U.S. patent application number 10/080276 was filed with the patent office on 2002-11-28 for laser scanning fluorescence microscopy with compensation for spatial dispersion of fast laser pulses.
Invention is credited to Bouzid, Ahmed, Lechleiter, James.
Application Number | 20020176076 10/080276 |
Document ID | / |
Family ID | 23427744 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020176076 |
Kind Code |
A1 |
Bouzid, Ahmed ; et
al. |
November 28, 2002 |
Laser scanning fluorescence microscopy with compensation for
spatial dispersion of fast laser pulses
Abstract
In laser scanning microscope systems using short pulsed laser
sources incorporating an acousto-optical deflector, compensation is
provided for spatial dispersion introduced by the deflector.
Spatial dispersion of short pulses, such as those provided by a
laser utilized in two photon fluorescence microscopy, occurs due to
the higher and lower wavelength components in the pulsed laser beam
as the beam is passed through an acousto-optical deflector or other
similar diffractive element. A dispersive prism is mounted adjacent
to the exit face of the acousto-optical deflector to spatially
recombine the components of the pulse. A mirror may be mounted
adjacent to the input face of the acousto-optical deflector and
adjusted to adjust the angle of incidence of the beam on the input
face of the deflector to match the Bragg condition at the center
wavelength and so that both sides of the spectrum of the pulses are
somewhat Bragg-mismatched and attenuated.
Inventors: |
Bouzid, Ahmed; (Suwanee,
GA) ; Lechleiter, James; (San Antonio, TX) |
Correspondence
Address: |
Patrick Stellitano
2803 Inridge Dr.
Austin
TX
78745
US
|
Family ID: |
23427744 |
Appl. No.: |
10/080276 |
Filed: |
February 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10080276 |
Feb 21, 2002 |
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09362840 |
Jul 28, 1999 |
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6449039 |
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Current U.S.
Class: |
356/318 |
Current CPC
Class: |
G02B 21/002 20130101;
G02B 21/06 20130101 |
Class at
Publication: |
356/318 |
International
Class: |
G01J 003/30 |
Claims
What is claimed is:
1. A laser scanning system for use with an optical microscope
comprising: (a) a laser light source emitting a pulsed light beam;
(b) a fluorescent light detector; (c) optical elements forming an
optical path from the light source to a position from which the
beam may be received by an objective lens of a microscope and
focussed on a specimen and such that light reflected or emitted
from the specimen is received by the objective lens and returned on
the optical path, the optical elements including an acousto-optical
deflector and a second deflector arranged to scan the laser light
beam in an X and Y direction onto the specimen, means for directing
fluorescent light emitted from the specimen at a different
wavelength than the wavelength of the laser light source on a path
to the fluorescent light detector, the acousto-optical deflector
having an input face which receives the light beam from the laser
light source and an exit face from which the light beam exits the
acousto-optical deflector, and a dispersive prism mounted to
receive the beam exiting from the exit face of the acoustic-optical
deflector and to converge the spectral components of the beam
exiting from the acousto-optical deflector caused by spatial
dispersion of wavelengths in the light beam.
2. The system of claim 1 wherein the optical elements forming the
optical path further includes a mirror positioned to deflect the
beam from the laser light source to the input face of the
acousto-optical deflector, and wherein the mirror and the
acousto-optical deflector are mounted for adjustment relative to
one another to allow the angle of incidence of the light beam on
the input face of the acousto-optical deflector to be adjusted
thereby to allow the beam incident on the input face of the
acousto-optical deflector to be at an angle to match the Bragg
condition for maximum diffraction efficiency for the center
wavelength of the light beam.
3. The system of claim 1 wherein the dispersive prism is a
triangular prism.
4. The system of claim 1 wherein the dispersive prism is arranged
such that the maximum correction is at the center of the scanned
angle.
5. The system of claim 1 further including a confocal aperture
plate positioned ahead of the fluorescent light detector to pass
fluorescent light in the optical path back to the detector that is
substantially only in the plane of focus of the objective lens.
6. The system of claim 1 wherein the second deflector comprises a
galvanometer driven mirror.
7. The system of claim 1 wherein the laser light source provides a
substantially monochromatic output beam having a wavelength in the
near infrared to red region of the spectrum and wherein the means
for directing fluorescent light includes a dichroic mirror arranged
in the optical path to direct the beam from the laser light source
on the optical path to the objective lens and specimen and formed
to reflect light at wavelengths in the red to near infrared region
of the spectrum and to pass light at a wavelength shorter than the
source light beam wavelengths.
8. The system of claim 1 wherein the laser light source provides an
output beam that is modulated in pulses with pulse widths of 5
picoseconds or less.
9. The system of claim 8 wherein the pulse width in the beam from
the laser light source is 500 femptoseconds or less.
10. The system of claim 1 further including a reflected light
detector selected to detect wavelengths of light in the beam from
the laser light source, and wherein the optical elements in the
optical path further include a polarizing beam splitter receiving
the light from the source and transmitting polarized light of a
first orientation onto the beam path to the specimen, and wherein
light returned from the specimen is orthogonally polarized and is
reflected by the polarizing beam splitter to the reflected light
detector.
11. The system of claim 10 further including a confocal aperture
plate with aperture positioned before the reflected light detector
to pass light through the aperture corresponding to light in the
focal plane of the objective lens of the microscope.
12. A laser scanning system for use with an optical microscope
comprising: (a) a laser light source emitting a pulsed light beam;
(b) a fluorescent light detector; (c) optical elements forming an
optical path from the light source to a position from which the
beam may be received by an objective lens of a microscope and
focussed on a specimen and such that light reflected or emitted
from the specimen is received by the objective lens and returned on
the optical path, the optical elements including an acousto-optical
deflector and a second deflector arranged to scan the laser light
beam in an X and Y direction onto the specimen, a light separation
element arranged in the optical path to direct the beam from the
laser light source on the optical path to the objective lens and
specimen and to direct fluorescent light emitted from the specimen
at a different wavelength than the wavelength of the laser light
source on a path to the fluorescent light detector, the
acousto-optical deflector having an input face which receives the
light beam from the laser light source and an exit face from which
the light beam exits the acousto-optical deflector, a dispersive
prism mounted to receive the beam exiting from the exit face of the
acoustic-optical deflector and to converge spectral components of
the beam exiting from the acousto-optical deflector caused by
spatial dispersion of wavelengths in the light beam, and a mirror
positioned to deflect the beam from the laser light source to the
input face of the acousto-optical deflector and wherein the mirror
and the acousto-optical deflector are mounted for adjustment
relative to one another to allow the angle of incidence of the
light beam on the input face of the acousto-optical deflector to be
adjusted, thereby to allow the beam incident on the input face of
the acousto-optical deflector to be at an angle to match the Bragg
condition for maximum diffraction efficiency for the center
wavelength of the light beam.
13. The system of claim 12 wherein the dispersive prism is a
triangular prism.
14. The system of claim 12 wherein the dispersive prism is arranged
such that the maximum correction is at the center of the scanned
angle.
15. The system of claim 12 further including a confocal aperture
plate positioned ahead of the fluorescent light detector to pass
fluorescent light on the optical path back to the detector that is
substantially only in the plane of focus of the objective lens.
16. The system of claim 12 wherein the second deflector comprises a
galvanometer driven mirror.
17. The system of claim 12 wherein the laser light source provides
a substantially monochromatic output beam having a wavelength in
the near infrared to red region of the spectrum and wherein the
light separation element is a dichroic mirror formed to reflect
light at wavelengths in the red to near infrared region of the
spectrum and to pass light at a wavelength shorter than the source
light beam wavelengths.
18. The system of claim 12 wherein the laser light source provides
an output beam that is modulated in pulses with pulse widths of 5
picoseconds or less.
19. The system of claim 18 wherein the pulse width in the beam from
the laser light source is 500 femptoseconds or less.
20. The system of claim 12 further including a reflected light
detector selected to detect wavelengths of light in the beam from
the laser light source, and wherein the optical elements in the
optical path further include a polarizing beam splitter receiving
the light from the source and transmitting polarized light of a
first orientation onto the beam path to the specimen, and wherein
light returned from the specimen is orthogonally polarized and is
reflected by the polarizing beam splitter to the reflected light
detector.
21. The system of claim 20 further including a confocal aperture
plate with an aperture positioned before the reflected light
detector to pass light through the aperture corresponding to light
in the focal plane of the objective lens of the microscope.
22. A laser scanning multi-photon microscope system comprising: (a)
a laser light source emitting a pulsed light beam, (b) a microscope
objective lens; (c) a reflected light detector; (d) a fluorescent
light detector; (e) optical elements forming an optical path from
the light source to a position from which the beam may be received
by the objective lens of the microscope and focussed on a specimen
and such that light reflected or emitted from the specimen is
received by the objective lens and returned on the optical path,
the optical elements including an acousto-optical deflector and a
second deflector arranged to scan the laser light beam in an X and
Y direction onto the specimen, and a light separation element
arranged in the optical path to direct the beam from the laser
light source on the optical path to the objective lens and specimen
and to direct fluorescent light emitted from the specimen at a
different wavelength than the wavelength of the laser light source
on a path to the fluorescent light detector, the acousto-optical
deflector having an input face which receives the light beam from
the laser light source and an exit face from which the light beam
exits the acousto-optical deflector, a dispersive prism mounted to
receive the beam exiting from the exit face of the acoustic-optical
deflector and to converge spectral components of the beam exiting
from the acousto-optical deflector caused by spatial dispersion of
wavelengths in the light beam, a mirror positioned to deflect the
beam from the laser light source to the input face of the
acousto-optical deflector and wherein the mirror and the
acousto-optical deflector are mounted for adjustment relative to
one another to allow the angle of incidence of the light beam on
the input face of the acousto-optical deflector to be adjusted
thereby to allow the beam incident on the input face of the
acousto-optical deflector to be at an angle to match the Bragg
condition for maximum diffraction efficiency, and a polarizing beam
Splitter receiving the light from the source and transmitting
polarized light of a first orientation on the optical path to the
specimen, and wherein reflected light returned from the specimen is
orthogonally polarized with respect to the first orientation and is
reflected by the polarizing beam splitter to the reflected light
detector.
23. The system of claim 22 wherein the dispersive prism is a
triangular prism.
24. The system of claim 22 wherein the dispersive prism is arranged
such that the maximum correction is at the center of the scanned
angle.
25. The system of claim 22 further including a confocal aperture
plate positioned ahead of the florescent light detector to pass
fluorescent light on the optical path back to the detector that is
substantially only in the plane of focus of the objective lens.
26. The system of claim 22 wherein the second deflector comprises a
galvanometer driven mirror.
27. The system of claim 22 wherein the laser light source provides
a substantially monochromatic output beam having a wavelength in
the near infrared to red region of the spectrum and wherein the
light separation element is a dichroic mirror formed to reflect
light at wavelengths in the red to near infrared region of the
spectrum and to pass light at a wavelength shorter than the source
light beam wavelengths.
28. The system of claim 22 wherein the laser light source provides
an output beam that is modulated in pulses with pulse widths of 5
picoseconds or less.
29. The system of claim 28 wherein the pulse width in the beam from
the laser light source is 500 femptoseconds or less.
30. The system of claim 22 further including a confocal aperture
plate with an aperture positioned before the reflected light
detector to pass light through the aperture corresponding to light
in the focal plane of the objective lens of the microscope.
31. A method of multi-photon fluorescence microscopy comprising:
(a) passing a pulsed light beam from a laser source through an
acousto-optical deflector to deflect the beam in a pattern over an
objective lens of a microscope to focus the light on a specimen and
excite multi-photon fluorescence emissions from a fluorophore in
the specimen; (b) collecting fluorescent light emitted from the
specimen and directing such fluorescent light to a fluorescent
light detector; and (c) passing the pulsed light beam exiting from
the acousto-optical deflector through a dispersive prism to
converge spectral components of the beam exiting from the
acousto-optical deflector caused by spatial dispersion of
wavelengths in the light beam introduced by the pulse modulation of
the light beam.
32. The method of claim 31 further comprising reflecting the light
beam with a mirror positioned adjacent to an input face of the
acousto-optical deflector, and adjusting the relative position of
the mirror and the input face of the acousto-optical deflector such
that the beam is incident on the input face of the acousto-optical
deflector at an angle which matches the Bragg condition for maximum
diffractive efficiency at the center wavelength of the light beam
from the laser source and such that the acousto-optical deflector
attenuates both sides of the spectrum of the light beam pulses
which are mismatched to the Bragg condition.
33. A method of multi-photon fluorescence microscopy comprising:
(a) passing a pulsed light beam from a laser source through an
acousto-optical deflector to deflect the beam in a pattern over an
objective lens of a microscope to focus the light on a specimen and
excite multi-photon fluorescence emissions from a fluorophore in
the specimen; (b) collecting fluorescent light emitted from the
specimen and directing such fluorescent light to a fluorescent
light detector; and (c) reflecting the light beam with a mirror
positioned adjacent to an input face of the acousto-optical
deflector, and adjusting the relative position of the mirror and
the input face of the acousto-optical deflector such that the beam
is incident on the input face of the acousto-optical deflector at
an angle which matches the Bragg condition for maximum diffractive
efficiency at the center wavelength of the light beam from the
laser source and such that the acousto-optical deflector attenuates
both sides of the spectrum of the light beam pulses which are
mismatched to the Bragg condition.
Description
FIELD OF THE INVENTION
[0001] This invention pertains generally to the field of laser
scanning microscopes and to laser scanning florescence
microscopy.
BACKGROUND OF THE INVENTION
[0002] Laser scanning microscopes and particularly confocal
microscopes are commonly used in research for the imaging of
structures such as cells. In such scanning microscopes, the light
from the laser source is focused to a point within the specimen by
the microscope objective, and the specimen and beam are moved
relative to one another, most commonly by deflecting the light beam
so that it scans across a stationary specimen. The light from the
specimen is collected by the objective and passed through the
microscope to the detector, such as a photomultiplier tube. Various
scanning systems have been developed to deflect the beam from the
source to scan across the specimen, including pairs of galvanometer
driven mirrors which provide both X and Y deflections of the beam.
Because such mechanically driven mirrors provide relatively slow
scanning of the beam, scanning systems have been developed which
provide the deflections of the beam in at least one direction by
faster devices, particularly acousto-optical deflectors (AODs). A
confocal laser scanning microscope having fast scanning capability
which incorporates an acousto-optical deflector is shown in U.S.
Pat. No. 4,863,226 to Houpt, et. al.
[0003] Scanning microscopes can also be utilized to detect
fluorescence induced by the illuminating light beam, which may be
carried out concurrently with the detection of the light reflected
from the specimen. In conventional fluorescence microscopy, the
fluorophores incorporated in the specimen are selected to absorb
the illumination light at a relatively short wavelength and to
fluorescently emit photons at a longer wavelength. These
fluorescent photons are passed back through the scanning optics to
a dichroic mirror which separates the fluorescent light from
reflected light and directs the fluorescent light to a detector
such as a photomultiplier tube.
[0004] Various fluorophores can absorb two or more photons of
relatively long wavelengths simultaneously when sufficiently
intense illumination light is applied to them, and will
fluorescently emit a photon at a shorter wavelength than the
wavelength of the incident light. In two photon laser scanning
microscopes an incident beam of relatively long wavelength light is
provided in short pulses (typically in the range of a few
picoseconds to a few hundred femptoseconds per pulse) from a laser.
The pulsed beam from the laser is focused onto a specimen so that
the light reaches an intensity at the focal point sufficient to
excite detectable two photon fluorescence. The emitted fluorescent
photons are collected by the objective lens of the microscope and
are passed back through the optical system of the scanning
microscope, either through the scanning optics to a dichroic mirror
which reflects light at longer wavelengths while passing the
shorter wavelength fluorescent light to a detector, or, by
bypassing the scanning system and directing the light from the
microscope objective lens to a dichroic mirror which passes the
shorter wavelength fluorescent light directly to a detector. Such
two photon systems are described in, e.g., Winfried Denk, et. al.,
"Two Photon Laser Scanning Fluorescence Microscopy," Science, Vol.
248, Apr. 6, 1990, pp. 73-76; Winfried Denk, et al., "Two-Photon
Molecular Excitation in Laser-Scanning Microscopy," Chapter 28,
Handbook of Biological Confocal Microscopy, Plenum Press, New York,
1995, pp. 445-448. If the incident light from the objective lens is
focused to a narrow spot or waist in a semi-transparent specimen
such that the intensity of the incident light is sufficient to
excite multi-photon excitation only at the focal spot within the
specimen, multi-photon fluorescence excitation will occur generally
only in the focal plane. The fluorescent light emitted by the
specimen can then be passed back and detected to obtain an image
corresponding only to the focal plane and not to structures above
and below the focal plane.
[0005] The laser light sources that are utilized to provide two (or
more) photon excitation are generally selected to provide very
short pulses of laser light, with a typical pulse width from a few
picoseconds to several hundred femptoseconds. At such narrow pulse
widths, the pulse modulation of the relatively long wavelength
(substantially monochromatic) laser light effectively introduces
higher and lower frequency components or sidebands in the pulse
modulated light beam. The occurrence of such spectral spread may
also be explained based on the uncertainty principle, which
predicts that the shorter the temporal extent of the pulse, the
wider its spectral content, regardless of how the pulse is
produced. Thus, the spectral content of, e.g., a femptosecond
(10.sup.-15 second) pulse is significantly larger than that of a
picosecond (10.sup.-12 second) pulse. When beams with such narrow
pulse widths are passed through an acousto-optic deflector (AOD), a
diffractive element, the various wavelengths within the pulse
modulated light tend to be spatially separated from one another by
the AOD element. Such spatial dispersion of the pulses reduces the
quality of the fluorescence image that can be obtained from such
scanning systems.
SUMMARY OF THE INVENTION
[0006] In accordance with the present invention, a laser scanning
microscope system utilizing short pulsed laser sources incorporates
an acousto-optical deflector with compensation for spatial
dispersion introduced by the deflector. In accordance with the
invention, spatial dispersion of the pulses passed through the
deflector is compensated by spatially compressing the pulses passed
through the deflector to provide a spatially recombined pulse to
the microscope optics.
[0007] In a laser scanning microscope system in accordance with the
invention, pulsed laser light from a source is provided on an
optical path defined by optical elements to the objective lens of
microscope which focuses the laser light onto a specimen. The
optical elements of the optical path include an acousto-optical
deflector (or other chromatically dispersive scanning element) for
deflecting the laser light selectively in one of the X or Y
directions with respect to the specimen and a second deflector,
such as a galvanometer driven mirror, to deflect the light in the
other direction. Multi-photon fluorescent light emissions from the
specimen are collected and directed to a fluorescent light
detector, such as by collecting fluorescent photons incident on the
objective lens and passing the fluorescent light back along the
optical path to a light separation element such as a dichroic
mirror which separates the fluorescent light from the reflected
excitation light and directs the fluorescent light to a detector.
Fluorescent light emitted in other directions, e.g., transmitted
through the sample away from the objective lens, may be collected
and detected. If desired, the system may also include elements for
conventional laser scanning microscopy, such as a polarizing beam
splitter for separating the reflected light at the excitation
wavelength from the incoming laser light and directing such
reflected light to a detector.
[0008] In the present invention, a dispersive prism is mounted
adjacent to the output face of the acousto-optical detector to
spatially compress the pulse which has been angularly spread by the
acousto-optical deflector because of the spectral content of the
pulse. The prism is preferably constructed so that the maximum
correction is obtained at the center of the scanned angle. The beam
exiting from the prism is directed to optical elements, such as
lenses, which receive the output beam from the prism and direct it
along the optical path to the objective lens of the microscope.
[0009] The optical path also preferably includes a mirror at the
input side of the acousto-optical deflector which receives the beam
on the optical path from the source and reflects it to the
deflector. The relative angle between the mirror and the input face
of the deflector are adjustable by mounting the mirror or the
acousto-optical deflector, or both, to allow adjustment of the
angular position between the face of the mirror and the input face
of the acousto-optical deflector. The input angle to the
acousto-optical deflector is preferably adjusted by appropriate
adjustment of the angles between the mirror and the deflector so
that the maximum diffraction efficiency (Bragg condition) is met at
the desired wavelength of operation of the laser. However, the side
wavelengths in the spectrum of the pulses will be somewhat
Bragg-mismatched and thus attenuated. In this manner, the
acousto-optical deflector also effectively acts as a spatial notch
filter while allowing for the Bragg-matching of the center
wavelength of the pulse. Utilization of an adjustable angle mirror
combined with the input face of the acousto-optical deflector, and
a dispersive prism mounted adjacent to the output face of the
acousto-optical deflector, provide spatial compensation of the
pulses with a minimum number of refractive elements and in a highly
efficient and effective manner.
[0010] Further objects, features and advantages of the invention
will be apparent from the following detailed description when taken
in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] In the drawings:
[0012] FIG. 1 is a simplified diagram of the optical layout of a
laser scanning microscope system in accordance with the
invention.
[0013] FIG. 2 is a simplified perspective view of the
acousto-optical deflector and compensation optics along a portion
of the optical path of the scanning system of FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
[0014] With reference to the drawings, a laser scanning microscope
system incorporating spatial compensation in accordance with the
invention is shown generally at 10 in FIG. 1. The scanning
microscope system 10 includes a light source 11 comprising a laser
that provides short pulses (e.g., 5 picoseconds pulse width or
less, and preferably 500 femptoseconds or less) of substantially
monochromatic light at a desired wavelength. An example is a
Ti:sapphire laser which puts out 100 to 200 femptosecond wide
pulses. The center wavelength of the pulses is tunable. Other
lasers with fixed and/or tunable wavelength output may also be
used. For two photon excitation, the laser source 11 may, for
example, provide excitation light at a nominal wavelength in the
range of 800 nm. The laser light is provided in an output beam 12
which is directed along an optical path 14 by various optical
elements to the objective lens 15 of a microscope which focuses the
light on a specimen 16 held on a slide 17. For exemplification, the
optical elements on the optical beam path may include a polarizing
beam splitter 20 which can be utilized for conventional reflection
scanning microscopy. A quarter-wave plate 21 would also be utilized
in the optical path in a conventional manner. The polarizing beam
splitter 20 transmits the input beam but reflects orthogonally
polarized reflected light to a detector 22 (e.g., a photomultiplier
tube), generally through an aperture in a confocal aperture plate
23. Although the present invention may be utilized with such a
reflected light confocal scanning system, it is not an essential
part of the present invention. The scanning system may be
constructed as described in the U.S. patent to Haupt, et al. U.S.
Pat. No. 4,863,226, the disclosure of which is incorporated by
reference.
[0015] The laser light along the optical path 14 is also passed
through a cylindrical lens 26 which expands the beam and directs it
to a lens 27 which provides a collimated and expanded output beam
28 to a mirror 30. The mirror 30 reflects the beam to an
acousto-optical deflector 31 which receives the beam at its input
face 32. The beam exiting from the acousto-optical deflector 31 at
its output face 33 is received by a compensating dispersive prism
35 which, as described further below, provides spatial compensation
for the pulses passed through the acousto-optical deflector 31. The
beam exiting from the prism 35 is passed through lenses 37 and 38
which refocus the laser beam back to a spherically symmetrical
form. A set of lenses 40 re-collimates the beam and images the
center of the acousto-optical deflector onto the microscope
objective pupil. The collimated beam 41 passed from the lenses 40
is directed to a dichroic mirror 43, which reflects the wavelengths
of the input beam light, and thence to a second deflector, e.g., a
galvanometer driven mirror 44, which redirects the light on a path
to the objective lens 15 (directly or via other optical elements
such as a planar mirror 45). The acousto-optical deflector 31 is
preferably utilized to provide rapid deflection of the light beam
in a raster scan along the lines of the scan, with the galvanometer
driven mirror 44 providing indexing of the lines of the raster scan
from one line to another at a much slower rate than the deflections
of the acousto-optical deflector 31.
[0016] The specimen 16 may contain fluorophores which absorb two
(or more) photons at the relatively long wavelength (typically in
the red to near-infrared region, e.g., 800 nm) of the light beam 12
and fluorescently emit a shorter wavelength photon. Some of the
fluorescent photons are collected by the microscope objective lens
15 and are directed back on the optical path 14 to the dichroic
mirror 43. The mirror 43 is selected to reflect the wavelength(s)
of the light beam 12 and the reflected light from the specimen at
the same wavelengths and to pass light at wavelengths shorter than
a cut-off wavelength which is longer than that of the fluorescent
photons emitted by the fluorophores. Generally, the laser light
source provides a substantially monochromatic center wavelength in
the near infrared to red region of the spectrum and the dichroic
mirror 43 is formed to reflect light with wavelengths in the red to
near infrared region of the spectrum and to pass light at a
wavelength shorter than the source excitation wavelengths. The
dichroic mirror 43 thus passes the fluorescent photons and directs
them to a fluorescent light detector 48. Equivalently, the mirror
43 may be formed to reflect the fluorescent light wavelengths and
pass the excitation beam wavelength. The mirror 43 is thus a light
separation element that directs the light beam from the source
along the beam path to the objective lens and directs the
fluorescent light to the fluorescent light detector. The dichroic
mirror 43 may be, e.g., a standard 700 nm Short Pass dichroic
mirror available from Chroma Technology, Inc., that, operating at
45.degree., reflects wavelengths longer than 700 nm and transmits
wavelengths shorter than 700 nm. Filters may also be used to
separate the reflected light at the source wavelengths from the
fluorescent light and such other light separation elements may be
substituted for dichroic mirrors. Additional optical elements such
as an excitation wavelength light barrier filter 49 and a lens 50
to focus the fluorescent light on the detector may also be used.
Although generally not necessary, a confocal aperture plate 51 with
a confocal aperture may be used to further block fluorescent light
from outside the focal plane from reaching the detector 48. The
dichroic mirror 43 may be located at other positions in the optical
path if desired, including a position between the objective 15 and
the galvanometer driven mirror 44. Fluorescent light emitted from
the specimen 16 in directions other than toward the objective 15
may also be collected and detected. For example only, fluorescent
light emitted through the transparent slide 17 may be collected and
detected by a detector 52, (e.g., a photomultiplier tube) with a
filter 53 used to filter out excitation (source) wavelength light.
A fluorescent light detector or detectors may be mounted adjacent
to the slide 17 to collect and detect fluorescent light or a fiber
optic cable, etc., may be used to direct fluorescent light from the
specimen to a detector.
[0017] It is understood that the components of the system 10 are
controlled and information from the detectors 22, 48 and 52 is
processed in a control module 54, typically incorporating a
computer workstation, in a conventional manner in commercially
available scanning systems (e.g., an Oz.TM. confocal laser scanning
microscope, Noran Instruments, Inc., Middleton, Wis.). The image
obtained from the information from the detectors 22, 48 and 52 may
be displayed on a video terminal 55, printed out on a printer,
stored for later display or processing, etc.
[0018] Conventional scanning systems that utilize AODs have lenses
or prisms at the input and output sides of the AOD. Normally,
efficient Bragg diffraction of a multichromatic light beam requires
some kind of chromatic correction, such as by the use of prisms, so
that all wavelengths nearly Bragg-match the angle of incidence for
nearly equal diffraction efficiencies. In the present invention,
conventional Bragg-matching prisms of the type located adjacent to
the input and output faces of the deflector 31 are preferably not
used. The mirror 30 and the acousto-optical deflector 31 preferably
are mounted in accordance with the invention so that the face of
the mirror and the input face 32 of the acousto-optical deflector
31 can be adjusted relative to each other. The mirror 30 is
preferably mounted about an axis for rotational adjustment, but the
AOD 31 may alternatively (or additionally) be mounted to rotate to
adapt its angular position. These elements can be adjusted, e.g.,
by rotation about an axis parallel to faces of the devices, to
positions such that the laser beam intersects the input face at an
angle to match the Bragg condition for maximum diffraction
efficiency at the center wavelength of the laser light. The
adjustability of the mirror 30 (or AOD 31 or both) allows the angle
of incidence on the AOD input face 32 to be selected so that the
maximum diffraction efficiency-the Bragg condition-is met at the
desired wavelength of operation. This is particularly useful for
tunable laser sources so that the same AOD arrangement can be used
to operate over a range of nominal laser wavelengths. For example,
the Bragg angle for pulses centered at 750 nm is 93.75% of the
Bragg angle for pulses centered at 800 nm, and the mirror 30 can be
rotated to adjust the angle of incidence accordingly.
Alternatively, the AOD may itself be rotated by the same amount in
the opposite direction. However, the Bragg mismatch at the
side-band wavelengths effectively acts as a spectral band-pass
filter at the input to the AOD and thus limits the extent of the
spatial (angular) spread of the AOD-deflected pulsed beam. In the
present invention, the use of dispersive elements, such as prisms,
on the input side of the AOD would not be preferred. If such an
element were used, for example, to spread the spectral components
apart to take advantage of the "band-pass" filter at the AOD
because of the resulting Bragg mismatching, doing so would also
increase the angular spread at the output side and therefore negate
the compressing role of the output side prism 35. If,
alternatively, a prism is used (in a conventional manner) to better
Bragg-match the various wavelengths at the input side of the AOD,
then the band-pass filtering provided by the AOD would be
reduced.
[0019] The utilization of the output side compensating prism 35 and
the input side mirror 30 does not significantly affect the
performance of the system 10 when used for conventional reflection
or fluorescence confocal microscopy using continuous wave (CW)
lasers. Since confocal microscopy generally uses illumination light
at different wavelengths than those used for multi-photon
fluorescence, the relative angular position of the mirror 30 and
the AOD deflector 31 need to be adjusted when a switch is made
between these modes. Alternatively, the mirror 30 or the
compensating prism 35 or both may be mounted to be displaced out of
the beampath for replacement by conventional elements, if
desired.
[0020] The prism 35 provides spectral compensation by converging
the spectrally spread parts of the pulse. The prism is preferably a
triangular shape so that the maximum correction is obtained at the
center of the scan angle. As an example of the present invention,
the laser source may provide a 20 nm wide pulse at 800 nm
wavelength. The deflection angle of the output of the
acousto-optical deflector is: 1 B f 2 V
[0021] where .lambda. represents the wavelengths in the pulse
(e.g., 789 nm<.lambda.<811 nm for a beam centered at 800 nm),
f is the acoustic modulation frequency of the acousto-optical
deflector, and V is the speed of the acoustic wave traversing the
acousto-optical deflector crystal. For a TeO.sub.2 acousto-optical
deflector, V=4322 m/s, and 200 MHz<f<400 MHz. At 300 MHz, a
20 nm-pulse will be spread across about 0.04.degree. by the
acousto-optical deflector. Utilizing a thin prism approximation,
the total deflection .delta. by a prism of angle .alpha., is:
.epsilon..apprxeq.(n(.lambda.)-1).alpha.
[0022] Thus, by utilizing the prism 35 to merge the angle of
deflection at the lower end of the prism, e.g. at the 790 nm side,
with that of the higher end of the pulse, e.g. 810 nm, using SF10
material from the Schott Glass Company, a triangular prism 35 with
a prism angle of 38.8.degree. (at the apex of a triangular prism)
is needed to correct the 0.04.degree. spread caused by the
acousto-optical deflector at the center of the scan. SF10 glass is
a highly dispersive (low V number, e.g., Vd=28.41) medium which
allows a relatively small prism angle. A smaller angle is preferred
since the smaller the prism angles, the less aberrations are
introduced in the optical beam. Other materials with appropriate
prism angles can be used if desired. For example, using SF11
material from Schott Glass Company an appropriate prism angle would
be 48.degree. to obtain the same correction.
[0023] For utilization of infrared pulses having pulse widths in
the range of a few picoseconds or less, it is also preferred that
the optics along the optical path be adapted for such systems. An
external pre-compensator 47 of conventional design may be utilized
to provide pre-compression of the pulses from the laser 11
temporally to counteract the temporal spread introduced by the
optical elements along the optical path 14. A typical
pre-compensator utilizes a two-prism and retroreflector arrangement
and commercially available units may be used. All of the various
optical elements are preferably ultrafast optics suited for use at
the near-infrared wavelengths in the light from the laser source
11. The various mirrors are preferably provided with highly
reflective near-infrared and visible coatings. The dichroic mirror
40 is selected to transmit visible and reflect near-infrared light
(or vice versa).
[0024] It is understood that the invention is not confined to the
embodiments set forth herein as illustrative, but embraces such
modified forms thereof as come within the scope of the following
claims.
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