U.S. patent application number 10/804258 was filed with the patent office on 2004-09-09 for arrangement for illumination and/or detection in a microscope.
Invention is credited to Schoeppe, Guenter, Simon, Ulrich, Stock, Michael, Weyh, Thomas, Wolleschensky, Ralf.
Application Number | 20040174593 10/804258 |
Document ID | / |
Family ID | 7876338 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040174593 |
Kind Code |
A1 |
Weyh, Thomas ; et
al. |
September 9, 2004 |
Arrangement for illumination and/or detection in a microscope
Abstract
A laser scanning microscope comprises at least one selectively
switchable micro-mirror arrangement (DMD) in the illumination beam
path and/or detection beam path which is used for the wavelength
selection of dispersively divided illumination and/or object light
such as reflection, fluorescence.
Inventors: |
Weyh, Thomas; (Stadtroda,
DE) ; Simon, Ulrich; (Rothenstein, DE) ;
Schoeppe, Guenter; (Jena, DE) ; Wolleschensky,
Ralf; (Schoeten, DE) ; Stock, Michael;
(Apolda, DE) |
Correspondence
Address: |
REED SMITH, LLP
ATTN: PATENT RECORDS DEPARTMENT
599 LEXINGTON AVENUE, 29TH FLOOR
NEW YORK
NY
10022-7650
US
|
Family ID: |
7876338 |
Appl. No.: |
10/804258 |
Filed: |
March 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10804258 |
Mar 18, 2004 |
|
|
|
09295555 |
Apr 21, 1999 |
|
|
|
Current U.S.
Class: |
359/388 ;
359/385 |
Current CPC
Class: |
G02B 26/0841 20130101;
G02B 21/0032 20130101; G02B 21/06 20130101; G02B 26/0833 20130101;
G02B 21/002 20130101 |
Class at
Publication: |
359/388 ;
359/385 |
International
Class: |
G02B 007/04; G02B
027/40; G02B 027/64; G02B 021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1998 |
DE |
198 35 072.4 |
Claims
What is claimed is:
1. In a laser scanning microscope, an arrangement comprising: a
micro-mirror arrangement acting as a confocal pinhole diaphragm in
a detection beam path; and a detector that receives a detection
beam from the micro-mirror arrangement.
2. The arrangement according to claim 1, wherein the micro-mirror
arrangement is programmable with respect to a size and shape of the
pinhole on the micro-mirror arrangement.
3. The arrangement according to claim 1, wherein the micro-mirror
arrangement is programmable with respect to a lateral position of
the pinhole on the micro-mirror arrangement.
4. The arrangement according to claim 1, wherein the micro-mirror
arrangement is programmable with respect to a size of the pinhole
on the micro-mirror arrangement in synchronization with a detected
spectral band.
5. The arrangement according to claim 1, further comprising: a
dispersive element positioned in the detection beam path between
the micro-mirror arrangement and the detector, and operable to
spatially disperse a detection beam from the micro-mirror
arrangement; wherein the micro-mirror arrangement is programmable
with respect to a size, shape and lateral position of the pinhole
on the micro-mirror arrangement in synchronization with a detected
spectral band.
6. The arrangement according to claim 1, further comprising: a
second micro-mirror arrangement and a second dispersive element
acting together as a beam splitter for wavelength-selective
coupling-in of an excitation beam in the direction of an object
under study and wavelength-selective coupling-out of a detection
beam in the direction of the detector.
7. In a laser scanning microscope, an arrangement comprising: a
micro-mirror arrangement acting as a slit pinhole in a detection
beam path so as to receive a scanned line of an object; and a
detector that receives the scanned line from the micro-mirror
arrangement.
8. The arrangement according to claim 7, further comprising a
dispersive element positioned in the detection beam path between
the micro-mirror arrangement and the detector; wherein the detector
is a two dimensional array of detector elements such that the
scanned line is imaged in one dimension of the detector and the
spectrum of the scanned line is imaged in the other dimension of
the detector.
9. In a laser scanning microscope, an arrangement comprising: a
dispersive element positioned in a detection beam path and operable
to spatially disperse a detection beam coming from an object under
study; a micro-mirror arrangement operable to switch selected
wavelengths of the dispersed detection beam; and a detector that
receives the switched wavelengths of the spatially dispersed
detection beam.
10. In a laser scanning microscope, an arrangement comprising: an
LCD arrangement acting as a confocal pinhole diaphragm in a
detection beam path; and a detector that receives a detection beam
from the LCD arrangement.
11. In a laser microscope, a combination comprising: at least one
dispersive element disposed in a detection beam path; and a
selectively switchable transmission diaphragm in the detection beam
path.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. parent
application Ser. No. 09/295,555, filed Apr. 21, 1999, which claims
priority of German Application No.198 35 072.4, filed Aug. 4, 1998,
all of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] a) Field of the Invention
[0003] The invention is directed to a laser scanning microscope
comprising a laser unit, scanning means, a microscope stand, a
detection unit and a control/receiving unit, wherein the spectral
characteristics of the detection unit can be freely programmed by a
switching mirror array integrated in a monochromator. The switching
mirror array (one- or two-dimensional) can be constructed, for
example, as a DMD (Digital Mirror Device or switchable-mirror
arrangement). The laser scanning microscope according to the
invention enables different operating modes. On the one hand, the
emitted spectrum can be detected with high resolution on every
scanned pixel (in the specimen); on the other hand, the emitted
spectrum can be divided into a quantity of spectral portions (zones
on the switching mirror array) and each of these portions can be
treated as a separate electronic detection channel (which is
advantageous, e.g., for receiving multiple fluorescences). Further,
a freely programmable confocal diaphragm (pinhole) can be realized
by means of an additional switching mirror array which is
introduced in a conjugate plane to the object plane.
[0004] b) Description of the Related Art
[0005] Modern laser scanning microscopes for examination of
(fluorescing) specimens generally comprise a detection unit which
divides the (fluorescent) radiation emitted from the specimen into
a certain quantity of (generally up to 4) detection channels by
means of dichroic splitter layers and filter layers (principle of
optical multichannel analyzer--OMA). These splitters/filters are
generally held in rotatable revolving wheels or linear-displaceable
disks. In this way, a spectral adaptation of the channels to the
spectral characteristics of the specimen radiation is possible up
to a certain degree. However, since only a finite number of
dielectric splitters and filters can be received in every holder
and the spectral characteristics of every individual
splitter/filter are determined in the process of their manufacture,
this arrangement is not flexible enough to be adapted to the
specimen spectrum in an optimum manner in many cases of application
(FIG. 1).
[0006] A confocal microscope with DMD mirror arrays for
illumination and/or detection is described in U.S. Pat. No.
5,587,832.
OBJECT AND SUMMARY OF THE INVENTION
[0007] The primary object of the invention is a laser scanning
microscope which is substantially more flexible with respect to its
excitation and/or detection.
[0008] The invention encompasses a laser scanning microscope with a
detection unit whose spectral characteristics are freely
programmable by means of a switching mirror array integrated in a
monochromator.
[0009] This laser scanning microscope, according to the invention,
comprises a laser unit, scanning means, a microscope stand, a
detection unit and a control/receiving unit. The (fluorescent)
radiation which is emitted from the specimen can be focused (in the
case of confocal applications) on a confocal pinhole that is
positioned in a conjugate object plane. This pinhole then
simultaneously represents the entrance aperture of a (grating)
monochromator which divides the specimen radiation into its
spectral components through its dispersive effect. An at least
one-dimensional switching mirror array on which the specimen
spectrum is optically imaged (FIG. 2) is located in the focal plane
of the dispersive medium (grating).
[0010] The at least one-dimensional switching mirror array
comprises a quantity of individually controllable switching
mirrors. While the scanning means essentially dwell on one specimen
point, one mirror after the other can now be individually
controlled (and therefore switched), e.g., sequentially, and the
individual spectral components of the specimen radiation are
accordingly reflected onto a suitably synchronized detector
sequentially for reception of the entire spectrum.
[0011] Alternatively, adjacent mirror zones corresponding to whole
spectral bands can be controlled in parallel in order to
simultaneously detect entire frequency bands in this way (one band
after the other or a plurality of bands simultaneously). In the
case of fluorescent radiation, those mirror pixels that correspond
to the excitation radiation can advantageously be omitted from the
detected spectrum.
[0012] In a variant, the excitation radiation can advantageously be
coupled into the system via the mirror pixels corresponding to the
excitation (FIGS. 3a, b). The need for a main beam splitter (DBS)
is therefore circumvented and an appreciably more compact system
construction can be realized. In addition, the excitation radiation
in this case automatically passes through the detection pinhole as
illumination pinhole, which leads to an improvement in beam quality
(spatial filtering) and therefore improved spatial resolution of
the entire microscope system. In addition, by means of oscillating
the mirror utilized for reflecting the excitation radiation between
two possible mirror positions, the illumination intensity can be
adjusted in an almost continuous manner and can be changed in a
synchronized manner with respect to the scan pixels (shading,
compensation, intensity modulation, etc.).
[0013] Further, a freely programmable confocal diaphragm (pinhole)
can be realized (FIG. 4) by means of another switching mirror array
which is introduced in a conjugate plane to the object plane. This
pinhole is freely programmable with respect to shape and size
(adjustable confocal volume) as well as with respect to lateral
position, which increases the flexibility of the system and is a
great advantage for the adjustment of the optical system
(autoadjustment of pinhole). In addition, in multichannel
applications, the pinhole size can be synchronized with the
spectral band detected at that instant in order that optical
sections independent from the wavelength can be realized with the
confocal microscope. In addition, the pinhole size can also be
advantageously adapted to the intensity of the fluorescent
radiation when various fluorescences of very different intensity
are present simultaneously in the specimen and are to be
detected.
[0014] In an advantageous variant, a curved grating can be used,
which curved grating likewise takes over the collimation and the
dispersion of the radiation to be detected and accordingly results
in a reduced quantity of optical components (FIG. 5). The
structural size of the arrangement according to the invention in
its entirety is accordingly advantageously reduced. Above all, this
leads to a greater optical stability of the detection system.
[0015] In a variant, the optical grating can be replaced by a
prism. In this way, an increase in the efficiency of the optical
system is potentially possible (FIG. 6), particularly in the
detection of unpolarized light.
[0016] In a variant, the spectroscopic system can also be adapted
to an existing state-of-the-art laser scanning microscope in order
to carry out a spectral characterization of the emission radiation
of the microscopic specimen. This can be carried out in particular
via a fiber coupling of the spectrometric unit to the laser
scanning microscope, wherein the fiber can be arranged, for
example, directly behind one of the confocal pinholes (FIG. 7).
[0017] In a variant which has as subject matter the excitation of
multiphoton fluorescence, a confocal diaphragm can be entirely
dispensed with due to the three-dimensionally spatially resolved
excitation (FIG. 8). In this case, in the arrangement according to
the invention, the confocal diaphragm in the monochromator entrance
diaphragm plane can be omitted and the emission light can be
radiated directly onto the dispersive medium.
[0018] In fluorescence microscopy, application in the biomedical
field is generally concentrated in the wavelength band from
approximately 350 to 800 nm. The wavelength resolution required for
application lies in the range of about 0.5 nm. At the present time,
switching mirror arrays can be obtained (also commercially) in a
wide variety of constructions (e.g., from Texas Instruments, Inc.,
Dallas, Tex.), also, for example, in 576.times.864 pixel.sup.2
display form. Accordingly, when imaging the spectrum along the 864
pixels, a resolution of about 0.5 nm can be realized. The
individual mirrors can be switched in a digitally controlled manner
between two highly-stable positions (.+-.10.degree.), wherein the
switching process is concluded within approximately 300 ns. The
reflecting mirrors can be digitally programmed optionally and
independently from one another.
[0019] Gratings, prisms or combinations thereof, for example, can
be used as dispersive media in the constructions described above.
By using these components in a double-pass configuration, the
effective dispersion can be doubled and a particularly compact
construction of the laser scanning microscope can accordingly
realized in an advantageous manner.
[0020] In principle, all of the arrangements described herein can
also be realized in transmission mode by using a transmission array
(LCD array; liquid crystal) instead of the switching mirror array
(FIG. 9).
[0021] A line-scanning laser scanning microscope can be realized in
that the laser beam is expanded to a line and scanned over the
object by means of only one scanner (one scanning axis) (FIG. 10).
When the punctiform pinhole in FIG. 2 is replaced with a slit
pinhole along the line-shaped laser beam, the DMD is replaced by a
two-dimensional switching mirror array and the detector is replaced
by a (sensitivity-enhanced) CCD, the system can be operated in line
scanning mode. The scanned line is imaged in one dimension of the
DMD array and the spectrum of this object line is imaged in the
other coordinate.
[0022] The resolving capacity of the spectrometric system can be
adapted to the needs of the application in question by exchanging
the optical grating or by displacing and rotating the grating
(monochromator size)
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In the drawings:
[0024] FIG. 1 is a schematic representation of a microscope M and
scan head S and which form an LSM;
[0025] FIG. 2 shows, also in schematic form, a further embodiment
of the laser scanning microscope in accordance with the
invention;
[0026] FIGS. 3a and 3b are optical arrangements indicating how the
excitation radiation can be coupled into the system in accordance
with the invention;
[0027] FIG. 4 illustrates in schematic representation how a freely
programmable diaphragm (pinhole) can be realized instead of PH1 in
FIG. 2;
[0028] FIG. 5 is a schematically represented further embodiment
using a curved grating;
[0029] FIG. 6, in another schematically represented embodiment,
uses a prism instead of the optical grating;
[0030] FIG. 7 is a schematically represented embodiment which
employs an additional fiber for carrying out a spectral
characterization of the emission radiation of the microscopic
specimen;
[0031] FIG. 8, also in schematic representation, shows an
arrangement in accordance with the invention in which the confocal
diaphragm as in FIG. 2 can be dispensed with;
[0032] FIG. 9 further schematically shows an arrangement as in FIG.
2 but which includes a transmissive modulator which passes only the
relevant wavelength of the dispersed spectrum; and
[0033] FIG. 10 shows in schematic representation how a line
scanning laser scanning microscope can be realized.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] FIG. 1 shows schematically a microscope unit M and a scan
head S which have a common optical interface via an intermediate
image and form an LSM.
[0035] The scan head S can be mounted on the phototube of an
upright microscope as well as on a lateral output of an inverse
microscope.
[0036] The drawings show a microscope beam path which is switchable
between incident light scanning and transmitted light scanning by
means of a swivelable mirror 14, with a light source 1,
illumination optics 2, beam splitter 3, objective 4, specimen stage
5, condenser 6, light source 7, receiver arrangement 8, a tube lens
9, an observation beam path with a tube lens 10 and an eyepiece 11,
and a beam splitter/mirror 12 for coupling in the scanning beam. A
laser module 13.1, 13.2 holds the laser and is connected via
monomode light-conducting fibers 14.1, 14.2 with the laser input
coupling unit of the scan head S.
[0037] The coupling of radiation into the light-conducting fibers
14.1, 14.2 is carried out by displaceable collimating optics and
beam deflecting elements 17.1, 17.2. A monitoring beam path is
stopped down by means of a partially reflecting mirror 18 in the
direction of a monitor diode 19, wherein line filters 21 and
neutral filters 20 are advantageously arranged in front of the
monitor diode 19 on a rotatable filter wheel, not shown.
[0038] The actual scan unit comprises a scanning objective 22, X/Y
scanner 23, main beam splitter 24 and common imaging optics 25 for
detection channels 26.1-26.4.
[0039] A deflecting prism 27 arranged behind the imaging optics 25
reflects the radiation coming from the object 5 in the direction of
dichroic beam splitters 28 in the convergent beam path of the
imaging optics 25, wherein pinholes 29 which are displaceable in
the direction of and vertical to the optical axis and which are
adjustable in diameter are arranged along with emission filters 30
and suitable receiver elements 31 (PMT) individually for each
detection channel following the beam splitters 28.
[0040] A control unit/computer unit 34 is connected, among others,
with the stage 5 and the scanners 23 and controls them.
[0041] FIG. 2 shows a possible embodiment form of the laser
scanning microscope, according to the invention, comprising a laser
unit L followed by shutter S, collimation optics KO, beam splitter
ST1 for the output coupling of a monitor beam path MO, a beam
splitter ST2 for conducting into scanning means SC and in the
direction of detection, a microscope analogous to FIG. 1, with
additional detection DT1 outside of the scanning beam path, wherein
mirror 12 is constructed in this case as a beam splitter, a
detection unit and a control/receiving unit. The radiation emitted
from the specimen can (in confocal applications) be focused on a
confocal pinhole PH1 positioned in a conjugate object plane. This
pinhole PH1 then simultaneously forms the entrance aperture of a
(for example, grating GT and imaging mirrors SP1, SP2)
monochromator which divides the specimen radiation into its
spectral components through its dispersive effect. An at least
one-dimensional switching mirror array DMD1 on which the specimen
spectrum is optically imaged is located in the focal plane of the
dispersive medium (for example, grating), wherein focusing optics
FO and a detector DT3 are arranged following the one-dimensional
switching mirror array DMD1.
[0042] FIGS. 3a,b: In an advantageous construction, the excitation
radiation can advantageously be coupled into the system via the
mirror pixels which correspond to the excitation. Accordingly, the
need for a main beam splitter (DBS) is circumvented and an
appreciably more compact system construction can be realized. In
addition, the excitation radiation in this case automatically
passes through the detection pinhole as illumination pinhole, which
leads to an improvement in the beam quality (spatial filtering) and
therefore leads to an improved spatial resolution of the entire
microscope system. In addition, in that the mirror utilized for
reflecting the excitation radiation is oscillated between two
possible mirror positions, the illumination intensity can be
adjusted in an almost continuous manner and can be changed in a
synchronized manner with respect to the scan pixels (shading
compensation, intensity modulation, etc.). A light-coupling fiber F
is shown schematically in FIG. 3a.
[0043] The coupled in laser light (in) reaches a grating GT2, is
spectrally divided and is reflected on a DMD arrangement, shown
schematically by a line extending along the dispersion direction,
via a field lens FL1 for parallelizing.
[0044] Through selective switching of individual mirror elements,
shown schematically with reference to FIG. 3b, determined
wavelengths or wavelength areas (excitation light) are reflected
back in the ON position in the direction of the grating, while
others, in the off position of the mirror, do not travel back to
the grating GT2 and are selected.
[0045] For the wavelength components (excitation light) reflected
back to the grating GT2, the dispersion is eliminated again and
they are imaged on a pinhole PH2 in the input of the microscope
(out-on) since they impinge on the grating in an offset manner.
[0046] The light returning from the object passes through PH2, GT2,
FL1, DMD, an additional field lens FL2 in the direction of a
detection unit DE shown schematically.
[0047] Reflection is carried out in a wavelength-selective manner
(out-off) in the direction of detection in the OFF position.
[0048] Splitting into individual wavelengths is carried out at the
grating GT2 and these individual wavelengths can be detected
individually.
[0049] FIG. 4: A freely programmable confocal diaphragm (pinhole)
can be realized instead of PH1 in FIG. 2 by means of another
switching mirror array DMD1 which is introduced in a conjugate
plane to the object plane. This pinhole is freely programmable with
respect to shape and size (adjustable confocal volume) as well as
with respect to lateral position, which increases the flexibility
of the system and is a great advantage for the adjustment of the
optical system (autoadjustment of pinhole). In addition, in
multichannel applications, the pinhole size can be synchronized
with the spectral band detected at that instant in order that
optical sections independent from the wavelength can be realized
with the confocal microscope. In addition, the pinhole size can
also be advantageously adapted to the intensity of the fluorescent
radiation when various fluorescences of very different intensity
are present simultaneously in the specimen and are to be
detected.
[0050] FIG. 5: In a variant, a curved grating GT3 can be used,
which likewise takes over the collimation and the dispersion of the
radiation to be detected and accordingly results in a smaller
quantity of optical components by doing away with the imaging
mirrors SP1, SP2 in FIG. 2. The structural size of the arrangement
according to the invention in its entirety is accordingly
advantageously reduced. This results, above all, in a greater
optical stability of the detection system.
[0051] FIG. 6: In another embodiment form, the optical grating can
also be replaced by a prism P. In this way, an increase in the
efficiency of the optical system is potentially possible,
particularly in the detection of unpolarized light.
[0052] FIG. 7 shows a state-of-the-art laser scanning microscope
similar to FIG. 1, without reference numbers in this case, at
which, by means of an additional fiber F1, a possible embodiment
form of the spectroscopic detection system according to the
invention as described, for example, in FIG. 5 is coupled out of
one of the detection beam paths (26.1 in FIG. 1) directly following
the pinhole in order to carry out a spectral characterization of
the emission radiation of the microscopic specimen.
[0053] FIG. 8: In the case of excitation of multiphoton
fluorescence, in the arrangement according to the invention, the
confocal diaphragm (PH1 in FIG. 2, DMD1 in FIG. 4) in the
monochromator entrance diaphragm plane can be dispensed with and
the emission light can be radiated directly onto the dispersive
medium as is shown with reference to the arrangement with curved
mirror according to FIG. 5.
[0054] FIG. 9 shows a further construction as in FIG. 2, but which
has, instead of the DMD following the grating GT1, a transmissive
modulator MT, for example, of an LCD array, which passes only the
relevant wavelengths of the dispersed spectrum to the detector.
[0055] A construction of an LCD array instead of a pinhole
analogous to the DMD in FIG. 4 in a transmission beam path is also
possible in an advantageous manner.
[0056] FIG. 10 shows that a line-scanning laser scanning microscope
can be realized in that the laser beam is expanded on a line, for
example, with a cylinder lens, and scanned over the object by means
of only one scanner (one scanning axis), for example, in the
Y-direction in this case. When the punctiform pinhole in FIG. 2 is
replaced with a slit pinhole along the line-shaped laser beam which
is realized by means of a two-dimensional switching mirror array
DMD1, represented by the dark mirrors in the shape of a stripe, and
the detector is replaced by a (sensitivity-enhanced) CCD, the
system can be operated in line scanning mode. The scanned line is
imaged in one dimension of the DMD array and the spectrum of this
object line is imaged in the other coordinate.
[0057] While the foregoing description and drawings represent the
preferred embodiments of the present invention, it will be obvious
to those skilled in the art that various changes and modifications
may be made therein without departing from the true spirit and
scope of the present invention.
* * * * *