U.S. patent number 4,863,226 [Application Number 07/166,226] was granted by the patent office on 1989-09-05 for confocal laser scanning microscope.
This patent grant is currently assigned to Nederlandse Organisatie Voor Toegepas - Natuurwetenschappelijk Onderzoek. Invention is credited to Arie Draaijer, Pieter M. Houpt.
United States Patent |
4,863,226 |
Houpt , et al. |
September 5, 1989 |
Confocal laser scanning microscope
Abstract
Confocal laser scanning microscope comprising a laser as point
light source, a deflection system for the line and frame scanning
and a lens system, at least one objective near the object, an
object stage, a spatial filter and a detector, and an electronic
control and imaging-processing system. The object is scanned point
by point by the light beam and measurement is made with the
detector only where the point light source is focused so that
out-of-focus light is not detected. As a result resolution and
contrast in three dimensions, in particular, axially to the image
plane can be improved considerably, and 3D information can be
derived from the object. For the more rapid line scanning, the
deflection system of the present invention comprises an
acousto-optical deflector and comprises for the slower frame
scanning, another deflector constructed in a manner such that at
least the frame-scanning movement of the return light beam is
completely eliminated, as a result of which the return light is
focused on the spatial filter.
Inventors: |
Houpt; Pieter M. (The Hague,
NL), Draaijer; Arie (Zwijndrecht, NL) |
Assignee: |
Nederlandse Organisatie Voor
Toegepas - Natuurwetenschappelijk Onderzoek (The Hague,
NL)
|
Family
ID: |
19849704 |
Appl.
No.: |
07/166,226 |
Filed: |
March 10, 1988 |
Foreign Application Priority Data
|
|
|
|
|
Mar 13, 1987 [NL] |
|
|
8700612 |
|
Current U.S.
Class: |
359/212.1;
250/566; 850/6; 850/63; 850/9 |
Current CPC
Class: |
G02B
21/0048 (20130101); G02B 21/0084 (20130101); G02B
21/0076 (20130101); G02B 21/0032 (20130101); G02B
21/0036 (20130101); G02B 21/0068 (20130101); G02B
21/0072 (20130101) |
Current International
Class: |
G02B
21/00 (20060101); G02B 026/10 () |
Field of
Search: |
;350/6.5,6.6,6.9,6.91,6.1,400,401,403,404,500,510,6.8,516,520,529
;250/566,572,310 ;346/108,109 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
J G. De La Rosa et al., "Wafer Inspection With a Laser Scanning
Microscope", AT&T Technical Journal, vol. 65, No. 1, Jan./Feb.
1986, pp. 68-77, Short Hills, New Jersey, US..
|
Primary Examiner: Arnold; Bruce Y.
Assistant Examiner: Ben; Loha
Attorney, Agent or Firm: Brumbaugh, Graves, Donohue &
Raymond
Claims
We claim:
1. A confocal laser scanning microscope for viewing an object and
producing a magnified image of the object comprising a plurality of
lines arranged in a frame, said microscope comprising:
a laser light beam source emitting a light beam;
deflection means to deflect the light beam along lines, and for
deflecting the line-deflected light beam into successive line
positions in the frame;
at least one objective in the path of the deflected light beam and
between the deflection means and the object, and located near the
object;
an object stage for holding the object;
diversion means for diverting, away from the laser light beam, the
light beam reflected from the object;
a spatial filter in the path of the diverted light beam for
spatially filtering the diverted light beam, thereby permitting
substantially only in-focus light reflected from the object to pass
beyond the spatial filter;
and a detector in the path of the spatially-filtered diverted light
beam;
wherein said deflection means comprises an acousto-optical
deflector for deflecting the light beam along the lines, and a
second deflector for deflecting the line-deflected light beam into
successive line positions in the frame, whereby resolution and
contrast considerably improve in three dimensions, in particular,
axially to the image plane, and three-dimensional information can
be derived from the object.
2. A confocal laser scanning microscope according to claim 1,
wherein said second deflector is a mirror galvanometer.
3. A confocal laser scanning microscope according to claim 1,
wherein the diversion means is a beam splitter; wherein the spatial
filter is a pinhole filter; and wherein the position of the
diversion means along the light beam is between the laser light
beam source and the deflection means whereby both the line-scanning
movement and the frame-scanning movement of the return light beam
are eliminated.
4. A confocal laser scanning microscope according to claim 3,
further comprising:
a quarter-wave plate located in the light beam between the
deflection means and the at least one objective, said quarter-wave
plate converting the light from the laser light beam source into
circularly polarized light, and said quarter-wave plate also
converting the circularly polarized light reflecting from said
object into linearly polarized light having a polarization
direction perpendicular to that of the light from the laser light
beam source; and
a polarizing filter located in the diverted light beam between the
diversion means and the spatial filter, the polarizing filter
oriented so that its direction of polarization is perpendicular to
the polarization direction of the light from the laser light beam
source, thereby transmitting substantially only light reflected by
the object and blocking substantially all light reflected elsewhere
along the light beam.
5. A confocal laser scanning microscope according to claim 4,
wherein the deflection means deflects light at an angle which
varies as a function of its wavelength, and further comprising:
displacement means for displacing said spatial filter in three
dimensions, said displacement means displacing said spatial filter
so as to transmit substantially only diverted light deflected at a
particular angle other than the angle through which light from the
laser light beam source is deflected, whereby light reflected or
emitted by the object that is of a particular wavelength other than
that of the light from the laser light beam source is transmitted;
and
wavelength filter means located along the path of the diverted
light to filter out diverted light of the same wavelength as that
of the light from the laser light beam source.
6. A confocal laser scanning microscope according to claim 1,
further comprising:
dichromatic reflection means located in the light beam between the
said acousto-optic deflector and the said second deflector, for
transmitting light of the wavelength of the light from the laser
light beam source and for reflecting light which has another
wavelength;
a line detector in the reflected light path; and
an objective in the reflected light path between said dichromatic
reflection means and said line detector.
7. A confocal laser scanning microscope according to claim 6,
wherein said line detector comprises a spatial slit filter and a
second detector.
8. In a confocal laser scanning microscope for viewing an object
and producing a magnified image of the object comprising a
plurality of lines arranged in a frame, said microscope
comprising:
a laser light beam source emitting a light beam;
deflection means to deflect the light beam along lines, and for
deflecting the line-deflected light beam into successive line
positions in the frame;
at least one objective in the path of the deflected light beam and
between the deflection means and the object, and located near the
object;
an object stage for holding the object;
diversion means for diverting, away from the laser light beam
source, the light beam reflected from the object;
a spatial filter in the path of the diverted light beam for
spatially filtering the diverted light beam, thereby permitting
substantially only in-focus light reflected from the object to pass
beyond the spatial filter, whereby the resolution and contrast
considerably improve in three dimensions, in particular, axially to
the image plane, and three-dimensional information can be derived
from the object;
and a detector in the path of the spatially-filtered diverted light
beam;
the improvement wherein:
said deflection means comprises an acousto-optic deflector for
deflecting the light beam along the lines, and a second deflector
for deflecting the line-deflected light beam into successive line
positions in the frame, whereby the frame-scanning movement of the
return light beam is eliminated, as a result of which the return
light beam is focused on the spatial filter.
9. A confocal laser scanning microscope according to claim 8,
wherein said second deflector is a mirror galvanometer.
10. A confocal laser scanning microscope according to claim 8,
wherein the diversion means is a beam splitter; wherein the spatial
filter is a pinhole filter; and wherein the position of the
diversion means along the light beam is between the laser light
beam source and the deflection means whereby both the line-scanning
movement and the frame-scanning movement of the return light beam
are eliminated.
11. A confocal laser scanning microscope according to claim 10,
further comprising:
a quarter-wave plate located in the light beam between the
deflection means and the at least one objective, said quarter-wave
plate converting the light from the laser light beam source into
circularly polarized light, and said quarter-wave plate also
converting the circularly polarized light reflecting from said
object into linearly polarized light having a polarization
direction perpendicular to that of the light from the laser light
beam source; and
a polarizing filter located in the diverted light beam between the
diversion means and the spatial filter, the polarizing filter
oriented so that its direction of polarization is perpendicular to
the polarization direction of the light from the laser light beam
source, thereby, transmitting substantially only light reflected by
the object and blocking substantially all light reflected elsewhere
along the light beam.
12. A confocal laser scanning microscope according to claim 11,
wherein the deflection means deflects light at an angle which
varies as a function of its wavelength, and further comprising:
displacement means for displacing said spatial filter in three
dimensions, said displacement means displacing said spatial filter
so as to transmit substantially only diverted light deflected at a
particular angle other than the angle through which light from the
laser light beam source is deflected, whereby light reflected or
emitted by the object that is of a particular wavelength other than
that of the light from the laser light beam source is transmitted;
and
wavelength filter means located along the path of the diverted
light to filter out diverted light of the same wavelength as that
of the light from the laser light beam source.
13. A confocal laser scanning microscope according to claim 8,
further comprising:
dichromatic reflection means located in the light beam between the
said acousto-optic deflector and the said second deflector, for
transmitting light of the wavelength of the light from the laser
light beam source and for reflecting light which has another
wavelength;
a line detector in the reflected light path; and
an objective in the reflected light path between said dichromatic
reflection means and said line detector.
14. A confocal laser scanning microscope according to claim 13,
wherein:
said line detector comprises a slit filter and a second detector.
Description
The invention relates to a confocal laser scanning microscope
provided with a laser as point light source, a deflection system
for the line and frame scanning and a lens system, at least one
objective near the object, an object stage, a spatial filter and a
detector, and an electronic control and image-processing system,
the object being scanned point by point by the light beam and
measurements being made with the detector only where the point
light source is focused so that out-of-focus light is not detected,
as a result of which resolution and contrast considerably improve
in three dimensions, in particular, axially to the image plane, and
3D information can be derived from the object. Such a confocal
laser scanning microscope is known from the book entitled "Theory
and Practice of Scanning Optical Microscopy" by T Wilson and C
Sheppard, Academic Press, 1984.
In such a microscope, the focused laser spot is scanned over the
stationary object or preparation. Use is generally made of
galvanometric deflection by means of mirror galvanometers in said
scanning movement of the focused laser spot both for the line
scanning and for the frame scanning. The object is also often
scanned by means of the moving object stage with respect to a
stationary focus laser spot. The disadvantage of such methods of
scanning is that they have a mechanical nature and are therefore
inherently slow. This also produces long frame times.
The object of the invention is to eliminate these problems and, in
a confocal laser scanning microscope, to achieve a deflection which
can operate with high speed and flexibility, i.e. with variable
scanning amplitude and various types of microscopy, and which has
an optically relatively simple embodiment. As a result of rapid
line and frame scanning, it is then possible, in a very short time,
to combine electronically a number of thin image sections to form
an image with an increased depth of focus.
In a confocal laser scanning microscope of the type mentioned in
the introduction, this is thus achieved according to the invention
in that, for the more rapid line scanning, the deflection system
comprises an acousto-optical deflector and, for the slower field
scanning, it comprises another deflector constructed in a manner
such that at least the frame-scanning movement of the return light
beam is completely eliminated, as a result of which the return
light is focussed on the spatial filter.
The use of an acousto-optical deflector is known per se in the case
of non-confocal laser scanning microscopy. If it is used in
confocal microscopy however, the known disadvantages of the
dispersive nature and the necessity of using several lenses then
occur, as a result of which, respectively, confocal microscopy
using light to be observed of a wavelength other than that of the
light incident on the object (fluorescence etc) is not readily
possible and the disturbing effect of reflections at surfaces of
the lenses used is considerable. In addition, the latter have to be
of good quality to avoid aberrations.
In an advantageous embodiment, the other deflector in the
deflection system may consist of a mirror galvanometer.
A further advantageous embodiment, in which a beam splitter is
incorporated in the light path in order to split off the return
light beam after the acousto-optical deflector and to direct it to
the detector via a further objective, is characterized in that the
deflection system with the acousto-optical deflector is also
constructed in a manner such that the line-scanning movement of the
return light beam is also completely eliminated, and in that the
spatial filter is a pin-hole filter which forms a point detector
with the subsequent detector.
Advantageously, in the confocal laser scanning microscope according
to the invention a quarter-wave plate may be incorporated in the
outward path in front of the objective in order to suppress the
disturbing effect of optical reflections in the outward path which
are produced by the use of the acousto-optical deflector with the
necessity, associated therewith, of using several lenses. A
polarizing filter, whose polarization direction is perpendicular to
the polarization direction of the outward light should then be
incorporated in the path of the reflected light beam. The outward
linearly polarized light is converted into circularly polarized
light by the quarter-wave plate. After reflection by the object,
said circularly polarized light again passes through the
quarter-wave plate so that light is linearly polarized with a
polarization direction perpendicular to that of the incident beam.
The polarization filter incorporated in the path of the reflected
light transmits only light having a polarization direction
perpendicular to that of the outward beam, and this can then be
detected by the point detector.
The invention will now be explained in more detail on the basis of
an exemplary embodiment with reference to the drawings, in
which:
FIG. 1 gives a diagrammatic view of a first embodiment of the
confocal laser scanning microscope according to the invention;
FIG. 2 gives a diagrammatic view of the manner in which the light
beam is incident on the objective;
FIG. 3 gives a detailed view of the spatial filter in the
microscope of FIG. 1; and
FIG. 4 gives a diagrammatic view of a second embodiment of the
confocal laser scanning microscope according to the invention.
The first embodiment is explained with reference to FIG. 1. The
laser light beam 1 first passes the lenses 2 and 3 forming together
a beam expansion optical system, followed by a beam splitter 4, an
acousto-optical deflector 5 having a planocylindrical lens 5.1 and
a planoconvex lens 5.2 both at the entrance and at the exit side, a
lens 6, a deflector 7, which may be a mirror galvanometer
deflector, a lens 8, a quarter-wave plate 16, and an objective 9.
In the object plane 10, an object, not shown, is further placed on
a stationary object stage. The reflected light traverses a return
path identical to the outward path up to the beam splitter 4 after
which it is split off to a polarizing filter 11, a further
objective 13, a spatial filter 14, a lens 17, a band pass or
cut-off filter 12, and finally a detector 15.
The expansion optical system mentioned, which has an expansion
factor of three, ensures, in combination with the other optical
elements, that the light completely fills the entrance pupil of the
objective 9. The beam splitter 4 ensures that light reflected by
the object is separated from the outward laser light. The
acousto-optical deflector 5 brings about the line scanning over the
object, and the mirror galvanometer 7 brings about the relatively
slower frame scanning across the object. In this connection, the
acousto-optical deflector can achieve a deflection of the laser
beam with such a high frequency that the latter takes place with
video speed or even higher speed.
The lens 6 increases the angle through which the laser beam is
deflected by the acousto-optical deflector. The focused spot of the
laser beam ends up in a position such that the objective is used in
the correct manner. The mirror galvanometer 7 is at the focal point
of said lens and is colinear with lens 8 and objective 9. As a
result of this, the laser beam is stationary both at the mirror
galvanometer and at the rear side of the objective so that, at
these positions, only the angle of incidence, but not the position
of incidence, of the laser beam changes. In this connection, see
FIG. 2 for comparison.
The objective 9 focuses the outward laser beam 18 on the object,
which may be, for example, a biological preparation or any other
object. The laser light 19 reflected or scattered by the object
follows the same optical path backwards up to the beam splitter 4.
After this, the reflected light follows the path already specified
through the elements 11, 13, 14, 17 and 12 up to the detector 15.
The scanning X-Y movement of the laser beam introduced by the
acousto-optical deflector and the mirror galvanometer is eliminated
again on the return path so that the reflected light is focused on
the stationary spatial filter 14, which is a pin-hole filter
(2-micron hole). This filter, with the detector immediately behind
it, forms a point detector. As a result of this the microscope has
confocal characteristics.
In this first embodiment according to the invention, the disturbing
reflections introduced in the outward path by the acousto-optical
deflector are eliminated by means of a quarter-wave plate 16
incorporated in front of the objective 9 and a polarizing filter 11
incorporated after the beam splitter. The outward linearly
polarized light is converted into circularly polarized light by the
plate 16, after which said light, after reflection, again passes
through said quarter-wave plate and is converted again into
linearly polarized light with a polarization direction
perpendicular to the incident beam. The polarization filter 11
incorporated after the beam splitter is also adjusted to this
polarization direction so that only reflections from the object and
the objective 9 are detected.
Further disadvantageous effects may possibly be caused by the
dispersive nature of the acousto-optical deflector, as a result of
which return light of a wavelength other than the laser light (for
example, light emitted by the object through fluorescence) no
longer passes through the spatial filter, are taken care of by
displacing the spatial filter 14 in those cases. Such a spatial
filter may advantageously be displaced by three piezoelectric
crystals, each for one of the three axes of the XYZ coordinate
system, as indicated in FIG. 3.
Advantageously, such a microscope can be used for examining
fluorescent preparations which have this property inherently or
have been marked for the purpose. The light emitted as a result
when light is incident on the object has a wavelength other than
that of the outward laser light. By using a band filter or cut-off
filter 12 in the return path which is matched to the expected
wavelength of the return light, the latter can be selectively
transmitted so that no disturbing effect is experienced from the
reflected laser light. Since the light of differing wavelengths
also undergoes a different deflection in the acousto-optical
deflector 5, the spatial filter 12 must be arranged in another
position corresponding to the angular dispersion of the
acousto-optical deflector.
FIG. 4 indicates a second embodiment. A dichromatic mirror 20 has
been incorporated in the light path between the planocylindrical
lens 5.1 and the lens 6. Said mirror transmits the (short-wave)
laser light and deflects the long-wave return light originating,
for example, from fluorescence. This light is passed through a
correction lens 21 and focused with the objective 22 on a special
spatial filter 23 which is a slit filter (330 microns.times.1
micron), as a result of which this system has confocal
characteristics. In this manner, a line detector is formed with the
subsequent lens 24 and detector 26. Between the lens 24 and the
detector 26 one more band pass or cut-off filter 25 has been
incorporated which has the same function as that of the band pass
or cut-off filter 12. With this embodiment, return light which has
a wavelength other than that of the outward light can be
advantageously examined if the acousto-optical deflector has too
low an efficiency for said light, i.e. brings about too large an
attenuation.
With this microscope it is possible to assemble, for example, 20
images per second at a line frequency of 20 kHz. That is to say,
each image contains 1000 lines. With such a number of 20,000 lines
per second and if there are to be 1000 image points or pixels per
line, the detector and the subsequent electronics which measure the
half tone of a pixel which have to have at least a response of not
more than 50 nsec. Advantageously, with such a fast image assembly,
an image with increased depth of focus can be built up by combining
a number of thin image sections. According to the above example, 20
sections situated underneath each other can be combined in one
second so that an object which is 20 sections thick can be
reproduced with complete sharpness.
In addition to being used in the field of application of biology,
such a microscope according to the invention having a line-scanning
frequency of, for example, 20-30 kHz and a frame-scanning frequency
of, for example, 90 Hz can also be used in forensic examination and
in the microelectronics industry. In all cases, the fact that it is
not necessary with this microscope to work with the object under
vacuum is of great advantage. In contrast to the scanning electron
microscope, for which the preparations have to be covered with a
thin conductive metal layer, this method is also non-destructive.
The fact that, in the microscope according to the invention, no
mechanical forces are exerted on the preparation is also an
advantage over systems in which the preparation is scanned. In the
microelectronics industry, this microscope can be used for
production control of LSI and VLSI chips, the production of "custom
design" chips and also the functional control of chips by means of
the so-called "optical beam induced current" method.
The present microscope can also be used advantageously for
examining optical memories.
* * * * *