U.S. patent application number 09/295554 was filed with the patent office on 2001-11-29 for adjustable coupling in and/or detection of one or more wavelengths in a microscope.
Invention is credited to SCHOEPPE, GUENTER.
Application Number | 20010046046 09/295554 |
Document ID | / |
Family ID | 26047897 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010046046 |
Kind Code |
A1 |
SCHOEPPE, GUENTER |
November 29, 2001 |
ADJUSTABLE COUPLING IN AND/OR DETECTION OF ONE OR MORE WAVELENGTHS
IN A MICROSCOPE
Abstract
A device for the adjustable coupling of wavelengths or
wavelength regions into the illumination beam path of a microscope,
preferably in the beam path of a confocal microscope, comprising at
least one dispersive element for wavelength separation of the
illumination light and at least one at least partially reflecting
element arranged in the wavelength-separated portion of the
illumination light for reflecting back a wavelength region in the
direction of the microscope illumination, and a device for the
adjustable detection of object light coming from an illuminated
object, preferably in a microscope beam path, comprising at least
one dispersive element for wavelength separation of the object
light and means arranged in the wavelength-separated portion of the
object light for the adjustable stopping down or cutting out of at
least one wavelength region and deflection in the direction of at
least one detector.
Inventors: |
SCHOEPPE, GUENTER; (JENA,
DE) |
Correspondence
Address: |
REED SMITH LLP
375 PARK AVENUE
NEW YORK
NY
10152
US
|
Family ID: |
26047897 |
Appl. No.: |
09/295554 |
Filed: |
April 21, 1999 |
Current U.S.
Class: |
356/318 |
Current CPC
Class: |
G01J 3/0205 20130101;
G01J 3/02 20130101; G01J 3/14 20130101; G01J 3/36 20130101; G01J
2003/1286 20130101; G01J 3/021 20130101; G02B 21/0064 20130101 |
Class at
Publication: |
356/318 |
International
Class: |
G01J 003/30 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1998 |
DE |
198 35 069.4 |
Sep 16, 1998 |
DE |
198 42 288.1 |
Claims
What is claimed is:
1. A device for the adjustable coupling of wavelengths or
wavelength regions into the illumination beam path of a microscope,
particularly in the beam path of a confocal microscope, comprising:
at least one dispersive element for wavelength separation of the
illumination light; and at least one at least partially reflecting
element arranged in the wavelength-separated portion of the
illumination light for reflecting back at least one wavelength
region in the direction of the microscope illumination.
2. The device according to claim 1, wherein one or more mirrors
with a width corresponding to the cut out wavelength region is/are
provided.
3. The device according to claim 1, wherein the mirrors are
exchangeable.
4. The device according to claim 1, wherein the mirrors are
arranged on a carrier.
5. The device according to claim 4, wherein the carrier is
constructed so as to be at least partially transparent to
light.
6. The device according to claim 4, wherein a plurality of mirrors
are provided adjacent to one another in a dispersion plane fixed by
the separated wavelength regions.
7. A device for the adjustable detection of object light coming
from an illuminated object, particularly in a microscope beam path,
comprising: at least one dispersive element for wavelength
separation of the object light; and means arranged in the
wavelength-separated portion of the object light for the adjustable
stopping down or cutting out of at least one wavelength region and
deflection in the direction of at least one detector.
8. The device according to claim 7, wherein the means comprise at
least one prism-shaped wedge made of light-transparent and
light-refracting material whose position is adjustable vertical to
the light direction in at least one direction.
9. The device according to claim 8, wherein the wedge or wedges has
or have a width which can be reduced at least partially vertical to
the light direction.
10. The device according to claim 8, wherein a plurality of wedges
are arranged in the object light.
11. The device according to claim 8, wherein a plurality of wedges
with different wedge angles and/or different alignment with respect
to their wedge orientation are arranged in the object light.
12. The device according to claim 8 with optics arranged subsequent
to at least one wedge for focusing the cut out wavelength regions
on at least one detector.
13. A device for the adjustable detection of object light coming
from an illuminated object, particularly in a microscope beam path,
comprising: at least one dispersive element for wavelength
separation of the object light; and at least one prism-shaped wedge
which is arranged in the wavelength-separated portion of the object
light, is made of light-transparent and light-refracting material
and whose position is adjustable vertical to the light direction in
at least one direction.
14. The device according to claim 13, wherein one or more wedges
has or have a width which can be reduced at least partially
vertical to the light direction.
15. The device according to claim 13, wherein one or more wedges
has or have a decreasing width vertical to the light direction.
16. The device according to claim 13, wherein a plurality of wedges
are arranged in the object light.
17. The device according to claim 13, wherein a plurality of wedges
with different wedge angles and/or different alignment of their
wedge orientation are provided.
18. The device according to claim 13, wherein optics are provided
following at least one wedge for focusing the cut out wavelength
regions on at least one detector.
19. The device according to claim 16, wherein the wedge or wedges
is or are displaceable in a first direction vertical to the
dispersion direction (dispersion plane) in order to change the
bandwidth of the cut out spectral region by means of its reducible
width and/or the wedge or wedges is or are displaceable at least in
a second direction in the direction of dispersion in order to
change the cut out spectral region and/or wherein the wedge or
wedges is or are changeable in their orientation, so that the
opening of the wedge angle is oriented electively in different
directions vertical to the optical axis.
20. A combination comprising: a device comprising at least one
dispersive element for wavelength separation of the illumination
light; and at least one at least partially reflecting element
arranged in the wavelength-separated portion of the illumination
light for reflecting back at least one wavelength region in the
direction of the microscope illumination; with at least one of a
device comprising: at least one dispersive element for wavelength
separation of the object light; and means arranged in the
wavelength-separated portion of the object light for the adjustable
stopping down or cutting out of at least one wavelength region and
deflection in the direction of at least one detector; and a device
comprising: at least one dispersive element for wavelength
separation of the object light; and at least one prism-shaped wedge
which is arranged in the wavelength-separated portion of the object
light, is made of light-transparent and light-refracting material
and whose position is adjustable vertical to the light direction in
at least one direction; wherein said combination is in a
microscope, particularly a confocal microscope.
21. The device according to claim 20, wherein at least one
dispersive element is used as a common element for coupling in the
illumination light and for detecting the object light.
22. The device according to claim 20, wherein different dispersive
elements are used for coupling in the illumination light and for
detecting the object light.
23. The device according to claim 20, wherein the reflecting
element for back reflection during the coupling in of the
illumination light is constructed so as to be partially transparent
in order to allow the light reflected from the object to pass.
24. A method of using a device as in claim 1 comprising the step of
using said device in a confocal microscope.
25. A method of using a device as in claim 7 comprising the step of
using said device in a confocal microscope.
26. A method of using a device as in claim 13 comprising the step
of using said device in a confocal microscope.
27. A method of using a device as in claim 1 comprising the step of
using said device in a laser scanning microscope.
28. A method of using a device as in claim 7 comprising the step of
using said device in a laser scanning microscope.
29. A method of using a device as in claim 13 comprising the step
of using said device in a laser scanning microscope.
30. A method of using a device as in claim 1 comprising the step of
using said device in a confocal fluorescence microscope.
31. A method of using a device as in claim 7 comprising the step of
using said device in a confocal fluorescence microscope.
32. A method of using a device as in claim 13 comprising the step
of using said device in a confocal fluorescence microscope.
Description
BACKGROUND OF THE INVENTION
[0001] a) Field of the Invention
[0002] The present invention is directed to an adjustable coupling
in and/or the detection of one or more wavelengths in a microscope.
In particular, the invention is directed to such coupling and
detection iin the beam path of a confocal microscope.
[0003] b) Description of the Related Art
[0004] WO Patent 95/07447 (DE 4330347C2) describes a device which
is arranged in the detection space of a laser scanning microscope.
A main beam splitter, as it is called, (reference number 8 in FIG.
2) must be used in this arrangement.
[0005] When a plurality of lasers are used, this same main beam
splitter is a very complicated optical layer system that reflects
the laser light with only limited selectivity and effectiveness
and, because of the non-optional level of edge steepness and
reflectivity, the light emitted by the fluorochromes is reflected
only with (high) losses.
[0006] In general, main beam splitters are those components of a
laser scanning microscope which most limit the efficiency and
selectivity.
[0007] U.S. Pat. No. 4,519,707 describes a multi-spectral detection
system with dispersion and separate detection.
[0008] JP 493915 describes a spectroscopic system for remote
sensing with a plurality of detector elements for
wavelength-selective detection of the sensed object.
[0009] JP 61007426 describes a photometer with a fluorescence
measurement filter in the dispersive light of the object.
[0010] DE 19510102 C1 describes a confocal fluorescence microscope
for evaluation of fluorescent light with two prism spectrometers in
the excitation light path, wherein the first prism spectrometer
fans out the excitation light exiting by means of a first stripe
diaphragm and the second prism spectrometer fans out the
fluorescent light exiting from a second stripe diaphragm and a
third prism spectrometer is provided in front of a detector.
[0011] Many locations on the object are illuminated and examined
simultaneously. In so doing, the entire object plane is illuminated
in parallel monochromatically. The achievable confocal effect and
contrast depends in these arrangements on the coverage of the
illuminated planes with transparent locations. In order to be able
to make effective use of these arrangements, there must be a
minimum coverage that limits the achievable contrast to 1:100 . . .
1:25. The use of slits leads to a textured confocal effect. This
arrangement has disadvantages in terms of application, especially
for multiple fluorescence.
[0012] The arrangement with three spectrometers which are connected
one behind the other and with the use of selection elements in the
form of gratings requires an enormous expenditure on adjustment and
high stability of adjustment. The use of three duplicate prism
spectrometers requires very close manufacturing tolerances. The use
of slits for field illumination and the guiding of fluorescent
light through the intermediate spaces either results in a
considerable portion of the fluorescent light being lost when there
is a high degree of coverage in that there is only a low
permissible dispersion of the spectrometers or results in a low
light yield or light efficiency in illumination at higher
dispersions because the slits must be at a distance from one
another corresponding at least to the spacing of the spectral
width. Multiple fluorescences can be analyzed simultaneously in
this arrangement only through increased expenditure.
OBJECT AND SUMMARY OF THE INVENTION
[0013] It is the primary object of the invention to replace the
conventional complicated main beam splitter with simpler components
while at the same time improving flexibility, selectivity and
efficiency, wherein the selected arrangement should also be
suitable for spectral separation of fluorescent light returning
from the object in a serially operating confocal laser scanning
microscope with the highest requirements with respect to contrast
and efficiency.
[0014] This is achieved in that the laser light which
advantageously, but not compulsorily, emerges from the end of a
fiber is sent through a spectrograph and a special band selector is
arranged in the image plane thereof, that is, a comb with narrow
fully-reflecting mirrors for fluorescence applications or with
partially reflecting mirror stripes for reflection applications.
The little mirrors are positioned at selected locations of the
wavelengths of the laser or lasers and reflect the light of the
desired wavelengths back into the spectrographs so as to be offset
by a small angle. In so doing, all illuminating light
advantageously returns to an individual point lying very close to a
fiber for coupling in the laser light, the pinhole of the laser
scanning microscope being located at this point.
[0015] Light which returns from the object with the wavelengths of
the illumination impinges on the little mirrors of the band
selector and, in the case of fluorescence, is fully reflected in
the direction of the fiber and therefore effectively separated from
the light to be detected; in the case of reflection, it passes
partially through the partially reflecting little mirrors as in
reflected-light microscopes and can be detected. This will be
discussed more closely later on.
[0016] In the case of fluorescence, the returning light has
wavelengths different than those of the illuminating light and
therefore impinges in the image plane of the spectrograph in the
neighborhood of the little mirrors.
[0017] Even wavelengths located next to the exciting wavelengths
around the resolution of the spectrographs can be detected by the
invention. These wavelengths are located much closer to the
exciting wavelengths than would be possible in the case of dichroic
splitters and losses are lower than with splitter layers because of
the possible higher transmission. In this case, a plurality of
regions of any width and spectral position can be cut out of the
spectrum and supplied to different receivers by means of suitably
shaped glass wedges such as those used in optical testing with
spectrographs for demonstrating subtractive and additive color
mixing.
[0018] A mirror unit comprises a transparent glass plate with small
parallel mirrors at the locations of the anticipated laser
wavelengths or at the locations of those laser wavelengths desired
for examination, these laser wavelengths enter together, are
dispersed, reflected at another location in the prism by the
mirrors and imaged on the pinhole.
[0019] The mirrors can also be partially reflecting in order to
enable detection (passage) of the illumination wavelength
(reflection applications).
[0020] In fluorescence applications, the mirrors are fully
reflecting in order to prevent the detection being influenced by
the illumination wavelengths.
[0021] The specimen light (fluorescent light) is spectrally
separated by at least one dispersive element, parallelized by a
field lens and imaged through small glass wedges at different
locations via a collector.
[0022] Without the wedges which cause a separation of the locations
of impingement behind the collector, all of the beams would land in
the focal point of the collector.
[0023] The spectral width (channel width) is changed by means of
vertical wedge displacement, the spectral region of concern
(channel position) is selected by horizontal wedge
displacement.
[0024] Since the wedge angle is constant, there is no change in the
deflection in the direction of the receiver.
[0025] The wedge angle determines the location of impingement and
can be changed by exchanging wedges.
[0026] Further, prismatic lenses formed, for example, by lenses
glued to wedges or decentered lenses formed by decentered lenses
could be used. In this case, the detectors would have to be
displaced along with the respective wedges when these lenses are
displaced.
[0027] The invention is described more fully hereinafter with
reference to the schematic illustrations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In the drawings:
[0029] FIG. 1 shows the principle, according to the invention, for
coupling in illumination;
[0030] FIG. 2 shows the wavelength selection according to the
invention in the detection of fluorescence light or reflected
light;
[0031] FIG. 2a shows the deflection of the detection light through
wedge-shaped elements; and
[0032] FIG. 3 shows the beam formation of the connected
microscope.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] In FIG. 1, the light exiting from the fiber F is directed in
parallel through the collector K and impinges on a dispersive
element DP, wherein a light staircase T is arranged in front of the
latter for guiding in the beam. When there is more than one
illumination fiber F, a beam combiner (not shown) can also be
provided.
[0034] The dispersive element DP, in this case a two-part Abbe
spectrometer prism, divides the illumination light into the
individual colors contained in the laser light, and the divided
light which is represented in the present case by 3 wavelengths
.lambda.1, .lambda.2, .lambda.3 reaches an element ST, a mirror
carrier, which has small mirrors S1, S2, S3 precisely at those
locations toward which the light of selected wavelengths is
focused, but is otherwise transparent, for example, a glass rod
carrying the mirrors; that is, there is always a small mirror
arranged where a wavelength of the laser light would be
focused.
[0035] The individual partial beams enter the upper part of the
lens L1 due to the fact that the illumination beam is introduced
through the spectrometer prism DP in the upper part so as to be
decentered and the individual partial beams are focused on the
little mirrors S1, 2, 3 so as to be inclined very slightly
diagonally downward, are reflected further downward by the
reflection, and return again through the lens L1 in the lower part
of the element DP.
[0036] The beam also passes through the field lens FL, whose focal
length corresponds in a good approximation to the spacing of
apparent intersections of all wavelengths in the space between the
field lens FL and prism DP, that is, the distance of an apparent
source point, i.e., all of the beams apparently come from virtually
one point and the focal plane of the lens must lie in this point,
so that the beams are all parallelized in the light direction.
[0037] On the return path of the light beams from the mirrors S1,
S2, S3, the dispersion is canceled again by passing through the
dispersive element DP and the light passes as a beam with a
plurality of wavelengths through a lens L2 and is collimated on a
pinhole PH which can be located, in a known manner, in the
intermediate image plane of a laser scanning microscope and enters
the microscope, i.e., via the microscope objective, to reach the
object as is shown schematically in FIG. 3.
[0038] The described excitation line selector (mirror carrier ST
with small mirrors S1, S2, S3. . .) can be exchangeable, so that
any wavelengths, either one or more than one, can be combined.
[0039] The light coming from the object can be reflected light or
fluorescent light.
[0040] For reflected light, the little mirrors S1, S2. . . must be
semi-reflecting so that the returning light travels into the
detecting beam path. This arrangement and manner of operation
corresponds in principle to that in a normal reflected-light
microscope.
[0041] Fluorescent light which has a wavelength displacement
relative to the excitation light arrives between the little mirrors
S1 S2 . . . This is described with reference to FIG. 2.
[0042] The light returning from the specimen passes through the
above described elements and is fanned out spectrally in a
dispersion plane DIE by the dispersive element DP and parallelized
by the field lens.
[0043] The fluorescent light with wavelengths different than those
of the excitation light passes next to the little mirrors S1-S3
through the light-transparent carrier ST.
[0044] Deflecting elements as wedge-shaped glass prisms GK, in this
case GK1, 2, 3, 4, whose quantity is equal to the quantity of
evaluating channels, are inserted in the beam path behind the field
lens.
[0045] The wedge-shaped prisms GK1, 2, 3, 4 are arranged so as to
taper downward or upward in the vertical direction relative to the
direction of the object light, i.e., are constructed in triangular
shape, and GK1, GK4 are arranged in a wedge-shaped manner, i.e.,
with increasing thickness, substantially in the horizontal
direction along the dispersion direction vertical to the optical
axis, specifically such that GK1 has a wedge shape extending
opposite to that of GK4. However, GK2 and GK3 are constructed so as
to be wedge-shaped in the vertical direction with increasing
thickness in opposite directions, wherein GK2 has a point at the
bottom and an upper rectangular surface at the top and GK3 has an
edge at the top.
[0046] Viewed from the front, the tapering edge of GK1 (not
visible) is arranged on the right-hand side and that of GK4 is
arranged on the left-hand side, whereas in the case of GK3 it forms
the upper edge.
[0047] The wedges become increasingly thick proceeding from the
edges GK1, 3, 4 or tips (GK2), wherein the light is deflected in
the direction in which the thickness of the wedge increases, that
is, with reference to the drawing, toward the left in the case of
GK1, toward the top in the case of GK2, toward the bottom in the
case of GK3 and toward the right in the case of GK4. This is shown
more clearly in FIG. 2a. In this view, the knife edge of the wedge,
i.e., the start of the wedge increase, is shown as lines K1-4,
respectively, wherein it can be seen that GK1, GK4 extend toward
the right and left, respectively, with decreasing thickness in the
direction of the wedge knife edge K1 and K4, respectively, in
horizontally opposite directions, whereas GK2 extends vertically
downward in the direction of the wedge knife edge K2 with
decreasing thickness in direction K2, whereas in the case of GK3
the upper wedge edge coincides with K3 with thickness decreasing
toward the top. The light direction L shown by an arrow undergoes
the deflections designated by a1-a4 after passing the wedges GK1-4,
a1 toward the left in the drawing, a2 upward, a3 downward, and a4
toward the right.
[0048] Orientations of the prismatic wedges other than those shown
in the drawings are also possible and conceivable. Due to the
different orientation of the wedges, detection paths lying far
apart from one another after the collector KO can be realized in an
advantageous manner. These elements GK have a deflecting effect due
to their wedge shape, and the wedge shape or wedge direction is
advantageously selected differently in order to be able to detect
channels DT separately. The light of every channel DT is located at
a different location behind the collector KO due to the different
wedge effect.
[0049] The different wedge angle of the prisms GK is responsible
for the light deflection and a horizontal displacement of the
prisms vertical to the optical axis alters the spectral position of
the evaluating channel DT. Further, a vertical displacement of the
prisms GK advantageously changes the width of the cut out spectral
region.
[0050] The light coming from the object is parallelized by the
field lens FL in the direction of the wedge prisms GK and the
parallel light band that is displaced laterally by the wedge prisms
GK is unified by the collector in a punctiform region, so that it
can be detected therein by means of a receiver DE1-DE4. Every band
is focussed on a different location, so that a plurality of
detectors DE can be arranged. If required, emission filters, not
yet shown, can be positioned between the collector KO and receiver
DE1-4.
[0051] The coupling of the device, according to the invention, to a
laser scanning microscope is shown by way of example in FIG. 3. The
light exiting from the pinhole PH, in this case 28, is imaged to
infinity by the collimator 21. The light impinges on the scanner
group 22 (shown here schematically as an individual mirror), is
imaged in the intermediate image plane 24 of the microscope by the
auxiliary objective 23 and, finally, in the specimen through the
tube lens 25 and microscope objective 26. The light coming from the
specimen takes the opposite optical path in the direction of
detection.
[0052] 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.
* * * * *