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Claims  |
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We claim:
1. A confocal scanning beam optical microscope for spectrally-resolved
measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to a diffraction-limited spot in a
prescribed specimen plane,
means for scanning the light beam to move the diffraction-limited spot in a
predetermined scan pattern on said specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
diffraction-limited spot in said specimen plane comprising
a pinhole and a focusing lens for obtaining a focal point for confocal
detection of the light returning from said specimen,
a detector placed behind said pinhole,
means for spectrally resolving said reflected, scattered or emitted light
passing from said specimen back toward said focusing lens, pinhole and
detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm,
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of diffraction gratings and prisms that can be
rotated to direct light of various wavelengths towards said focusing lens,
whereby the diffraction-limited spot at the specimen acts like the
entrance aperture of an integrated monochromator, and the pinhole in front
of the detector acts like its exit aperture,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
2. A confocal scanning beam optical microscope for spectrally-resolved
measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to a diffraction-limited spot in a
prescribed specimen plane,
means for scanning the light beam to move the diffraction-limited spot in a
predetermined scan pattern on said specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
diffraction-limited spot in said specimen plane comprising
a pinhole and a focusing lens for obtaining a focal point for confocal
detection of the light returning from said specimen,
a detector placed behind said pinhole,
means for spectrally resolving said reflected, scattered or emitted light
passing from said specimen back toward said second focusing lens, pinhole
and detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm,
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of Fabry-Perot interferometers and Fourier
Transform spectrometers,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
3. A confocal scanning stage optical microscope for spectrally-resolved
measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to a diffraction-limited spot in a
prescribed specimen plane,
means for translating the specimen such that said diffraction-limited spot
moves relative to the specimen in a raster scan confined to said
prescribed specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
diffraction-limited spot in said specimen plane comprising
a pinhole and a focusing lens for obtaining a focal point for confocal
detection of the light returning from said specimen,
a detector placed behind said pinhole,
means for spectrally resolving said reflected, scattered or emitted light
passing from said specimen back toward said focusing lens, pinhole and
detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm,
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of diffraction gratings and prisms that can be
rotated to direct light of various wavelengths towards said focusing lens,
whereby the diffraction-limited spot at the specimen acts like the
entrance aperture of an integrated monochromator, and the pinhole in front
of the detector acts like its exit aperture,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
4. A confocal scanning stage optical microscope for spectrally-resolved
measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to a diffraction-limited spot in a
prescribed specimen plane,
means for translating the specimen such that said diffraction-limited spot
moves relative to the specimen in a raster scan confined to said
prescribed specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
diffraction-limited spot in said specimen plane comprising
a pinhole and a focusing lens for obtaining a focal point for confocal
detection of the light returning from said specimen,
a detector placed behind said pinhole,
means for spectrally resolving said reflected, scattered or emitted light
passing from said specimen back toward said focusing lens, pinhole and
detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm,
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of Fabry-Perot interferometers and Fourier
Transform spectrometers,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
5. A scanning beam optical microscope or mapping system for
spectrally-resolved measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to an illuminated spot in a prescribed
specimen plane,
means for scanning the light beam to move said illuminated spot in a
predetermined scan pattern on said specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
illuminated spot in said specimen plane comprising
an aperture and focusing lens,
a detector placed behind said aperture,
means for spectrally-resolving said reflected, scattered or emitted light
passing form said illuminated spot in said specimen plane back towards
said focusing lens, aperture and detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of diffraction gratings and prisms that can be
rotated to direct light of various wavelengths towards said focusing lens,
whereby the illuminated spot at the specimen acts like the entrance
aperture of an integrated monochromator, and said aperture acts like its
exit aperture,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
6. A scanning beam optical microscope or mapping system for
spectrally-resolved measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to an illuminated spot in a prescribed
specimen plane,
means for scanning the light beam to move said illuminated spot in a
predetermined scan pattern on said specimen plane,
a detection arm receiving light reflected, scattered or emitted from said
illuminated spot in said specimen plane comprising
an aperture and focusing lens,
a detector placed behind said aperture,
means for spectrally-resolving said reflected, scattered or emitted light
passing from said illuminated spot in said specimen plane back towards
said focusing lens, aperture and detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of Fabry-Perot interferometers and Fourier
Transform spectrometers,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
7. A scanning stage optical microscope or mapping system for
spectrally-resolved measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to an illuminated spot in a prescribed
specimen plane,
means for translating the specimen such that said illuminated spot moves
relative to the specimen in a raster scan in the prescribed specimen
plane,
a detection arm receiving light reflected, scattered or emitted from said
illuminated spot in said specimen plane comprising
an aperture and focusing lens,
a detector placed behind said aperture,
means for spectrally-resolving said reflected, scattered or emitted light
passing from said illuminated spot in said specimen plane back towards
said focusing lens, aperture and detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of Fabry-Perot interferometers and Fourier
Transform spectrometers,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light.
8. A scanning stage optical microscope or mapping system for
spectrally-resolved measurements comprising
means for supporting a specimen to be observed and measured,
an illumination source producing a light beam directed along an optical
path toward said specimen,
means for focusing the light beam to an illuminated spot in a prescribed
specimen plane,
means for translating the specimen such that said illuminated spot moves
relative to the specimen in a raster scan in the prescribed specimen
plane,
a detection arm receiving light reflected, scattered or emitted from said
illuminated spot in said specimen plane comprising
an aperture and focusing lens,
a detector placed behind said aperture,
means for spectrally-resolving said reflected, scattered or emitted light
passing from said illuminated spot in said specimen plane back towards
said focusing lens, aperture and detector,
a beamsplitter reflecting light returning from said specimen into said
detection arm
wherein said spectrally-resolving means in said detection arm is selected
from the group consisting of diffraction gratings and prisms that can be
rotated to direct light of various wavelengths towards said focusing lens,
whereby the illuminated spot at the specimen acts like the entrance
aperture of an integrated monochromator, and said aperture acts like its
exit aperture,
means for measuring the intensity distribution with respect to wavelength
of said reflected, scattered or emitted light. |
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Claims |
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Description |
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TECHNICAL FIELD
This invention relates to the fields of scanning optical microscopy,
photoluminescence analysis and photoluminescence imaging, fluorescence
analysis and fluorescence imaging, as well as many other fields, including
photon scattering experiments used to map subsurface defects in
semiconductor wafers, and Raman Effect measurements.
BACKGROUND OF THE INVENTION
Photoluminescence analysis and photoluminescence imaging are particularly
valuable for characterizing semiconductor materials, wafers, epitaxial
layers, and devices. In photoluminescence analysis, most measurements to
date have been made at a single point on a specimen, especially when the
specimen is held at low temperature in a Dewar. Because the signal
strength is low, considerable effort has been made to increase the
throughput of the grating spectrometer, including a method to shape the
beam from the laser (or other light source) at the point of impingement on
the specimen so that the illuminated area has the same shape as the
entrance slit of the grating monochromator, and when imaged on the
entrance slit, slightly overfills it, but provides for very efficient
collection of the photoluminescence light produced by the specimen, as
described by Gerry Auth in U.S. Pat. No. 4,572,668.
Spectrally-resolved photoluminescence mapping of semiconductor wafers with
high spatial resolution has recently been described by Tajima,
"Characterization of Semiconductors by Photoluminescence Mapping at Room
Temperature", Journal of Crystal Growth 103, 1-7 (1990); by Moore et al,
"A Spatially Resolved Spectrally Resolved Photoluminescence Mapping
System", Journal of Crystal Growth 103, 21-27 (1990); and in Waterloo
Scientific Inc. Application Notes on Photoluminescence #1 (1989) and #2
(1990), Waterloo Scientific Inc., 419 Phillip St., Waterloo, Ont. Canada
N2L 3X2. In this application it is known to use apparatuses which are
essentially scanning-stage non-confocal laser microscopes, in which the
exciting laser wavelength is blocked in the detector path, and the
remaining light collected from the specimen is focussed on the entrance
slit of a grating spectrometer, which is used to measure the wavelength
and intensity of the photoluminescence signal at each specimen position.
In this situation, the highest spatial resolution is achieved when the
focused spot at the point of impingement on the specimen is as small as
possible. The technique of shaping the spot described above reduces the
spatial resolution, and is therefore not appropriate when the highest
possible spatial resolution is required.
In the field of fluorescence microscopy, the confocal microscopes and
techniques presently in use have recently been described in "The Handbook
of Biological Confocal Microscopy", IMR Press, Madison, Wiss. (1989),
edited by Pawley, and in a review paper by Shotton, "Confocal Scanning
Optical Microscopy and its Applications for Biological Specimens", Journal
of Cell Science 94, 175-206 (1989). Since there may be more than one
source of fluorescence at the focal spot of the confocal microscope, it is
important to be able to separate the different wavelengths of the two
sources. In addition, a particular fluorescence source may emit different
wavelengths, and/or intensities, depending on its local environment, so it
is important to be able to map changes in spectra with position in the
specimen.
In both photoluminescence and fluorescence, it is known that measurement of
lifetimes is important. Photoluminescence or fluorescence decay is usually
measured using a pulsed or modulated light source, and the decay of the
fluorescence or photoluminescence signal is monitored with a high speed
detector. In many cases, more than one lifetime signal is detected, and
those signals are mixed together in the detected signal. It is important
to be able to separate these lifetimes, and good spatial resolution is
also important. In the case of fluorescence measurements, fluorescence
recovery after bleaching is also important. In all of these cases, high
spectral and spatial resolution in the instrument used to make the
measurements, as well as good photon collection efficiency, are important.
A simple prior art confocal scanning laser microscope is shown in FIG. 1.
In this implementation the beam from laser 102 is focused by lens 104 on
pinhole 106, and the light passing through the pinhole passes through
beamsplitter 108 and is focused by objective lens 110 to a focal spot 111
which is diffraction-limited at the surface of (or inside) specimen 112.
Light reflected from or emitted by the specimen at focal spot 111 is
collected by objective lens 110, and part of this light is reflected by
beamsplitter 108 to be focused at detector pinhole 114, which is confocal
with focal spot 111 at the specimen and pinhole 106. Light passing through
detector pinhole 114 is collected by detector 116. The combination of
detector pinhole 114 and detector 116 is a confocal detector. Light from
focal spot 111 at specimen 112 passes through detector pinhole 114, but
light from any other point on the specimen runs into the edges of detector
pinhole 114, and is not collected. Thus, out-of-focus signals are
rejected. This gives the confocal microscope the ability to do optical
tomography, which allows it to record true three dimensional images. The
microscope shown in FIG. 1 uses scanning stages 118 to move the specimen
under the stationary laser beam to record the image, but it is also
possible to scan the beam instead of scanning the specimen. Microscopes
using infinity-corrected optics are also common, both with scanning stages
and in scanning-beam configurations. These configurations are described in
"The Handbook of Biological Confocal Microscopy" edited by pawley. In
addition, detector pinhole 114 and detector 116 behind it can be replaced
by a small detector whose area is the same as that of detector pinhole
114.
Confocal Scanning Laser Microscopes have been used to record
photoluminescence and fluorescence images with high spatial resolution (in
three dimensions) using filters to block the exciting wavelength, but
accepting all (or substantially all) wavelengths of the luminescence
signal. One possible prior art configuration for such a microscope is
shown in FIG. 2, where dichroic beamsplitter 200 transmits light at the
wavelength of the incoming laser beam but reflects most of the longer
wavelength photoluminescence or fluorescence emitted from specimen 112
towards detector pinhole 114. To further reduce the small amount of
reflected laser light, blocking filter 202 which blocks light at the laser
wavelength can be placed in the detection arm of the microscope, as shown.
Three known implementations of a confocal microscope that can measure
spectrally-resolved data are as follows. It is known that a bandpass
filter can be placed in the detection arm of the microscope, either in
front of or behind the detector pinhole as described by Stelzer in
"Considerations on the intermediate optical system in confocal
microscopes", a chapter in "The Handbook of Biological Confocal
Microscopy", edited by Pawley. This may allow the operator to separate the
emission bands of two fluorophores by using two different bandpass
filters, or to measure a crude spectrum by changing filters each time a
new wavelength is to be measured, but this technique is impractical for
measuring a complete spectrum with good spectral resolution.
A second known implementation is to focus the light emitted from the
detector pinhole of a confocal photoluminescence or fluorescence
microscope onto the entrance slit of a grating monochromator (or to place
the monochromator in a position such that it's entrance slit replaces the
detector pinhole). These solutions both pas to the detector only a
fraction of the photoluminescence light collected by the microscope, and
are expensive because a complete grating monochromator is required.
A third known implementation uses a lens to focus light from the detector
pinhole onto the input aperture of a Fourier Transform Infrared
Spectrometer, or any other type of spectrometer that is appropriate for
the wavelength range involved.
Presently, the simplest confocal fluorescence microscopes usually use a
dichroic beamsplitter to separate the longer fluorescence wavelengths from
the exciting wavelength, and detect all of the fluorescence wavelengths at
once (one such microscope is shown in FIG. 2). This implementation does
not allow the operator to make spectrally-resolved measurements.
The present non-confocal photoluminescence mapping system sold by Waterloo
Scientific Inc. works by focusing the light from the non-confocal
microscope onto the entrance slit of a grating monochromator, which is
expensive, and all of the light collected by the objective lens does not
reach the grating of the monochromator.
An object of this invention is to provide a scanning microscope or mapping
system that has both good spatial resolution and good spectral resolution,
and at the same time is very efficient in collecting light emitted from
the specimen.
A further object of this invention is to reduce the cost of the
spectrally-resolved microscope or mapping system by integrating the
spectrally-resolving element into the detection arm of the microscope.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a simplified side view of a scanning stage confocal microscope of
the prior art.
FIG. 2 is a simplified side view of a scanning stage confocal fluorescence
microscope of the prior art.
FIG. 3 shows a simple embodiment of the present invention in an
infinity-corrected scanning stage confocal microscope (simplified side
view).
FIG. 4 shows a simplified side view of a scanning stage spectrally-resolved
confocal transmission microscope.
FIG. 5 is a simplified perspective view of a spectrally-resolved confocal
scanning-beam optical microscope. This is the preferred embodiment of the
present invention.
FIG. 5a is a simplified side view showing the specimen and focal spot of
the microscope of FIG. 5.
FIG. 6 is a simplified side view of a different embodiment of the detection
arm of a microscope like the microscope shown in FIG. 3 or FIG. 5.
FIG. 7 is a further embodiment of the present invention in which a linear
array of pinholes are illuminated at the top of the microscope to produce
a linear array of focal spots on the specimen, which in turn produce a
linear array of spectra at the position of a linear array of detectors.
FIG. 8 is a simplified side view of another implementation of a detection
arm of a microscope like the microscope shown in FIG. 3 or FIG. 5. This
implementation incorporates a Fabry Perot interferometer.
FIG. 9 is a simplified side view of yet another implementation of a
detection arm of a microscope like the microscope shown in FIG. 3 or FIG.
5. This implementation incorporates a Fourier Transform spectrometer.
DESCRIPTION OF THE INVENTION
We have invented a spectrally-resolved confocal scanning optical microscope
that incorporates one or more of a class of generalized confocal
microscopes. The present invention is a microscope with good spatial
resolution and good spectral resolution and is very efficient at
collecting light from the specimen. This is achieved by integrating a
monochromator or spectrometer (several kinds are possible) into the
detection arm of a confocal microscope, in front of the detector pinhole.
In the case of a confocal fluorescence or photoluminescence microscope,
this allows the confocal properties of the microscope to be maintained
while providing an efficient light path for detecting spatially-resolved
fluorescence or photoluminescence spectra. Non-confocal implementations
are also disclosed.
The invention can be implemented in several ways. First, several
embodiments will be described in which a grating has been added to
scanning laser microscopes of various optical designs to form an
integrated monochromator. In this embodiment, the illuminated spot on the
specimen acts like the entrance aperture of the integrated grating
monochromator. (A prism monochromator could also be integrated into the
microscope in a similar way.) The scanning laser microscope is used for
illustration only. Any instrument that measures spectra resulting from
excitation of the specimen by a focused beam of radiation (or two focused
beams that are confocal, or a focused and an unfocused beam) could benefit
from the invention.
Next, embodiments will be described in which the grating is replaced by
spectrometers using interference effects, including, but not necessarily
limited to, Fabry Perot interferometers and Fourier Transform
spectrometers. In these cases the exciting beam need not be focused, since
the illuminated spot on the sample does not act as an entrance aperture
for these devices.
FIG. 3 shows an infinity-corrected scanning stage confocal scanning laser
microscope in which the spectrally-resolved detection system is
implemented. In this embodiment, an incoming parallel beam 302 of laser
light (or light from some other source, not shown) passes through
beamsplitter 304 to enter objective lens 306, which focuses the beam to a
small focal spot 310 at the surface of, or inside, specimen 308. In order
to achieve the smallest possible focal spot, the incoming beam 302 has
been expanded to fill objective lens 306, and a high quality, high
numerical aperture (high NA) lens (infinity-corrected in this example) is
used for objective lens 306 so that a very small diffraction-limited spot
(focal spot 310) will be formed. Light reflected back from (or emitted or
scattered from) the region of the specimen enclosed in a small volume
enclosing focal spot 310 is collected by objective lens 306, passes back
up the microscope and is partially reflected by beamsplitter 304 into
detection arm 322 of the microscope. This parallel beam 311 strikes
diffraction grating 312 and is diffracted towards detector lens 314 of
focal length f.sub.1, placed a distance f.sub.1 in front of detector
pinhole 316. The diffraction grating 312 separates the incoming parallel
beam 311 into its spectral components (three are shown in the diagram,
317, 318 and 319), and only a very narrow band of wavelengths (centered at
the wavelength equivalent to beam 318 in the diagram), will pass through
detector pinhole 316 to reach detector 320. For that narrow band of
wavelengths, detector pinhole 316 is confocal with focal spot 310 at the
focal point of objective lens 306, so the confocal properties of the
microscope are preserved. In this embodiment, the three arms of the
microscope (a, b and c) act like a grating monochromator, in which the
illuminated focal spot 310 at the specimen position is the source of light
entering the monochromator (and acts like the entrance slit of an ordinary
grating monochromator), objective lens 306 focuses the light to a parallel
beam which impinges on plane grating 312, and light at one wavelength
(beam 318) is diffracted by the grating in exactly the correct direction
to be focused on detector pinhole 316 by detector lens 314. Light at other
wavelengths hits the area surrounding the pinhole, and is not detected.
Detector pinhole 316 performs the function of the exit slit that would be
used in an ordinary grating monochromator. A simple beam-expanding
telescope can be used to adjust the diameter of the beam where it hits
diffraction grating 312 (if required) and the diameter of the beam leaving
the grating can be readjusted to fill detector lens 314 in front of
detector pinhole 316 if necessary (beam shaping optics can also be added
before and after diffraction grating 312 if necessary). All lenses in the
return path of the beam should be achromatic over the range of wavelengths
of interest, or alternatively reflecting optics can be used. Other optical
combinations, which might include a concave grating, are also possible.
FIG. 4 shows the spectrally-resolved detector integrated into a scanning
stage confocal transmission microscope. In this embodiment, incoming
parallel beam 302 (from a laser or other light source) is focused to a
focal spot 310 at the surface of or inside specimen 308 by objective lens
306. Light transmitted through the specimen is collected by second
objective lens 402, is then diffracted by diffraction grating 404, is
focused by detector lens 406 and a small range of wavelengths passes
through detector pinhole 408 and is detected by transmitted-light detector
410. Light of other wavelengths will not pass through pinhole 408 to be
detected. In this embodiment objective lenses 306 and 402 are usually a
matched pair of infinity-corrected microscope objectives, and the specimen
is moved in a raster scan on translation stages (not shown).
FIG. 5 illustrates a further embodiment of the invention, the preferred
embodiment, in which a grating monochromator has been integrated into the
detection arm of a scanning beam confocal optical microscope. Light beam
503 from laser 502 (or other light source) passes through narrow bandpass
filter 504 and is focused on pinhole 508 by lens 506. The expanding beam
exiting pinhole 508 is focused to a parallel beam by lens 510. (Lens 506,
pinhole 508 and lens 510 constitute a spatial filter and beam expander.)
The parallel beam passes through beamsplitter 512 and is deflected in the
x-y plane by first scanning mirror 514, which rotates about an axis
parallel to the z-direction. Lenses 516 and 518 of focal length f.sub.2
return the deflected light beam to the center of second scanning mirror
520, which rotates about an axis parallel to the x-direction and imparts a
deflection in the y-z plane. Lenses 522 and 524 of focal length f.sub.3
return the deflected beam (which now has been deflected by both scanning
mirrors) to enter objective lens 526 centered on its entrance pupil.
Objective lens 526 focuses the light to a focal spot 527 (see FIG. 5a) at
the surface of or inside specimen 528. The focus position is set by focus
stage 530, which moves in the z-direction. Light reflected back from or
emitted by the tiny volume of the specimen at focal spot 527 is collected
by objective lens 526 and passes back through the scan system of the
microscope. Part of this returning beam is reflected by beamsplitter 512
towards beam expander 532 (optional) and scanning grating 534. Scanning
grating 534 diffracts light towards lens $36, and a small range of
wavelengths will be focused by lens 536 to pass through detector pinhole
538 and will reach detector 540. Light with other wavelengths will hit the
opaque surface around pinhole 538 and will not be detected.
Our invention (as disclosed in FIGS. 3, 4 and 5 and described above), has
several advantages over simply focusing the light from the detector
pinhole onto the entrance slit of a grating monochromator (or placing the
monochromator so that its entrance slit replaces the detector pinhole).
First, it is a much more efficient optical arrangement, allowing more
light to reach the detector. All of the light entering the detection arm
of the microscope reaches the diffraction grating, whereas in the other
arrangement only a fraction of the light passes through the entrance slit
of the monochromator. Second, it is a simpler optical configuration, since
for best optical efficiency with the grating monochromator it would be
necessary to match the input NA of the grating monochromator with the
output from the microscope, and third, it results in a more compact and
less expensive microscope or mapping system.
This microscope measures the intensity of light reflected or emitted from
the specimen as a function of x,y,z and wavelength. Data can be collected
by scanning the grating to record a full spectrum at each position on the
specimen (wavelength scanning), or the grating can be held in a single
position while the specimen (or beam) is moved in a raster scan, storing a
complete raster scan at only one wavelength. Another raster scan can then
be performed at the next grating position (next wavelength), and so on.
Any combination of single or multiple point, line, area or volume scans in
position can be performed, measuring the light intensity at a single
wavelength, several discrete wavelengths, or over the whole range of
wavelengths measurable with that grating.
A further embodiment of the detection arm of the microscope (outlined with
dashes in FIG. 3) is shown as detection arm 604 in FIG. 6. Here detector
pinhole 316 and detector 320 (or single small detector) are replaced by a
linear array 602 of N.sub.1 small detectors. (In the figure, N.sub.1 =3.)
This enables the microscope to simultaneously measure N.sub.1 different
wavelengths without scanning the grating.
Other optical arrangements may be appropriate in special situations, e.g.
detector pinhole 316 (shown in FIG. 3) can be replaced by a slit parallel
to the rulings on the grating, and in such a case it may be appropriate to
use an evenly-illuminated slit in place of the pinhole in the spatial
filter, resulting in a slit-shaped illuminated spot on the specimen. This
slit should be fabricated with a width that is about the same as the
diameter of the original spatial filter pinhole, so that the bright area
on the specimen is as narrow as possible for good spectral resolution
(since this slit image acts just like the real slit at the entrance of a
grating monochromator), and should be oriented so that its image on the
grating is oriented with its long dimension parallel to the rulings on the
grating. In this kind of slit-scanning microscope, the specimen (or beam)
is scanned in a direction parallel to the short dimension of the image of
the rectangular slit at the specimen position. Scanning slit microscopes
have some confocal qualities, and are described in the literature by
Wilson, "The Role of the Pinhole in Confocal Imaging Systems", in "The
Handbook of Biological Confocal Microscopy", edited by Pawley.
A further possible arrangement would be to replace the pinhole at the
spatial filter position by a linear array of pinholes illuminated with
equal intensity, and then to focus these to a linear array of bright spots
in the focal plane of the instrument (see FIG. 7). A linear array of small
detectors 722, placed in confocal positions parallel to the direction of
rulings on diffraction grating 714, allows the microscope to collect data
from several positions on the specimen simultaneously when the specimen
(or beam) is scanned in a direction perpendicular to the line of bright
spots. If the linear detector array is replaced by a two-dimensional array
with the same array spacing as just described above in the direction
parallel to the ruling direction on the grating, then a spectral range can
be collected from each spot as it scans across the specimen.
In all of the embodiments described so far, the illuminated focal spot
acted like the entrance slit of the grating monochromator that has been
integrated into the microscope, and it was therefore necessary to excite
or illuminate the specimen with a focused beam of radiation. In the two
embodiments described next, an entrance slit or pinhole is not required
for the monochromator, and it is not necessary to illuminate or excite the
specimen with a focused beam.
FIG. 8 shows a further embodiment of a detection arm 810 that can replace
detection arm 322 shown in FIG. 3. In FIG. 8, the grating has been
replaced by Fabry-Perot interferometer 802. In this case a bandpass filter
set is often used in front of the interferometer (at position b) to limit
the range of wavelengths entering the interferometer to the range that it
was designed to measure. The monochromatic light leaving the
interferometer is then focused onto pinhole 806 (or small detector) by
lens 804. Only light originating from a tiny volume around the focal point
of the objective lens of the microscope (see FIG. 3) will be focused On
pinhole 806, so this is a confocal microscope. Light originating from all
other points in the specimen will be blocked by the metal surrounding
pinhole 806. The microscope can be used in two different data collection
modes. If the spacing of the interferometer plates is kept constant, a
raster scan across the specimen allows data to be collected at a single
wavelength. The spacing of the interferometer plates can then be changed
to let a second wavelength through, and the raster scan repeated. This
sequence is repeated until the required spectral information is collected
at each scan position. In the second data collection mode, the measurement
position is held fixed (by holding the specimen in a fixed position in a
stage-scanning microscope, or by holding the beam-scanning components
fixed in a beam-scanning microscope) while the complete spectrum is
measured by varying the distance between plates in Fabry-Perot
interferometer 802. The specimen or beam is then moved to the next
position, and the spectrum is measured at that position, and so on until
spectral information has been measured at each position in the raster
scan. We contemplate using the Fabry-Perot interferometer to enable
spectrally resolved measurements in yet a further class of scanning
optical microscope, the Nipkow Disk microscope described by Kino in
"Efficiency in Nipkow Disk Microscopes", an article in "The Handbook of
Biological Confocal Microscopy", edited by Pawley.
Both of the embodiments described in FIGS. 3 and 8 are also very useful for
fluorescence or photoluminescence lifetime experiments. In these
experiments, two or more lifetimes are often measured simultaneously,
making it difficult to separate the signals, especially if the measured
lifetimes have nearly the same value. If different sources of
photoluminescence or fluorescence are involved, they will likely emit
radiation with different spectral components, so it is now possible to
separate lifetimes from different sources (e.g. different fluorophores) by
performing a lifetime measurement at different wavelengths, which not only
separates the different lifetimes, but also helps identify the source of
the signal. In these measurements the lifetime is measured at a single
wavelength at each sample position, the detection wavelength is then
changed, a second measurement is performed, and so on.
In FIG. 9, the detection arm 322 of the microscope shown in FIG. 3 has been
replaced by a new detection arm 920 containing a two-arm (or two-beam)
interferometer composed of beamsplitter 902, fixed mirror 904 and movable
mirror 906, detector lens 908 to focus the light onto pinhole 910, and
detector 912 to detect the light transmitted through pinhole 910. With its
associated electronics, this forms the basis of a Fourier Transform (FT)
spectrometer. In an ordinary FT spectrometer, light leaving the
spectrometer is focused with a simple collector lens onto a detector which
has an area that is much bigger than the pinhole used in this embodiment,
and it is the addition of detector lens 908 and pinhole 910 that allows
the FT spectrometer to be integrated into the detection arm of a confocal
microscope, since the pinhole blocks light coming from any position in the
specimen except from a tiny volume around the focal point of the
microscope's objective lens. Since a Fourier Transform spectrometer
measures a spectrum each time the movable mirror 906 is scanned, and
cannot measure only one wavelength at a time, in this particular
embodiment of the scanning laser microscope, a spectrum must be measured
at each pixel position, so this embodiment is not appropriate for lifetime
measurements.
The light source shown in all of the microscopes described is a laser,
however other light or radiation sources can also be used.
Having described preferred embodiments of the new and improved
spectrally-resolved scanning optical microscope or mapping system
constructed in accordance with the present invention, it is believed that
other modifications, variations, and changes will be suggested to those
skilled in the art in view of the teachings set forth herein. It is
therefore to be understood that all such variations, modifications and
changes are believed to fall within the scope of the present invention as
defined by the appended claims.
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