US20060245047A1 - Illumination module for evanescent illumination and microscope - Google Patents
Illumination module for evanescent illumination and microscope Download PDFInfo
- Publication number
- US20060245047A1 US20060245047A1 US11/415,041 US41504106A US2006245047A1 US 20060245047 A1 US20060245047 A1 US 20060245047A1 US 41504106 A US41504106 A US 41504106A US 2006245047 A1 US2006245047 A1 US 2006245047A1
- Authority
- US
- United States
- Prior art keywords
- microscope
- illumination
- illumination module
- measuring
- light
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
Definitions
- the invention relates to a microscope with a light source that produces an illumination light beam for evanescently illuminating a sample.
- the invention further relates to an illumination module with a light source that produces an illumination light beam.
- the task of the present invention is to disclose a microscope that enables variable adjustment of the penetration depth of illumination light during evanescent illumination of a sample.
- This task is solved by a microscope wherein an adjustment mechanism is provided with which the polarization of the illumination light beam may be changed.
- a further task of the present invention is to disclose an illumination module for a microscope that enables illumination of a microscopic sample during evanescent sample of illumination with adjustable penetration depth.
- the further task is solved by an illumination module wherein the illumination module can be coupled to a microscope for the purpose of evanescent illumination of a sample, and wherein an adjustment mechanism is provided with which the polarization of the illumination light beam may be changed.
- the penetration depth (and the light power) of an evanescent illumination field in a sample is dependent on the polarization of the illumination light that strikes the cover glass-sample interface or the sample holder-sample interface, respectively.
- sample objects such as molecules, cell components, etc.
- the orientation of sample objects may also advantageously be determined, as well as the corresponding isotropy of the refracted space.
- the microscope exhibits an objective with an objective pupil, whereby the illumination light beam for evanescently illuminating the sample exhibits a focus in the area of the objective pupil.
- an adjustable beam deflector is provided with which the position of the focus within the objective pupil may be moved. This creates an additional possibility for changing the penetration depth.
- the penetration depth of an evanescent illumination field in a sample is dependent on the angle at which total reflection occurs at the cover glass interface or at the sample holder interface, respectively.
- This angle is directly correlated with the angle relative to the optical axis at which the illumination light beam provided for evanescent sample illumination exits the objective via the front lens.
- This angle is dependent on the distance to the optical axis at which the illumination light beam passes through the rear focal plane of the objective (pupil).
- the illumination light beam In order to have available a largely parallel illumination light beam for evanescently illuminating a sample, the illumination light beam must exhibit a focus in the rear focal plane of the objective. Finally, the distance of the focus to the optical axis of the objective determines said angle, and thereby the penetration depth of the evanescent field in the sample that is to be tested.
- an adjustable beam deflector is arranged in the beam path of the illumination light beam.
- the beam deflector preferably comprises at least one galvanometric mirror.
- the beam deflector preferably comprises two galvanometric mirrors, which cause deflection of the illumination light beam in different lateral directions (e.g., x- and y-direction).
- the beam deflector may also comprise rotatable or tippable prisms and/or rotatable or tippable mirrors. The use of acousto-optical or electro-optical deflection elements can also be envisioned.
- the adjustment mechanism with which the polarization of the illumination light beam may be adjusted preferably comprises a phase plate, preferably a rotatable ⁇ /2 plate—preferably motorized.
- the adjustment mechanism may also comprise a Faraday rotator and/or a Pockels cell and/or a double-refractive material and/or a liquid crystal cell.
- the adjustment mechanism is the control element of a regulator, which adjusts the polarization according to settings input by the user.
- storage memory is provided in which—preferably sample-specific—polarization settings are stored to achieve different penetration depths. In this manner, adjustment, according to the invention, is quantifiable and reproducible.
- a control mechanism is preferably provided to measure and/or monitor the polarization of the illumination light beam.
- the control mechanism is the measuring element of the regulator.
- control mechanism may comprise a beam splitter that outcouples the measuring light from the illumination light beam.
- control element comprises at least one detector that detects the light power of at least a part of the measuring light.
- control mechanism comprises at least one polarization analyzer, which is preferably arranged in the beam path of the measuring light before the minimum of one detector. It may, for example, be a polarization foil, a double-refractive prism (e.g., a Glan-Thomson prism), or a polarization beam splitter, which may, for example, be implemented as a cube.
- the polarization beam splitter splits the measuring light into an s-polarized measuring beam and a p-polarized measuring beam.
- two detectors are provided, of which one detector receives the s-polarized measuring beam and the other the p-polarized measuring beam. In this manner, precise conclusions may be drawn from the light power measured by both detectors regarding the polarization of the illumination light beam.
- a processing module is provided with which the measurement data are processed. The processing module may also be a component of the regulator.
- At least the light source and the adjustment mechanism are preferably integrated into an illumination module that may be detachably coupled to a microscope stand or to an already existent microscope.
- the illumination module preferably also comprises the control mechanism.
- a bayonet coupling is preferably provided.
- the microscope preferably comprises a camera and/or a CCD element and/or an EMCCD element for the purpose of imaging.
- a power adjustment mechanism is provided to change the light power of the illumination light beam.
- This may, for example, be a mechanical beam attenuator, an LCD module, or an electro-optical—or acousto-optical—component (e.g., AOTF).
- the microscope preferably comprises a scanning microscope, in particular a confocal scanning microscope.
- At least the light source and the adjustment mechanism are integrated into an illumination module, which preferably may be coupled to a microscope and/or to a microscope stand.
- the illumination module according to the invention provides the advantage that it may be coupled as a retrofit to an already existent microscope or microscope stand.
- This task is solved by a microscope, wherein an adjustment mechanism is provided with which the spatial position of the focus within the plane of the objective pupil may be changed.
- a further task of the present invention is to disclose an illumination module for a microscope that enables illumination of a microscopic sample, in particular for evanescent sample illumination with adjustable penetration depth.
- the further task is solved by an illumination module, wherein the illumination module may be coupled to a microscope such that the illumination light beam in the plane of the objective pupil of the microscope exhibits a focus, and wherein the illumination module comprises an adjustment mechanism with which the spatial position of the focus within the plane of the object pupil may be changed.
- the penetration depth of an evanescent illumination field in a sample is dependent on the angle at which total reflection at the cover glass interface or at the sample holder interface occurs.
- This angle is directly correlated with the angle relative to the optical axis at which the illumination light beam which is provided for evanescent sample illumination exits from the objective via the front lens.
- This angle is dependent upon the distance from the optical axis at which the illumination light beam passes through the rear focal plane of the objective (pupil).
- the illumination light beam In order to have available a largely parallel illumination light beam for the purpose of evanescent sample illumination, the illumination light beam must exhibit a focus in the rear focal plane of the objective. Finally, the distance of the focus to the optical axis of the objective determines the aforementioned angle, and therewith the penetration depth of the evanescent field in the sample to be tested.
- the adjustment mechanism comprises an adjustable beam deflector that is arranged in the beam path of the illumination light beam.
- the beam deflector comprises at least one galvanometric mirror.
- the beam deflector preferably comprises two galvanometric mirrors, which cause deflection of the illumination light beam in different lateral directions (e.g., x- and y-direction).
- the beam deflector may also comprise rotatable or tippable prisms and/or rotatable or tippable mirrors.
- the use of acousto-optical or electro-optical deflection elements can also be envisioned.
- the adjustment mechanism comprises a light-conducting fiber which is at least partially movable.
- mechanical positioners are preferably provided that enable the light outgoing end of the light-conducting fiber to be positioned precisely within the objective pupil.
- the illumination light beam in this further development of the invention is focused onto the light incoming end of the light-conducting fiber, conveyed through the light-conducting fiber, and de facto again exhibits a focus at the outcoupling end that is positioned within the objective pupil, because of the small diameter of customarily used light-conducting fibers.
- the adjustment mechanism can be particularly advantageous for certain applications to drive the adjustment mechanism such that the focus describes a selectable curve path within the objective pupil plane.
- particularly homogeneous illumination can, for example, be achieved.
- the curve path is a circular path.
- An embodiment of the invention in which the curve path is a circular path the midpoint of which lies on the optical axis of the objective is very particularly preferred.
- the penetration depth remains constant while the focus describes the circle of the curve path, whereby, however, the illumination light beam that exits from the objective is continuously coupled to the cover glass or to the sample holder from various directions. It is also possible to select different coupling directions in order to compare the resultant, possibly different, images of the sample.
- a compensating optic is provided in order to compensate for unevennesses in the objective pupil plane.
- the microscope objective is exchangeable (e.g., objective turret), whereby a compensating optic is provided to compensate for the various pupil positions of different objectives.
- the distances between the front focal plane and the rear focal plane may differ from objective to objective, which may lead to problems because in order to achieve optimal evanescent sample illumination, the focus of the illumination light beam must lie more or less exactly in the objective pupil.
- the aforementioned compensating optic which may for example, be a zoom optic or several exchangeable optics arranged on a turret, compensates for these differences in distance.
- a light trap is provided to eliminate unused illumination light. Only a portion of the illumination light coupled to the cover glass or sample holder actually evanescently illuminates the sample. The light, which again exits from the cover glass or from the sample holder after several total reflections, many return to the microscope again and lead to imaging disturbances (as a result, for example, of scattered light). This is avoided, according to the invention, by a suitably arranged light trap.
- a switch is provided for switching between classic incident illumination and evanescent sample illumination.
- the switch may, for example, comprise a wing mirror.
- the light cone is variable, in particular for changing the azimuth.
- An aperture optic such as an iris optic arranged in the intermediate image plane may be provided in order to set the diameter of the illumination light beam that exits from the microscope objective.
- a camera is provided for imaging.
- the camera may, in a particularly preferred variant, be implemented as a color camera, or as a CCD camera.
- the objective exhibits a numeric aperture that is greater than 1.4, in particular greater than 1.45, in particular greater than 1.6.
- the numeric aperture of the objective is 1.45 or 1.65.
- At least the light source and the adjustment mechanism are incorporated in a single illumination module, which can preferably be coupled to a microscope and/or a microscope stand.
- the illumination module offers the advantage that it may be coupled as a retrofit to a preexistent microscope or microscope stand.
- FIG. 1 a microscope according to the invention:
- FIG. 2 a further microscope according to the invention
- FIG. 3 a further microscope according to the invention with an illumination module.
- FIG. 1 shows a microscope 1 according to the invention with an objective 3 and a light source 5 , which is implemented as a laser 7 and which produces an illumination light beam 9 .
- the illumination light beam 9 emitted by the light source 5 enables evanescent illumination of a sample 11 that is positioned on a sample holder 13 .
- the illumination light beam 9 exhibits a focus 19 , which is represented by a point, on the plane 15 of the objective pupil 17 .
- Several optical elements for directing and shaping the beam are located in the beam path of the microscope 1 . There may, for example, be a first optic 21 , a second optic 23 , and an optic 25 , which produce a first intermediate image plane 27 and a second intermediate image plane 29 .
- An adjustment mechanism 31 is provided with which the polarization of the illumination light beam 9 may be changed.
- the adjustment mechanism 31 is implemented as a ⁇ /2 plate 33 that is rotatable around the optical axis. For each rotational position of the ⁇ /2 plate 33 there is an associated resultant polarization setting of the illumination light beam 9 .
- the penetration depth in the sample 11 and the light power of the evanescent field may be varied with the adjustment mechanism 31 .
- the ⁇ /2 plate 33 is rotated by a servomotor 51 .
- the detection light 35 issuing from the sample 11 passes through the objective 3 as well as through the beam splitter 39 , which directs the illumination light beam 9 to the objective 3 , and through it to a detector 41 , which is implemented as a CCD camera.
- the beam splitter 39 is implemented as a dichroic beam splitter, and designed such that the light at the wavelength of the illumination light beam is reflected, whereas light at the wavelength of the detection light 35 may pass through.
- the detection light data are transferred to a data processing module 45 . Correlation of image objects with various layer depths of the sample from the first and the second detection light data ensues in the data processing module 45 , and a 3-D data stack is produced, which is displayed as a three-dimensional image of the sample 11 or of the illuminated area of the sample on a display monitor 47 of a PC 49 .
- a beam splitter 53 is arranged in the further beam path of the illumination light beam 9 , which splits off a small portion of the illumination light beam 9 as a measuring beam 55 for polarization measurement.
- the measuring beam 55 is split by a polarization beam splitter 57 into an s-polarized partial beam 63 , which is detected by a first detector 59 , and a p-polarized partial beam 65 , which is detected by a second detector 61 .
- Conclusions may be drawn about the polarization of the illumination light beam 9 from the ratio of the light power measured by the first detector 59 and by the second detector 61 .
- the rotational position of the ⁇ /2 plate 33 is set according to user input via a feedback system, which is not shown.
- the processing module 67 receives the measurement signals from the first detector 59 and the second detector 61 , as well as the user input settings from the PC, and adjusts the rotational position of the ⁇ /2 plate 33 with the help of the servomotor 51 .
- Memory is provided in the PC 49 in which are stored the sample-specific polarization settings for achieving various penetration depths such that the user may input penetration depths directly without having to determine the associated polarization setting.
- FIG. 2 shows a further microscope according to the invention in which in addition to the adjustment mechanism 31 for setting the polarization of the illumination light beam, the spatial position of the focus 19 within the plane 15 of the objective pupil 17 may also be adjusted with the help of an adjustable beam deflector 69 .
- the adjustable beam deflector 69 comprises a cardanically suspended rotating mirror, which is not shown. The distance of the focus 19 to the optical axis 71 of the objective 3 may be adjusted, and the penetration depth of the illumination light beam in the sample 11 thereby varied using the adjustment mechanism 31 .
- FIG. 3 shows a further microscope according to the invention with an illumination module 73 that has already been coupled to an existent microscope 75 to achieve adjustable evanescent illumination with regard to penetration depth and with regard to illumination light power.
- the illumination module 73 exhibits a bayonet mount, which is not shown, for optical mounting to the microscope 75 . Furthermore, the illumination module 73 also exhibits plugs, also not shown, for the electrical and electronic connections.
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/435,208 US7746552B2 (en) | 2003-09-25 | 2009-05-04 | Illumination module for evanescent illumination and microscope |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEDE10344410.6 | 2003-09-25 | ||
DE10344410A DE10344410A1 (de) | 2003-09-25 | 2003-09-25 | Rastermikroskop mit evaneszenter Beleuchtung |
DE102004044309 | 2004-09-10 | ||
DEDE102004044309.2 | 2004-09-10 | ||
PCT/EP2004/052268 WO2005031432A1 (de) | 2003-09-25 | 2004-09-22 | Beleuchtungsmodul zur evaneszenten beleuchtung und mikroskop |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2004/052268 Continuation WO2005031432A1 (de) | 2003-09-25 | 2004-09-22 | Beleuchtungsmodul zur evaneszenten beleuchtung und mikroskop |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/435,208 Division US7746552B2 (en) | 2003-09-25 | 2009-05-04 | Illumination module for evanescent illumination and microscope |
Publications (1)
Publication Number | Publication Date |
---|---|
US20060245047A1 true US20060245047A1 (en) | 2006-11-02 |
Family
ID=34395048
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/415,041 Abandoned US20060245047A1 (en) | 2003-09-25 | 2006-05-01 | Illumination module for evanescent illumination and microscope |
US12/435,208 Expired - Fee Related US7746552B2 (en) | 2003-09-25 | 2009-05-04 | Illumination module for evanescent illumination and microscope |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/435,208 Expired - Fee Related US7746552B2 (en) | 2003-09-25 | 2009-05-04 | Illumination module for evanescent illumination and microscope |
Country Status (5)
Country | Link |
---|---|
US (2) | US20060245047A1 (fi) |
EP (1) | EP1690122B8 (fi) |
AT (1) | ATE413620T1 (fi) |
DE (1) | DE502004008431D1 (fi) |
WO (1) | WO2005031432A1 (fi) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080151226A1 (en) * | 2003-09-25 | 2008-06-26 | Leica Microsystems Cms Gmbh | Method for analyzing a sample and microscope for evanescently illuminating the sample |
US20130170785A1 (en) * | 2010-01-28 | 2013-07-04 | Peiliang GAO | Multifunctional integrated optical device |
JP2017522603A (ja) * | 2014-07-22 | 2017-08-10 | ライカ マイクロシステムズ シーエムエス ゲゼルシャフト ミット ベシュレンクテル ハフツングLeica Microsystems CMS GmbH | 試料を顕微鏡検査する方法及び装置 |
US11378793B2 (en) * | 2018-03-09 | 2022-07-05 | Carl Zeiss Microscopy Gmbh | Camera module for a microscope, and method for operating same |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102005023768B4 (de) | 2005-05-19 | 2017-06-29 | Leica Microsystems Cms Gmbh | Verfahren zur Ermittlung der Orientierung von Molekülen in biologischen Proben |
DE102009049050B4 (de) * | 2009-10-12 | 2011-07-21 | Leica Microsystems CMS GmbH, 35578 | Verfahren und Vorrichtung zum Stabilisieren einer Lichtleistung eines Beleuchtungslichtstrahls und Mikroskop |
DE102012214568A1 (de) * | 2012-08-16 | 2014-02-20 | Leica Microsystems Cms Gmbh | Optische Anordnung und ein Mikroskop |
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2004
- 2004-09-22 WO PCT/EP2004/052268 patent/WO2005031432A1/de active Application Filing
- 2004-09-22 EP EP04766837A patent/EP1690122B8/de not_active Not-in-force
- 2004-09-22 DE DE502004008431T patent/DE502004008431D1/de active Active
- 2004-09-22 AT AT04766837T patent/ATE413620T1/de not_active IP Right Cessation
-
2006
- 2006-05-01 US US11/415,041 patent/US20060245047A1/en not_active Abandoned
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2009
- 2009-05-04 US US12/435,208 patent/US7746552B2/en not_active Expired - Fee Related
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Publication number | Priority date | Publication date | Assignee | Title |
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US20080151226A1 (en) * | 2003-09-25 | 2008-06-26 | Leica Microsystems Cms Gmbh | Method for analyzing a sample and microscope for evanescently illuminating the sample |
US7633622B2 (en) * | 2003-09-25 | 2009-12-15 | Leica Microsystems Cms Gmbh | Method for analyzing a sample and microscope for evanescently illuminating the sample |
US20130170785A1 (en) * | 2010-01-28 | 2013-07-04 | Peiliang GAO | Multifunctional integrated optical device |
JP2017522603A (ja) * | 2014-07-22 | 2017-08-10 | ライカ マイクロシステムズ シーエムエス ゲゼルシャフト ミット ベシュレンクテル ハフツングLeica Microsystems CMS GmbH | 試料を顕微鏡検査する方法及び装置 |
US11378793B2 (en) * | 2018-03-09 | 2022-07-05 | Carl Zeiss Microscopy Gmbh | Camera module for a microscope, and method for operating same |
Also Published As
Publication number | Publication date |
---|---|
US20090213456A1 (en) | 2009-08-27 |
EP1690122A1 (de) | 2006-08-16 |
EP1690122B8 (de) | 2009-02-18 |
DE502004008431D1 (de) | 2008-12-18 |
EP1690122B1 (de) | 2008-11-05 |
US7746552B2 (en) | 2010-06-29 |
WO2005031432A1 (de) | 2005-04-07 |
ATE413620T1 (de) | 2008-11-15 |
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