sample holder
310 has a base 315 supporting a reflector 320 that, in turn,
supports the glass slide 305 and sample 300.
[0025] Excitation light 350 illuminates the sample 300 and
interacts with the sample 300, sample support 305 and reflector
320. For example, the excitation light 350 may be back-scattered
from the sample, shown as ray 352, or may be back-scattered from
the sample support 305, shown as ray 354, or may be backscattered
from the reflector, shown as ray 356. Some of the back-scattered
light 352 354 356 is collected by the objective lens (not shown)
and transmitted through to the imaging lens. The back-scattered
light 352 354 356 collected by the imaging lens contributes to the
background noise level of the image and therefore reduces the S/N
ratio of the image.
[0026] A small fraction of the excitation light 350 interacts with
the fluorescent tags 302 causing the fluorescent tags 302 to emit
fluorescent light 360 362. Some of the emission light 360 is
collected by the objective lens and imaged by the imaging lens
thereby forming the image of the biological structures of interest.
Less than one-half of the emission light 360 362 is directly
collected by the objective lens because at least one-half of the
emission light is emitted in a direction away from the objective
lens as represented by ray 362.
[0027] In a preferred embodiment, the reflector 320 is a dichroic
mirror that allows the short wave-length excitation light 351 to
pass through the mirror 320 while reflecting the longer wave-length
emission light 362. Selection of the reflector 320 to match the
excitation and emission frequencies of the specific fluorescent tag
molecule used to prepare the sample is well known to one of
ordinary skill in the fluorescent microscopy art.
[0028] The novel feature of the reflector 320 is that, unlike the
dichroic mirror commonly used in typical epifluorescent
microscopes, the reflector 320 reflects the emission light instead
of the excitation light. In the preferred embodiment, the reflector
320 transmits or absorbs most of the excitation light 350 and
therefore reduces the amount of back-scattered excitation light 356
that may be collected by the objective lens. Reducing the amount of
back-scattered excitation light 356 also reduces the noise in the
image and results in a higher contrast image of the sample. In
addition, the reflected emission light 362 may be collected by the
objective lens and contribute to the "signal portion" of the image
and thereby create a higher contrast image.
[0029] The reflected emission light 362 is reflected from the
surface of the reflector 320. The reflector surface is behind, with
respect to the direction of the excitation light, the tag molecule
in the sample and therefore will not be in the same focal plane 370
as the sample. The resulting image will have a higher intensity due
to the reflected emission light but will have a lower resolution
due to the spatial displacement of the reflector surface with
respect to the plane of the sample. In many situations, the higher
intensity image is more important than the slight loss of
resolution. For example, if the emission light is used to detect
the presence of a rare cell in a sample, a brighter image is
preferred because a bright image is easier to detect. The slight
loss in resolution in this example is not as important because the
detection of the rare cell depends primarily on image brightness,
not image resolution.
[0030] In another embodiment of the present invention, the sample
is placed directly on the reflector 320. Placing the sample
directly on the reflector 320 eliminates the need for a sample
support 305 and reduces the distance between the plane of the
reflector and the plane of the sample 370 thereby reducing the
focal mismatch between the image formed by the emission light
collected directly from the sample and the image formed by the
reflected emission light 362.
[0031] The reflection surface may also be used as a reference plane
for automatically focusing the image using laser tracking such as
the Teletrac LTAF8000 series Laser Tracking Autofocus from Axsys
Technologies of Rocky Hill, Conn. In typical auto-focusing methods,
the image is focused based on the reflected light from a surface,
usually a cover slide. In typical laser autofocusing systems, the
frequency of the laser light is usually in the infrared portion of
the spectrum and has a longer wavelength than the light emitted by
the fluorescent tags. The amount of reflected light is usually less
than about 5% of the incident light. The small signal strength of
the reflected light causes the microscope to lose focus if the
sample is perturbed. In an embodiment where the reflector acts as a
high-pass filter allowing the higher frequency excitation light
through the filter while reflecting the lower frequency emission
and autofocusing light back through the objective lens. Although
the reflector may not reflect all of the infra-red focusing light,
a sufficient amount of focusing light will be reflected for the
laser auto-focus system to maintain focus on the top surface of the
reflector.
[0032] FIG. 4 is a side view of another embodiment of the present
invention. An infra-red reflecting film 410 is deposited on the top
surface of a sample support 420 and a dichroic film 430 reflecting
emission light while transmitting excitation light is deposited on
the bottom surface of the sample support. The sample support may be
a single-use disposable glass slide. The sample 405 is placed
directly on the infra-red reflector 410 and illuminated by both the
excitation light 450 and a focusing beam 460. The focusing beam 460
is preferably an infra-red beam, characterized by a wavelength
between 700-800 nm, that is part of a laser auto-focus system such
as the one described above. The focusing beam 460 is reflected
(indicated by ray 465) by the infra-red reflecting film 410 back to
the laser auto-focus system that automatically focuses the
microscope on the infra-red reflecting film 410. In most
situations, the biological structures of interest usually settle
onto the surface of the infra-red reflecting film 410. Therefore,
focusing on the reflecting film 410 will likely bring the
biological structures of interest into focus. The dichroic film 430
on the bottom surface of the sample support 420 will reflect the
emission light (indicated by ray 455) back through the sample for
collection by the objective lens while transmitting or absorbing
the excitation light (indicated by ray 451).
[0033] In a preferred embodiment, the infra-red reflecting film 410
is metal film, such as for example titanium, between 0.6-90 nm. The
metal film may be deposited using any of the known techniques for
depositing thin films such as physical deposition. In a preferred
embodiment, magnetron sputtering may be used to apply the infra-red
reflecting film to the glass slide. The sputtering composition, in
a preferred embodiment, is substantially titanium with impurities
such as carbon, nitrogen, iron, oxygen, and hydrogen cumulatively
comprising less than 1% of the sputtering composition. Other
sputtering compositions comprising metals different than titanium
may be used to form the metal film.
[0034] The selection of the sputtering composition and film
thickness may determined by one of skill in the art by measuring
the intensity of the reflected auto-focus beam from the reflecting
film. In one embodiment of the present invention, the thickness and
composition of the film is adjusted to reflect between 4-8% of the
incident infra-red auto-focus beam. In a preferred embodiment, the
thickness and composition of the film is adjusted to reflect
between 5.5-7% of the incident auto-focus beam.
[0035] In other situations, however, a high contrast, high
resolution image is preferred. In another embodiment of the present
invention, the reflector is shaped into a concave surface having a
focal point in the plane (defined by the excitation light ray) of
the sample. This has the advantage of being able to focus both the
direct and reflected emission light on the same focal plane.
[0036] In another embodiment of the present invention, more than
one kind of fluorescent tag may be used to image different
biological structures of the sample.
[0037] A mixture of different kinds of fluorescent tag molecules is
used to prepare the sample. Each kind of fluorescent tag attaches
to different biological structures. The light emitted by the
fluorescent tags may have a different frequency and the excitation
light required to cause the tags to fluoresce may have a different
frequency depending on the kind of fluorescent tag. Each tag may
require its own set of excitation and emission filters selected for
the excitation and emission light frequencies of the specific tag.
The sample holder reflector is chosen to transmit or absorb all the
excitation frequencies of the fluorescent tags while reflecting all
the emission frequencies of the fluorescent tags.
[0038] The invention described herein is not to be limited in scope
by the preferred embodiments herein described, since these
embodiments are intended as illustrations of several aspects of the
invention. Any equivalent embodiments are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. For example, instead of transmitting the excitation
light, the sample reflector may absorb the excitation light.
Another example includes the use of a laser as the excitation light
source. Since the laser produces essentially monochromatic light,
using a laser as the excitation light source eliminates the need
for an excitation filter. Such modifications are also intended to
fall within the scope of the invention.
* * * * *