U.S. patent application number 09/769215 was filed with the patent office on 2001-09-13 for microscope and segmenting device for a microscope.
This patent application is currently assigned to Leica Microsystems Heidelberg GmbH. Invention is credited to Knebel, Werner.
Application Number | 20010021063 09/769215 |
Document ID | / |
Family ID | 7629074 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021063 |
Kind Code |
A1 |
Knebel, Werner |
September 13, 2001 |
Microscope and segmenting device for a microscope
Abstract
A microscope, in particular for confocal scanning microscopy,
having a light source (1) for illuminating an object (6) to be
investigated. An optical device (9, 12) is provided for splitting
transmitted light (15) passing through the object (6) and
fluorescent light (10, 13) produced in the object (6). A segmenting
device (17) acting on the transmitted light (15) is configured with
regard to a detection-light path which is as short as possible in
such a way that the segmenting device (17) is arranged between the
object (6) and the light-splitting device (9, 12).
Inventors: |
Knebel, Werner; (Kronau,
DE) |
Correspondence
Address: |
Simpson, Simpson & Snyder, L.L.P.
5555 Main Street
Williamsville
NY
14221
US
|
Assignee: |
Leica Microsystems Heidelberg
GmbH
Am Friedensplatz 3
Mannheim
DE
|
Family ID: |
7629074 |
Appl. No.: |
09/769215 |
Filed: |
January 24, 2001 |
Current U.S.
Class: |
359/385 ;
250/459.1; 359/368; 359/370; 359/386 |
Current CPC
Class: |
G02B 21/0032 20130101;
G02B 21/0052 20130101; G02B 21/0076 20130101; G02B 21/082 20130101;
G02B 21/18 20130101 |
Class at
Publication: |
359/385 ;
250/459.1; 359/368; 359/370; 359/386 |
International
Class: |
G01N 021/64; G02B
021/00; G02B 021/06; G01T 001/10; G01J 001/58 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 28, 2000 |
DE |
100 03 762.3 |
Claims
What is claimed is:
1. A microscope comprising: a light source (1) for illuminating an
object (6) to be investigated, a an optical device (9, 12) for
splitting transmitted light (15) passing through the object (6) and
fluorescent light (10, 13) generated in the object (6), a
segmenting device (17) acting on the transmitted light (15),
wherein the segmenting device (17) is arranged between the object
(6) and the optical device (9, 12).
2. Microscope according to claim 1, characterized in that a
condenser (7) for the transmitted light (15) and the fluorescent
light (10, 13) is arranged on the side of the object (6) averted
from the light source (1).
3. Microscope according to claim 2, characterized in that the
segmenting device (17) is arranged between the condenser (7) and
the optical device (9, 12).
4. Microscope according to claim 3, characterized in that the
segmenting device (17) is arranged in a Fourier plane between the
condenser (7) and the optical device (9, 12).
5. Microscope according to claim 1, characterized in that the
segmenting device (17) is arranged on a device for inserting
optical components into a beam path.
6. Microscope according to claim 1, characterized in that the
segmenting device (17) has a segment stop, a segment phase stop or
a segment phase filter.
7. Microscope according to claim 1, characterized in that the
segmenting device (17) has a transparent substrate (19) with a
colour-selective coating (20).
8. Microscope according to claim 7, characterized in that the
coating (20) is opaque to the transmitted light (15) and
transparent to the fluorescent light (10, 13).
9. Microscope according to claim 1, characterized in that the
segmenting device has a colour LCD matrix.
10. Microscope according to claim 1, characterized in that the
optically active part of the segmenting device (17) is arranged
perpendicular to an optical axis of the microscope.
11. A confocal microscope comprising: a laser light source (1) for
illuminating an object (6) to be investigated, a an optical device
(9, 12) for splitting transmitted light (15) passing through the
object (6) and fluorescent light (10, 13) generated in the object
(6), a segmenting device (17) acting on the transmitted light (15),
wherein the segmenting device (17) is arranged between the object
(6) and the optical device (9, 12).
12. The confocal microscope according to claim 11, characterized in
that a condenser (7) for the transmitted light (15) and the
fluorescent light (10, 13) is arranged on the side of the object
(6) averted from the light source (1).
13. The confocal microscope according to claim 12, characterized in
that the segmenting device (17) is arranged between the condenser
(7) and the optical device (9, 12).
14. The confocal microscope according to claim 11, characterized in
that the segmenting device (17) is arranged on a device for
inserting optical components into a beam path.
15. The confocal microscope according to claim 11, characterized in
that the segmenting device (17) has a transparent substrate (19)
with a colour-selective coating (20).
16. The confocal microscope according to claim 15, characterized in
that the coating (20) is opaque to the transmitted light (15) and
transparent to the fluorescent light (10, 13).
17. The confocal microscope according to claim 11 characterized in
that a multiband detector arranged to the optical device (9,
12).
18. The confocal microscope according to claim 11, characterized in
that at least one fluorescent light detector (11, 14) for detecting
the fluorescent light (10, 13) and at least one transmitted-light
detector (16) for detecting the transmitted light (15) are
arranged, for the purpose of simultaneously detecting fluorescent
and transmitted light (10, 13; 15), on the side of the object (6)
averted from the light source (1).
19. A segmenting device (17) comprising: a transparent substrate
(19) and a colour-selective coating (20) formed on said transparent
substrate (19).
20. The segmenting device according to claim 19, characterized in
that the coating (20) is opaque to the transmitted light (15) and
transparent to the fluorescent light (10, 13).
21. Segmenting device according to claim 19, characterized in that
the coating (20) is dielectric.
22. Segmenting device according to claim 19, characterized in that
the coating (20) is vapour-deposited onto the substrate (19).
23. Segmenting device, according to claim 19, characterized in that
the segmenting device has a suitably configured, preferably
tailored, colour filter.
24. Segmenting device according to claim 23, characterized in that
the colour filter is produced from plastic.
25. Segmenting device, according to claim 19, characterized in that
the segmenting device has a colour LCD matrix.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority of a German patent
application DE P 100 03 762.3 filed Jan. 28, 2000 which is
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to a microscope. In particular the
invention relates to a confocal scanning microscopy having a
segmenting device.
[0003] Furthermore, the invention relates to a segmenting device
which is used, in particular, in the abovenamed microscope or
confocal microscope.
BACKGROUND OF THE INVENTION
[0004] A microscope generally comprises a light source and a
focussing optical system with the aid of which the light from the
source is focussed onto a pinhole stop. In this case, a beam
splitter, a scanning device for beam control, a microscope optical
system, a detection stop and detectors for detecting detection
and/or fluorescent light are provided.
[0005] In a confocal microscope, the illuminating light is mostly
coupled in via the beam splitter. The focus of the light beam is
moved with the aid of the scanning device in a sample plane. For
this purpose, it is customary to use two mirrors which are tilted,
the deflection axes mostly being perpendicular to one another, such
that one mirror deflects in the X-direction and the other deflects
in the Y-direction.
[0006] It is also possible in a transmitted-light arrangement for
the fluorescent light or the transmitted light--the transmission of
the exciting light--, for example, to be detected on the condenser
side, that is to say on the side of a condenser arranged downstream
of the object. The detection-light beam then does not pass via the
scanning mirrors to the detector. Such an arrangement is denoted as
a non-descanning arrangement.
[0007] In order to detect the fluorescent light, there would be a
need in the transmitted-light arrangement for a condenser-side
detection stop in order--as in the descanning arrangement
described--to achieve a three-dimensional resolution. In the case
of two-photon excitation, however, it is possible to dispense with
a condenser-side detection stop, since the probability of
excitation is a function of the square of the photon density or the
intensity, which is naturally much higher at the focus than in the
neighbouring regions. The fluorescent light to be detected
therefore originates with high probability in overwhelming
proportion from the focussing region, and this renders superfluous
further differentiation of fluorescence photons from the focussing
region from fluorescence photons from the neighbouring regions with
the aid of a stop arrangement.
[0008] Particularly against the background of a yield of
fluorescence photons which is low in any case for two-photon
excitation, a non-descanning arrangement in which less light is
generally lost on the detection-light path is of interest.
Nevertheless, because of the lack of marking in living
preparations, cell contours, for example, also cannot be detected
sufficiently well in the case of this type of observation of
fluorescent light, and so it would be desirable to be
simultaneously able to observe the transmitted light, which would
permit clear conclusions.
[0009] A microscope for simultaneously detecting fluorescent and
transmitted light is already known to the applicant from an earlier
patent application. The known microscope comprises downstream of a
condenser a light-splitting device in the form of at least one
colour beam splitter which spatially separates or splits the
fluorescent light from the transmitted light.
[0010] For the purpose of transmitted-light contrast microscopy, in
the case of the known microscope the segmenting device required for
contrast is arranged in the form of a segment stop in a Fourier
plane of the transmitted light downstream of the light-splitting
device or downstream of the colour beam splitter. This requires a
long detection-light path downstream of the light-splitting device,
which path also has to be set up in addition to the standard
microscope equipment.
BRIEF SUMMARY OF THE INVENTION
[0011] It is therefore the object of the present invention to
specify a microscope having a detection-light path which is as
short as possible and can be implemented with the aid of
structurally simple means.
[0012] The above object is achieved by a microscope which
comprises: a light source for illuminating an object to be
investigated, a an optical device for splitting transmitted light
passing through the object and fluorescent light generated in the
object, a segmenting device acting on the transmitted light,
wherein the segmenting device is arranged between the object and
the optical device.
[0013] It is a further object of the present invention to specify a
confocal microscope having a detection-light path which is as short
as possible and can be implemented with the aid of structurally
simple means.
[0014] The above object is achieved by a confocal microscope which
comprises: a laser light source for illuminating an object to be
investigated, a an optical device for splitting transmitted light
passing through the object and fluorescent light generated in the
object, a segmenting device acting on the transmitted light,
wherein the segmenting device is arranged between the object and
the optical device.
[0015] It is an additional object of the invention to provide a
segmenting device which allows a detection-light path to be as
short as possible and simple to implement.
[0016] The above object is achieved by a segmenting device which
comprises: a transparent substrate and a colour-selective coating
formed on said transparent substrate.
[0017] The advantage of the invention is, that an optimized
arrangement of the segmenting device solves the above object in a
surprisingly simple way. For this purpose, the segmenting device is
no longer arranged downstream of the light-splitting device, but
between the object and the light-splitting device. A
detection-light path, already prescribed by the arrangement of the
light-splitting device, between the object and the light-splitting
device is employed in this case, in addition, by virtue of the
arrangement of the segmenting device on this detection-light path.
The creation of an additional detection-light path is thereby
avoided.
[0018] Consequently, the microscope according to the invention has
a detection-light path which is as short as possible with the aid
of structurally simple means.
[0019] In a concrete embodiment of the microscope, a condenser for
the transmitted light and the fluorescent light could be arranged
on the side of the object averted from the light source. The
segmenting device could then be arranged in a simple way between
the condenser and the optical device, in which case with regard to
segmenting which is as effective as possible, it is advantageous to
arrange the segmenting device in a Fourier plane between the
condenser and the optical device. In any case, the segmenting
device could act exclusively on the transmitted light, in which
case the fluorescent light can pass through the segmenting device
without being influenced.
[0020] The device for inserting optical components into a beam path
could have a revolver mechanism or a sliding magazine in order to
ensure a particularly reliable insertion of the optical components
into the beam path. Not only a very reliable, but also a very
simple insertion of a segmenting device into the beam path is
achieved thereby.
[0021] In a particularly versatile way, different segmenting
devices could be arranged in the device. Depending on the required
investigation methods, it is therefore possible to insert the
suitable segmenting device into the beam path.
[0022] The most varied components come into consideration as
segmenting device. In particular, the segmenting device could have
a segment stop, a segment phase stop or a segment phase filter.
[0023] In a structurally particularly simple configuration, the
segmenting device could have a transparent substrate with a
colour-selective coating. This implements not only a particularly
simple segmenting device, but also an exceptionally thin segmenting
device which can be inserted without difficulty into a microscope
of the type mentioned at the beginning. The coating could in this
case be opaque to the transmitted light and transparent to the
fluorescent light.
[0024] The coating could advantageously be a dielectric coating. In
any case, in a particularly suitable way, the coating could be
vapour-deposited onto the substrate. Reliable adhesion of the
coating on the substrate can thereby be achieved.
[0025] In an alternative refinement, the segmenting device could
have a suitably configured colour filter. The colour filter could,
in particular, be tailored in accordance with a known segment stop.
Various materials come into consideration as colour filter
material. In this case, a configuration of the colour filter from
plastic, preferably plastic film, is particularly easy to handle.
However, it is also conceivable for the colour filter to be
configured from glass.
[0026] In a further alternative refinement, the segmenting device
could have a colour LCD matrix. Such a colour LCD matrix permits
variation of the segment or stop shape and variation of the colour
properties without further redesigning.
[0027] In the simplest case, the optically active part of the
segmenting device could be arranged perpendicular to an optical
axis of the microscope. In a conventional segment stop, the
optically active part is formed by an essentially flat region. In
the case, for example, of a coated substrate, the coating would be
the optically active part of the segmenting device.
[0028] In order to avoid reflections which could run back into the
light source, the optically active part of the segmenting device
could be arranged in a fashion deviating from the perpendicular to
an optical axis of the microscope. In this case, the aim is, in
particular, a slight deviation which does not influence the optical
action of the segmenting device.
[0029] The segmenting device or the optically active part of the
segmenting device can have different geometrical shapes, depending
on the application. In a particularly simple way, the optical
device is configured as a light-splitting device and could have at
least one colour beam splitter. In this case a plurality of colour
beam splitters could, specifically, be arranged in series to enable
different wavelengths or wavelength regions to be split.
[0030] As an alternative to this, the light-splitting device could
have at least one partially transparent mirror. A bandpass or
blocking filter could be arranged downstream of this mirror or
these mirrors. In the case of the use of mirrors as splitting
component as well, a plurality of such mirrors could be arranged in
series, if appropriate with a downstream bandpass or blocking
filter. It is also possible thereby to split the fluorescent light
into a plurality of spectral regions.
[0031] It is possible as an alternative to the use of colour beam
splitters or mirrors to make use for splitting of a multiband
detector which is described, for example, in DE 199 02 625 A1. Such
a multiband detector can also be used to split the fluorescent
light into a plurality of spectral regions.
[0032] With regard to reliable detection of the transmitted light
and the fluorescent light, it would be possible to arrange at least
one fluorescent-light detector for detecting the fluorescent light
and at least one transmitted-light detector for detecting the
transmitted light, for the purpose of simultaneously detecting
fluorescent and transmitted light, on the side of the object
averted from the light source. In a particularly compact refinement
of the microscope, the fluorescent light and the transmitted light
could be detected in the same detector. With regard to as clear a
differentiation as possible, the fluorescent light and the
transmitted light could, however, also be detected in different
detectors.
[0033] A laser could be used in a particularly advantageous way as
light source. However, it is also conceivable to use other suitable
light sources.
[0034] The segmenting device could, for example, operate using the
Dodt principle or using the Hoffinan principle. In this case, the
segmenting device does not --as is usual--comprise a solid opaque
segment stop. In the case of a segmenting device designed as a
coated substrate, the coating is transparent to the fluorescent
light, while it is opaque to the transmitted light--two--or
multiphoton exciting light.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0035] There are various possibilities of configuring and
developing the teaching of the present invention in an advantageous
way. For this purpose, reference is made, to the following
description of preferred exemplary embodiments of the invention. In
the drawing:
[0036] FIG. 1 shows a diagrammatic illustration of an exemplary
embodiment of a microscope according to the invention, and
[0037] FIG. 2 shows a diagrammatic illustration of an exemplary
embodiment of a segmenting device according to the invention with a
suitably coated substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0038] FIG. 1 shows an exemplary embodiment of a microscope
according to the invention in a diagrammatic illustration. The
microscope is a confocal laser scanning microscope. The microscope
has a light source 1 designed as a laser. The light source 1 emits
an illuminating light beam 2 which is reflected to a scanning
device 4 via a main beam splitter 3. The scanning device 4 leads
the illuminating light beam 2 through a microscope optical system
or an objective 5 via an object 6. Both the transmitted light
passing through the object 6 and the fluorescent light produced in
the object 6 reach via a condenser 7 and a deflecting mirror 8 a
first colour beam splitter 9 which splits the spectrally lower-wave
region 10 of the fluorescent light and reflects it to a
fluorescent-light detector 11. Via a colour beam splitter 12, the
spectrally higher-wave region 13 of the fluorescent light is
reflected to a further fluorescent-light detector 14. The
transmitted light 15 reaches a transmitted-light detector 16
arranged in the straight ahead direction.
[0039] The microscope consequently has a light source 1 for
illuminating an object 6 to be investigated. Furthermore, the
microscope has an optical device which is configures as a
light-splitting device with two colour beam splitters 9 and 12 for
splitting transmitted light 15 passing through the object 6 and
fluorescent light 10 and 13 produced in the object. Finally, the
microscope comprises a segmenting device, in the form of a colour
segment stop 17, which acts on the transmitted light 15. With
regard to a detection-light path which is as short as possible, the
segmenting device is arranged between the object 6 or the condenser
7 and the light-splitting device. The microscope is suitable for
transmitted-light contrast microscopy on account of the segmenting
device.
[0040] The microscope shown in FIG. 1 further has a detector 18
which is arranged on the objective side and is normally not used in
transmitted-light contrast microscopy.
[0041] Both fluorescent-light detectors 11 and 14 are arranged on
the side of the object 6 averted from the light source 1.
Furthermore, the transmitted-light detector 16 is arranged on the
side of the object 6 averted from the light source 1.
[0042] The segmenting device in the form of the colour segment stop
17 is arranged in a Fourier plane between the condenser 7 and the
light-splitting device, more precisely the deflecting mirror 8. A
revolver mechanism or a sliding magazine can be used to insert the
colour segment stop 17.
[0043] FIG. 2 shows a diagrammatic representation of an exemplary
embodiment of a segmenting device according to the invention. The
segmenting device has a flat substrate 19 with a coating 20. The
coating 20 is preferably dielectric and vapour-deposited onto the
substrate 19. The coating 20 vapour-deposited onto the transparent
substrate is opaque to the transmitted light and transparent to the
fluorescent light. In particular, the dielectric coating 20 is
transparent to light of wavelengths which are less than 790 nm.
Light of higher wavelengths is reflected. The wavelength limit with
regard to the transmission and reflection of light can, however,
also be at another suitable wavelength.
[0044] With regard to further advantageous refinements and
developments of the microscope according to the invention, and to
the segmenting device according to the invention, reference may be
made for the purpose of avoiding repetition to the general part of
the description and to the attached patent claims.
[0045] Finally, it may be pointed out expressly that the
above-described exemplary embodiments of the microscope according
to the invention and of the segmenting device according to the
invention serve merely to discuss the teaching claimed but do not
limit the latter to these exemplary embodiments.
PARTS LIST
[0046] 1 Light source
[0047] 2 Illuminating light beam
[0048] 3 Main beam splitter
[0049] 4 Scanning device
[0050] 5 Objective
[0051] 6 Object
[0052] 7 Condenser
[0053] 8 Deflecting mirror
[0054] 9 Colour beam splitter
[0055] 10 Wavelength region
[0056] 11 Fluorescent-light detector
[0057] 12 Colour beam splitter
[0058] 13 Wavelength region
[0059] 14 Fluorescent-light detector
[0060] 15 Transmitted-light beam
[0061] 16 Transmitted-light detector
[0062] 17 Colour segment stop
[0063] 18 Detector
[0064] 19 Substrate
[0065] 20 Coating
* * * * *