US20020159144A1 - Scanning microscope and coupling-out element - Google Patents
Scanning microscope and coupling-out element Download PDFInfo
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- US20020159144A1 US20020159144A1 US10/128,858 US12885802A US2002159144A1 US 20020159144 A1 US20020159144 A1 US 20020159144A1 US 12885802 A US12885802 A US 12885802A US 2002159144 A1 US2002159144 A1 US 2002159144A1
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- coupling
- detection
- out element
- light
- scanning microscope
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
Definitions
- 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.
- At least one first detector for descan detection of the detection light proceeding from the specimen at least one first detector for descan detection of the detection light proceeding from the specimen
- the invention has the advantage of making it possible to switch over easily between descan detection and non-descan detection.
- 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.
- FIG. 1 shows a known arrangement for fluorescence microscopy, having several interchangeable modular filter blocks in a turret;
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- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Computer Vision & Pattern Recognition (AREA)
- General Engineering & Computer Science (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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.
Description
- This invention claims priority of the
German patent application 101 20 424.8-42 which is incorporated by reference herein. - 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.
- The invention further concerns a coupling-out element for a scanning microscope.
- 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.
- In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- The aforesaid object is achieved by means of a scanning microscope comprising:
- a light source for emitting illuminating light for illumination of a specimen (19),
- 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
- 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.
- 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.
- The invention has the advantage of making it possible to switch over easily between descan detection and non-descan detection.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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:
- FIG. 1 shows a known arrangement for fluorescence microscopy, having several interchangeable modular filter blocks in a turret;
- FIG. 2 is a plan view of the arrangement from FIG. 2;
- FIG. 3 shows a scanning microscope according to the present invention;
- FIG. 4 is a plan view of the scanning microscope according to the present invention from FIG. 4;
- FIG. 5 shows a further scanning microscope according to the present invention;
- FIG. 6 shows a further scanning microscope according to the present invention;
- FIG. 7 shows a coupling-out element; and
- FIG. 8 shows a further coupling-out element.
- FIG. 1 schematically shows an incident-light fluorescence microscope1 known from the existing art, having a fluorescent incident-
light illuminator 3.Excitation filter 11 is used to filter out from light 9 coming fromlight source 5, which is embodied as anarc lamp 7, those components having the desired wavelengths (i.e. excitation light 13).Excitation light 13 is then reflected fromdichroic beam splitter 15 towardmicroscope objective 17, which focusesexcitation light 13 ontospecimen 19.Fluorescent light 21 proceeding from the specimen travels back through the microscope objective todichroic beam splitter 15, passes through it, and is incident through blockingfilter 23 andeyepiece 25 into the user'seye 27.Excitation filter 11, blockingfilter 23, anddichroic beam splitter 15 are arranged in an interchangeable modularfirst filter block 29. -
First filter block 29 is arranged, together with afurther filter block 31 that contains anexcitation filter 33, adetection filter 35, and abeam splitter 37, in aturret 41 that can rotate aboutshaft 39.Excitation filter 33,detection filter 35, andbeam splitter 37 have different spectral properties fromexcitation filter 11,detection filter 23, andbeam splitter 15 offirst filter block 29.Filter block - FIG. 2 shows incident-light fluorescence microscope1 in a plan view, in which it is evident that the turret contains not only filter
blocks - FIG. 3 shows a
scanning microscope 47 according to the present invention. Illuminating light 53 coming from alight source 49, which is embodied as a mode-locked titanium-sapphire laser 51, has a wavelength of approx. 800 nm and is focused byoptical system 55 ontoexcitation pinhole 57, and is then reflected by abeam splitter 59 tobeam deflection device 61 which contains a gimbal-mountedmirror 63. Scanningoptical system 65, tubeoptical system 67, and objective 69 define anillumination beam path 71 and a detection beam path 72, along which illuminatinglight 53, shaped into a beam, is guided over or throughspecimen 19. Located between tubeoptical system 67 and objective 69 is coupling-outelement 73, containing adichroic beam splitter 77 and anexcitation 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 detectedlight 75 is reflected (out of the plane of the drawing) towardfurther detectors 80, 81 (not shown in this Figure). Coupling-outelement 73 is arranged in aturret 85 that can rotate aboutshaft 83, and is aligned in such a way that by rotation ofturret 85, it can be positioned in the excitation and detection beam path. Arranged inturret 85 is a further coupling-outelement 87 that contains anexcitation filter 89, a detection filter 91, and abeam splitter 93, these elements having spectral properties different from those of first coupling-outelement 73. The coupling-outelement detection beam path 71, 72 by rotation ofturret 85. For descan detection,turret 85 can additionally be rotated into anopen position 99, 101 (not depicted in this Figure) so that both illuminatinglight 53 and detected light 75 pass unimpeded. In descan detection mode, detected light 75 travels viabeam deflection device 61 back tobeam splitter 59, passes through the latter, through blockingfilter 79 which suppresses residual radiation from the excitation light, and throughdetection pinhole 95, and then strikesfirst detector 97. - 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. Detectedlight 75 emerges laterally from coupling-outelement 73 and encounters filterblock 104, which contains adichroic beam splitter 103 and two blockingfilters dichroic beam splitter 103, which is embodied as a color beam splitter, detectedlight 75 is divided in accordance with the spectral distribution intobeam segments detectors filters detectors Open positions - FIG. 5 shows, in a perspective view, a
confocal scanning microscope 47 that comprises a conventionallight microscope 109 and ascanner module 111.Scanner module 111 contains alight source 49 for generating illuminatinglight 53, abeam deflection device 61, afirst detector 97 for descan detection, and abeam splitter 59 that reflects illuminating light 53 tobeam deflection device 61 and, in descan detection mode, allows the detected light to pass tofirst 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 aspecimen slide 113, on a microscope stage (not shown). Conventionallight microscope 109 comprises a housing (not depicted) onto whichscanner module 111 is flange-mounted. Conventionallight microscope 109 furthermore contains a fluorescent incident-light illuminator 3 having alight source 5 that is embodied as anarc lamp 7, and aturret 41 that is rotatable aboutshaft 39. A coupling-outelement 73 for scanning microscopy with non-descan detection, and a first and asecond filter block Turret 41 also comprises anopen position 99 that can be rotated into illumination anddetection beam path 71, 72. This position is used for scanning microscopy with descan detection. The housing of conventionallight microscope 109 has a lateral opening through which, in non-descan detection mode, detectedlight 75 emerges and travels to furtherdetectors - FIG. 6 shows a
scanning microscope 47 according to the present invention which is constructed for the most part exactly like scanningmicroscope 47 shown in FIG. 5. What is used for non-descan detection in this embodiment, however, is notfurther detectors first detector 97 that is actually provided for descan detection. Detectedlight 75 is delivered to the latter via a light-guidingfiber 115 and amirror 117, bypassing the detection pinhole. - FIG. 7 shows a coupling-out
element 73 according to the present invention that contains anexcitation filter 7, adetection filter 119, and adichroic beam splitter 77. Coupling-outelement 73 comprises ahousing 121 having threeopenings light 53 and detected light 75 pass. Mounted onhousing 121 are guidance and/orbanking elements detection beam path 71, 72. - FIG. 8 shows a coupling-out
element 73 according to the present invention having an integratedfurther detector 137 that is configured as a photodiode. Anoptical system 120 is arranged in front of the detector. - 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.
Claims (20)
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.
Priority Applications (1)
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US10/766,260 US6977773B2 (en) | 2001-04-26 | 2004-01-27 | Scanning microscope and coupling-out element |
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DE10120424A DE10120424B4 (en) | 2001-04-26 | 2001-04-26 | Scanning microscope and decoupling element |
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US20090194715A1 (en) * | 2008-01-31 | 2009-08-06 | Harald Schadwinkel | Laser scanning microscope and assembly for non-descanned detection |
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DE102008015720B4 (en) | 2008-03-26 | 2018-01-25 | Carl Zeiss Microscopy Gmbh | Illuminating device for a microscope and fluorescence microscope with the same |
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Also Published As
Publication number | Publication date |
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US20040190133A1 (en) | 2004-09-30 |
DE10120424B4 (en) | 2004-08-05 |
US6977773B2 (en) | 2005-12-20 |
DE10120424A1 (en) | 2002-11-07 |
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