U.S. patent application number 10/128858 was filed with the patent office on 2002-10-31 for scanning microscope and coupling-out element.
This patent application is currently assigned to Leica Microsystems Heidelberg GmbH. Invention is credited to Engelhardt, Johann, Knebel, Werner, Ulrich, Heinrich.
Application Number | 20020159144 10/128858 |
Document ID | / |
Family ID | 7682777 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020159144 |
Kind Code |
A1 |
Engelhardt, Johann ; et
al. |
October 31, 2002 |
Scanning microscope and coupling-out element
Abstract
A scanning microscope having a light source that emits
illuminating light for illumination of a specimen, having at least
one first detector for detection of the detected light proceeding
from the specimen, having an objective being arranged in both an
illumination beam path and a detection beam path, and having a
coupling-out element that is selectably for descan detection and
non-descan detection positionable in the illumination and detection
beam path, is disclosed.
Inventors: |
Engelhardt, Johann; (Bad
Schoenborn, DE) ; Knebel, Werner; (Kronau, DE)
; Ulrich, Heinrich; (Heidelberg, DE) |
Correspondence
Address: |
DAVIDSON, DAVIDSON & KAPPEL, LLC
485 SEVENTH AVENUE, 14TH FLOOR
NEW YORK
NY
10018
US
|
Assignee: |
Leica Microsystems Heidelberg
GmbH
Am Friedensplatz 3
Mannheim
DE
68165
|
Family ID: |
7682777 |
Appl. No.: |
10/128858 |
Filed: |
April 23, 2002 |
Current U.S.
Class: |
359/385 ;
359/368; 359/388 |
Current CPC
Class: |
G02B 21/0032 20130101;
G02B 21/008 20130101 |
Class at
Publication: |
359/385 ;
359/368; 359/388 |
International
Class: |
G02B 021/00; G02B
021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 26, 2001 |
DE |
101 20 424.8 |
Claims
What is claimed is:
1. A scanning microscope comprising: a light source for emitting
illuminating light for illumination of a specimen, at least one
first detector for descan detection of the detection light
proceeding from the specimen, an objective being arranged in both
an illumination beam path and a detection beam path, a coupling-out
element being insertable into the illumination and detection beam
path for non-descan detection, and removable from the illumination
and detection beam path for descan detection
2. The scanning microscope as defined in claim 1 further
comprising: at least one guidance element for positioning the
coupling-out element.
3. The scanning microscope as defined in claim 1 further
comprising: at least one banking element for positioning the
coupling-out element.
4. The scanning microscope as defined in claim 1, further
comprising: a turret or a sliding carriage, on which the
coupling-out element is mounted.
5. The scanning microscope as defined in claim 1, wherein the
coupling-out element comprises a beam splitter.
6. The scanning microscope as defined in claim 1, wherein the
coupling-out element comprises an excitation filter for blocking
light of at least one wavelength out of the illuminating beam
path.
7. The scanning microscope as defined in claim 1, wherein the
coupling-out element comprises a detection filter for blocking
light of at least one wavelength out of the detection beam
path.
8. The scanning microscope as defined in claim 1 further
comprising: a further detector for non-descan detection receiving
the detection light coupled out by coupling-out element.
9. The scanning microscope as defined in claim 8, wherein the
coupling-out element contains the further detector.
10. The scanning microscope as defined in claim 1 further
comprising: a light-guiding fiber transporting at least a portion
of the detecting light from the coupling-out element to the first
detector.
11. The scanning microscope as defined in claim 1 further
comprising: a fluorescent incident-light illuminator in which the
coupling-out element is positionable.
12. A coupling-out element for a scanning microscope, which is
insertable into an illumination and detection beam path of the
scanning microscope for non-descan detection, and which is
removable from the illumination and detection beam path for descan
detection.
13. The coupling-out element as defined in claim 12, wherein the
coupling-out element is positionable in a turret.
14. The coupling-out element as defined in claim 12, wherein the
coupling-out element is positionable in a sliding carriage.
15. The coupling-out element as defined in claim 12, wherein the
coupling-out element is positionable in a fluorescent
incident-light illuminator.
16. The coupling-out element as defined in claim 12 further
comprising guidance and/or banking elements for positioning of the
coupling-out element.
17. The coupling-out element as defined in claim 12 further
comprising: an excitation filter for blocking light of at least one
wavelength out of the illumination beam path.
18. The coupling-out element as defined in claim 12 further
comprising: a detection filter for blocking light of at least one
wavelength out of the detection beam path
19. The coupling-out element as defined in claim 12 further
comprising: a further detector for non-descan detection receiving
the detection light coupled out by the coupling-out element.
20. The coupling-out element as defined in claim 12 further
comprising: an optical system.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This invention claims priority of the German patent
application 101 20 424.8-42 which is incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The invention concerns a scanning microscope having a light
source that emits illuminating light for illumination of a
specimen, having at least one first detector for detection of the
detected light proceeding from the specimen, and having an
objective by means of which the specimen can be illuminated and
detected, the objective being arranged in both an illumination beam
path and a detection beam path.
[0003] The invention further concerns a coupling-out element for a
scanning microscope.
BACKGROUND OF THE INVENTION
[0004] In scanning microscopy, a specimen is illuminated with a
light beam in order to observe the detected light (in the form of
reflected or fluorescent light) emitted by the specimen. The focus
of an illuminating light beam is moved in a specimen plane by means
of a controllable beam deflection device, generally by tilting two
mirrors; the deflection axes are usually perpendicular to one
another, so that one mirror deflects in the X and the other in the
Y direction. Tilting of the mirrors is brought about, for example,
by means of galvanometer positioning elements. The power level of
the detected light coming from the specimen is measured as a
function of the position of the scanning beam. The positioning
elements are usually equipped with sensors to ascertain the current
mirror position.
[0005] In confocal scanning microscopy specifically, a specimen is
scanned in three dimensions with the focus of a light beam.
[0006] A confocal scanning microscope generally comprises a light
source, a focusing optical system with which the light of the
source is focused onto a pinhole (called the "excitation pinhole"),
a beam splitter, a beam deflection device for beam control, a
microscope optical system, a detection pinhole, and the detectors
for detection of the detected or fluorescent light. The
illuminating light is coupled in via a beam splitter. The
fluorescent or reflected light coming from the specimen travels via
the beam deflection device back to the beam splitter, passes
through the latter and is then focused onto the detection pinhole,
behind which the detectors are located. This detection arrangement
is called a "descan" arrangement. Detected light that does not
originate directly from the focus region takes a different light
path and does not pass through the detection pinhole, so that a
point datum is obtained which, by sequential scanning of the
specimen with the focus of the illuminating light beam, results in
a three-dimensional image. A three-dimensional image is usually
achieved by acquiring image data in layers. Commercial scanning
microscopes usually comprise a scan module that is flange-mounted
onto the stand of a conventional light microscope and contains all
the aforesaid elements additionally necessary for scanning a
specimen.
[0007] Commercial scanning microscopes usually contain a microscope
stand like the one also used in conventional light microscopy. As a
rule, confocal scanning microscopes in particular are also usable
as conventional light microscopes. In conventional fluorescent
incident-light microscopy, the portion of the light of a light
source (for example of an arc lamp) that comprises the desired
wavelength region for fluorescent excitation is coupled into the
microscope beam path with the aid of a color filter called the
excitation filter. Coupling into the beam path of the microscope is
accomplished using a dichroic beam splitter, which reflects the
excitation light to the specimen while allowing the fluorescent
light proceeding from the specimen to pass largely unimpeded. The
excitation light scattered back from the specimen is held back with
a blocking filter that is, however, transparent to the fluorescent
radiation. Combining mutually matched filters and beam splitters
optimally to yield an easily interchangeable modular filter block
has been common for some time. The filter blocks are usually
arranged in a turret within the microscope as a part of so-called
fluorescent incident-light illuminators, thus enabling rapid and
easy interchanging.
[0008] In confocal scanning microscopy, a detection pinhole can be
dispensed in the case of two-photon (or multi-photon) excitation,
since the excitation probability depends on the square of the
photon density and thus on the square of the illuminating light
intensity, which of course is much greater at the focus than in the
adjacent regions. The fluorescent light being detected therefore
very probably originates almost exclusively from the focus region,
which renders superfluous any further differentiation, using a
pinhole arrangement, between fluorescent photons from the focus
region and fluorescent photons from the adjacent regions.
[0009] Especially given that the yield of fluorescent photons in
two-photon excitation is in any case low, a non-descan arrangement,
in which the detected light does not arrive at the detector via the
beam deflection device (descan arrangement) and the beam splitter
for coupling in the illuminating light, but rather is deflected
directly after the objective by means of a dichroic beam splitter
and detected, is of interest because less light is generally lost
when the detected light is guided in this fashion. In addition,
when descan detection is used in two-photon excitation, scattered
components of the detected light contribute significantly to the
signal, whereas with non-descan detection they play only a greatly
reduced role. Arrangements of this kind are known, for example,
from the publication by David W. Piston et al., "Two-photon
excitation fluorescence imaging of three-dimensional calcium ion
activity," Applied Optics, Vol. 33, No. 4, February 1996, and from
Piston et al., "Time-Resolved Fluorescence Imaging and Background
Rejection by Two-Photon Excitation in Laser Scanning Microscopy,"
SPIE Vol. 1640.
[0010] One problem with the known arrangements is that of arranging
a beam splitter, to deflect the detected light out of the
microscope beam path after the objective, within a scanning
microscope for non-descan deflection, and aligning it precisely.
This requires the implementation of complex additional arrangements
that necessitate massive physical modifications to the scanning
microscope and in particular to the microscope stand. Retrofitting
to a scanning microscope with descan detection is usually
impossible or very complex.
SUMMARY OF THE INVENTION
[0011] It is therefore the object of the invention to propose a
scanning microscope which is operable selectably with descan
detection or with non-descan detection and with which it is
possible to switch over easily and reliably between descan
detection and non-descan detection.
[0012] The aforesaid object is achieved by means of a scanning
microscope comprising:
[0013] a light source for emitting illuminating light for
illumination of a specimen (19),
[0014] at least one first detector for descan detection of the
detection light proceeding from the specimen,
[0015] an objective being arranged in both an illumination beam
path and a detection beam path,
[0016] a coupling-out element being insertable into the
illumination and detection beam path for non-descan detection, and
removable from the illumination and detection beam path for descan
detection
[0017] A further object of the invention is to disclose a
coupling-out element which easily enables a scanning microscope to
be operated selectably with descan detection or with non-descan
detection.
[0018] This object is achieved by means of a coupling-out element
for a scanning microscope, which is insertable into an illumination
and detection beam path of the scanning microscope for non-descan
detection, and which is removable from the illumination and
detection beam path for descan detection.
[0019] The invention has the advantage of making it possible to
switch over easily between descan detection and non-descan
detection.
[0020] In a preferred embodiment, the coupling-out element contains
a beam splitter that is preferably configured as a dichroic beam
splitter or color beam splitter. The beam splitter is preferably
configured to be transparent to the illuminating light and
reflective to the detected light.
[0021] In a further preferred embodiment, the coupling-out element
comprises filters that act as excitation filters on the light in
the illumination beam path or as detection filters on the light in
the detection beam path. In a very particularly preferred
embodiment, the coupling-out element contains both an excitation
filter and a detection filter. The detection filter is preferably
configured in such a way that it allows only the detected light
proceeding from the specimen, and in particular no light of the
wavelength of the illuminating light, to pass, thus advantageously
preventing any undesirable and falsifying detection of that light
in particular. The excitation filter serves to filter out from the
spectrum of the light source those light components having the
wavelengths of interest for illumination.
[0022] In a variant embodiment, the coupling-out element can be
introduced from outside into the illumination and detection beam
path in order to switch over from descan detection to non-descan
detection, guidance elements such as guide rails, slide bars, or a
bayonet mount, which make possible simple and reliable introduction
and positioning, being provided. Also provided are banking
elements, which define a working position of the coupling-out
element in the illumination and detection beam path and which are
configured so that the positioned coupling-out element is
automatically aligned with respect to the detection beam path, and
no further alignment of the coupling-out element is necessary after
positioning.
[0023] In another preferred embodiment, a turret or a sliding
carriage which comprises at least one element receptacle is
provided for positioning the coupling-out element, the coupling-out
element being mounted on or in the element receptacle in such a way
that the coupling-out element can be positioned in the illumination
and detection beam path by simply rotating the turret or sliding
the sliding carriage. Alignment of the coupling-out element is
performed only once, when the coupling-out element is mounted in or
on the turret or sliding carriage. The latter advantageously has a
snap-in apparatus that releasably immobilizes the turret or sliding
carriage when the coupling-out element is positioned in the
illumination and detection beam path. In a further variant
embodiment, the turret or sliding carriage comprises several
element receptacles in which other optical elements, for example
filters or additional optical systems, are mounted. The turret or
sliding carriage also comprises at least one open position that can
be introduced into the illumination and detection beam path in such
a way that the illuminating light and detected light can pass
unimpeded. This manner of achieving the object of the invention is
economical and highly flexible, since several coupling-out elements
having different beam splitters with different spectral properties
can be held in readiness and easily interchanged.
[0024] In a very particularly preferred embodiment, the
coupling-out element is a component of a fluorescent incident-light
illuminator. This embodiment is very particularly advantageous in
scanning microscopes that are also suitable for conventional
incident-light microscopy, which contain a fluorescent
incident-light illuminator with a turret. This has the advantage of
utilizing or putting to a different purpose apparatuses which
usually are present in any case, thereby avoiding any physical
modification to the microscope or the microscope stand. By simply
rotating the turret of the fluorescent incident-light illuminator,
it is possible to switch between conventional fluorescence
microscopy, descan detection, and non-descan detection.
[0025] The coupling-out element delivers at least a portion of the
detected light to a further detector. In an embodiment, this
detector is mounted externally on the scanning microscope, the
scanning microscope having an opening in the housing through which,
in the context of non-descan detection, the detected light emerges
and arrives at the further detector. In another embodiment, the
further detector is a component of the coupling-out element.
Semiconductor detectors, such as photodiodes or avalanche
photodiodes, are particularly advantageous in this context because
of their small overall size. Photomultipliers are also usable. In a
particularly preferred embodiment, the further detector contains at
least two further individual detectors for separate detection of
different spectral regions of the detected light. Beam splitters,
which advantageously are housed in a filter block, are provided in
order to divide the detected light. Further filters and/or optical
systems can be inserted into the filter block. It is very
particularly advantageous, especially in terms of integrated
manufacture, if the filter block has the same dimensions and
guidance and/or banking elements as the coupling-out element.
[0026] In a particular variant embodiment, in non-descan detection
mode the detected light coupled out by means of the coupling-out
element is delivered to the detector actually provided for descan
detection. Mirror arrangements or light-guiding fibers are provided
for this purpose. This variant embodiment has the particular
advantage that a single detector alone can be used for both descan
detection and non-descan detection, which makes the entire scanning
microscope much simpler and less expensive to manufacture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The subject matter of the invention is schematically
depicted in the drawings and will be described below with reference
to the Figures, identically functioning elements being labeled with
the same reference characters. In the drawings:
[0028] FIG. 1 shows a known arrangement for fluorescence
microscopy, having several interchangeable modular filter blocks in
a turret;
[0029] FIG. 2 is a plan view of the arrangement from FIG. 2;
[0030] FIG. 3 shows a scanning microscope according to the present
invention;
[0031] FIG. 4 is a plan view of the scanning microscope according
to the present invention from FIG. 4;
[0032] FIG. 5 shows a further scanning microscope according to the
present invention;
[0033] FIG. 6 shows a further scanning microscope according to the
present invention;
[0034] FIG. 7 shows a coupling-out element; and
[0035] FIG. 8 shows a further coupling-out element.
DETAILED DESCRIPTION OF THE INVENTION
[0036] FIG. 1 schematically shows an incident-light fluorescence
microscope 1 known from the existing art, having a fluorescent
incident-light illuminator 3. Excitation filter 11 is used to
filter out from light 9 coming from light source 5, which is
embodied as an arc lamp 7, those components having the desired
wavelengths (i.e. excitation light 13). Excitation light 13 is then
reflected from dichroic beam splitter 15 toward microscope
objective 17, which focuses excitation light 13 onto specimen 19.
Fluorescent light 21 proceeding from the specimen travels back
through the microscope objective to dichroic beam splitter 15,
passes through it, and is incident through blocking filter 23 and
eyepiece 25 into the user's eye 27. Excitation filter 11, blocking
filter 23, and dichroic beam splitter 15 are arranged in an
interchangeable modular first filter block 29.
[0037] First filter block 29 is arranged, together with a further
filter block 31 that contains an excitation filter 33, a detection
filter 35, and a beam splitter 37, in a turret 41 that can rotate
about shaft 39. Excitation filter 33, detection filter 35, and beam
splitter 37 have different spectral properties from excitation
filter 11, detection filter 23, and beam splitter 15 of first
filter block 29. Filter block 29, 31 having the desired optical
properties can be brought into the microscope's beam path by
rotating the turret.
[0038] FIG. 2 shows incident-light fluorescence microscope 1 in a
plan view, in which it is evident that the turret contains not only
filter blocks 29 and 31 but also two further filter blocks 43,
45.
[0039] FIG. 3 shows a scanning microscope 47 according to the
present invention. Illuminating light 53 coming from a light source
49, which is embodied as a mode-locked titanium-sapphire laser 51,
has a wavelength of approx. 800 nm and is focused by optical system
55 onto excitation pinhole 57, and is then reflected by a beam
splitter 59 to beam deflection device 61 which contains a
gimbal-mounted mirror 63. Scanning optical system 65, tube optical
system 67, and objective 69 define an illumination beam path 71 and
a detection beam path 72, along which illuminating light 53, shaped
into a beam, is guided over or through specimen 19. Located between
tube optical system 67 and objective 69 is coupling-out element 73,
containing a dichroic beam splitter 77 and an excitation filter 74,
for coupling out detected light 75 proceeding from the sample.
Dichroic beam splitter 77 is configured so that illuminating light
53 having a wavelength of approx. 800 nm can pass unimpeded, and
detected light 75 is reflected (out of the plane of the drawing)
toward further detectors 80, 81 (not shown in this Figure).
Coupling-out element 73 is arranged in a turret 85 that can rotate
about shaft 83, and is aligned in such a way that by rotation of
turret 85, it can be positioned in the excitation and detection
beam path. Arranged in turret 85 is a further coupling-out element
87 that contains an excitation filter 89, a detection filter 91,
and a beam splitter 93, these elements having spectral properties
different from those of first coupling-out element 73. The
coupling-out element 73, 87 that has the particular desired optical
properties can be positioned in illumination and detection beam
path 71, 72 by rotation of turret 85. For descan detection, turret
85 can additionally be rotated into an open position 99, 101 (not
depicted in this Figure) so that both illuminating light 53 and
detected light 75 pass unimpeded. In descan detection mode,
detected light 75 travels via beam deflection device 61 back to
beam splitter 59, passes through the latter, through blocking
filter 79 which suppresses residual radiation from the excitation
light, and through detection pinhole 95, and then strikes first
detector 97.
[0040] FIG. 4 shows a plan view of the scanning microscope depicted
in FIG. 3. Illuminating light 53 is incident onto the plane of the
drawing. Detected light 75 emerges laterally from coupling-out
element 73 and encounters filter block 104, which contains a
dichroic beam splitter 103 and two blocking filters 106, 108. At
dichroic beam splitter 103, which is embodied as a color beam
splitter, detected light 75 is divided in accordance with the
spectral distribution into beam segments 105 and 107 and conveyed
to detectors 80 and 81, which are embodied as photomultipliers. The
two blocking filters 106, 108 are configured so that only detected
light of the respectively desired wavelength reaches detectors 80,
81. Open positions 99, 101 for descan detection are drawn with
dotted lines.
[0041] FIG. 5 shows, in a perspective view, a confocal scanning
microscope 47 that comprises a conventional light microscope 109
and a scanner module 111. Scanner module 111 contains a light
source 49 for generating illuminating light 53, a beam deflection
device 61, a first detector 97 for descan detection, and a beam
splitter 59 that reflects illuminating light 53 to beam deflection
device 61 and, in descan detection mode, allows the detected light
to pass to first detector 97. Scanner module 111 furthermore
contains elements (not shown) for beam guidance and shaping, as
well as an excitation pinhole and a detection pinhole, which in the
interest of clarity also are not shown. Specimen 19 rests, together
with a specimen slide 113, on a microscope stage (not shown).
Conventional light microscope 109 comprises a housing (not
depicted) onto which scanner module 111 is flange-mounted.
Conventional light microscope 109 furthermore contains a
fluorescent incident-light illuminator 3 having a light source 5
that is embodied as an arc lamp 7, and a turret 41 that is
rotatable about shaft 39. A coupling-out element 73 for scanning
microscopy with non-descan detection, and a first and a second
filter block 29, 31 for conventional fluorescent incident-light
microscopy, are arranged in the turret. Turret 41 also comprises an
open position 99 that can be rotated into illumination and
detection beam path 71, 72. This position is used for scanning
microscopy with descan detection. The housing of conventional light
microscope 109 has a lateral opening through which, in non-descan
detection mode, detected light 75 emerges and travels to further
detectors 80 and 81 that are mounted on the housing.
[0042] FIG. 6 shows a scanning microscope 47 according to the
present invention which is constructed for the most part exactly
like scanning microscope 47 shown in FIG. 5. What is used for
non-descan detection in this embodiment, however, is not further
detectors 80, 81 but rather first detector 97 that is actually
provided for descan detection. Detected light 75 is delivered to
the latter via a light-guiding fiber 115 and a mirror 117,
bypassing the detection pinhole.
[0043] FIG. 7 shows a coupling-out element 73 according to the
present invention that contains an excitation filter 7, a detection
filter 119, and a dichroic beam splitter 77. Coupling-out element
73 comprises a housing 121 having three openings 123, 125, 127
through which illuminating light 53 and detected light 75 pass.
Mounted on housing 121 are guidance and/or banking elements 129,
131, 133, 135 which make possible simple and reproducible
positioning in excitation and detection beam path 71, 72.
[0044] FIG. 8 shows a coupling-out element 73 according to the
present invention having an integrated further detector 137 that is
configured as a photodiode. An optical system 120 is arranged in
front of the detector.
[0045] The present invention has been described with reference to a
particular embodiment. It is nevertheless self-evident that changes
and modifications can be made without thereby leaving the range of
protection of the following claims.
* * * * *