Method and arrangement for the controlled actuation of a microscope, in particular of a laser scanning microscope
Method for actuation control of a microscope, in particular of a Laser Scanning Microscope, in which, at least one first illumination light, preferably moving at least in one direction, as well as at least one second illumination light moving at least in one direction, illuminate a sample through a beam combination, a detection of the light coming from the sample takes place, whereby, at least one part of the illumination light is generated through the splitting of the light from a common illuminating unit, characterized in that, by means of a common control unit, a controlled splitting into the first and the second illumination light takes place, in which the intensity of the first illuminating light, specified by the user or specified automatically, is assigned a higher priority (is prioritized) compared to the specified value for the second illumination light, and an adjustment for the second illumination light takes place until a maximum value is obtained, which is determined by the value specified for the first illumination light.
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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTNot Applicable
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMNot Applicable
BACKGROUND OF THE INVENTION(1) Field of the Invention
The present invention relates to a method and apparatus for the controlled actuation of a microscope, in general, and to controlled actuation of a laser scanning microscope having multiple light sources, in particular.
(2) Description of Related Art
Confocal microscopy is, among other things, the tool for defined controlled actuation of micro-objects. Based on that, numerous methods for examination and influencing of microscopic objects were proposed, thus, for instance, by Denk in U.S. Pat. No. 5,034,613, by Liu in U.S. Pat. No. 6,159,749, or by Karl Otto Greulich in “Micromanipulation by Light in Biology and Medicine” in 1999.
A combination comprising an image-forming point scanning or line scanning system and a “manipulator” system is increasingly finding more and more interest in the professional circles.
The interest in the observation and analysis of fast microscopic processes has brought forth new devices and methods (for example Carl Zeiss Line Scanner LSM 5 LIVE), which, in combination with the above mentioned methods of manipulation, lead to new insights. Thereby, the simultaneous microscopic observation of radiation-induced manipulation of the samples with spatial resolution by means of a suitable imaging system stands especially in the foreground (See for example U.S. Pat. No. 6,094,300 and DE 102004034987 A1). Therefore the modern microscopes attempt to offer as many flexible and optically equivalent decoupling and coupling ports as far as possible (See: DE 102004016433 A1).
The availability at the same time of at least two coupling ports for independent scan systems is thereby of special importance in order to avoid limitations in the temporal resolution due to the slowness of mechanical switching processes. Besides the tube interface, other coupling ports on the sides of the microscope stand are possible (preferably in the extended infinite space between the microscope objective and the tube lens; the so-called “sideports”) as well as on the rear side of the stand (typically optically modified incident light axis or transmitted light axis with suitable tube lens; the “rearports”) as well as on the bottom side (the “baseport”).
Thereby, arrangements with a common direction of the incident light (either reflected or transmitted light) or with a direction opposite to incident light (transmitted light and reflected light) are possible in principle. Apart from the viewpoint of the applicability, a common direction of incidence is frequently preferred from the device-technical viewpoint.
In that case, use of at least one element is necessary, which combines the beam paths of both devices in the space between the scanners of the scan systems that are to be operated simultaneously and the objective. Thereby, according to the state-of-the-art, a diverse variety of beam-combining elements are conceivable, such as, for example, the optomechanical components, like suitably coated beam combiner flat plates and beam combiner wedges, beam combiner cubes and polarization splitters. Conceivable are further beam combining acousto-optical modulators and deflectors.
In the following, reference is made in particular to DE 102004034987 A1, which is incorporated by reference herein as if reproduced in full and which forms a part of the subject matter of the present publication.
In
In a preferred embodiment, the electronic actuation of the microscope stand and the coupled manipulation and the imaging module are suitably equipped using a real-time electronic control system with an integrated real-time computer for the processing of the high data rates. Thereby, such embodiments are conceivable in which the scan systems of the manipulations and the imaging modules coupled with the microscope stand can be actuated in synchronous or asynchronous manner. Thus simultaneous scan modes of both the modules are possible in which manipulation and imaging in the different regions of the sample (ROIs; “regions of interest” DE19829981 C2) with variable scanning rates takes place as in
Both for the manipulating system as well as for the imaging system, the useful spectral range can be extended, depending on the respective application, from the ultraviolet to the infrared spectral range. Manipulation wavelengths typically found in the applications are, for instance, 351, 355 and 364 nm (photo-uncaging), 405 nm (photoconversion, Kaede, Dronpa, PA-GFP), 488 and 532 nm (photobleaching, FRET, FRAP, FLIP) as well as 780-900 nm (multiphoton bleaching, for example MPFRAP, 2-photon uncaging; and direct multiphoton stimulation).
Since in many applications, both the manipulating as well as the imaging system employ the same laser wavelengths, it is reasonable to feed both the scan modules with a common laser source. In DE 102004034987 A1 different suitable arrangements for variably adjustable division of the beam between two independent scan modules are described:
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- a. Laser-specific, variable beam splitting with a rotatable λ/2-plate and polarization beam splitters (ref.
FIG. 2 ):- By using a motorized rotatable λ/2-plate before each laser and a polarization beam splitter cube in the combined beam path of all lasers, a variable, loss-free beam splitting into two illumination canals takes place. Thereby, by rotating the
λ 2-plate by an angle Θ, the polarization of the incident polarized laser is rotated by angle 2Θ. The horizontally and the vertically polarized components of the field amplitude are split by the subsequent polarization beam splitter cube (Glan-Taylor prism). Thereby the horizontally polarized light is transmitted and the vertically polarized light is reflected. By rotating the λ/2-plate from 0° to 45° the polarization of the incident beam is rotated from 0° to 90° and the beam intensity is thus divided continuously and variably between the split partial beams. The intensity of the split laser beams can be modulated in any of the illumination canals individually with the help of an appropriate light modulator (for example graduated, acousto-optical modulators like Pockels cells). When different laser sources are used in which their beams are combined as inFIG. 2 , this method of variable beam splitting is particularly practicable, if the individual beam combiners of the laser module are largely independent of the polarization. - In addition to that, the fact that a finite switching time is necessary for the rotation of the λ/2-plate must be taken into account. Therefore a limitation from the viewpoint of the applications arises in the case of this method precisely then, when the manipulation and the fast imaging take place sequentially at time intervals of less than this switching period for the same wavelength and, in addition to that, the sum of laser power required for both partial processes exceeds the total available. The described method can be employed with advantage especially then, when the same laser line can be used simultaneously in the manipulating as well as in the imaging system. This is true particularly in photobleaching applications, such as, for instance, FRET, FRAP and FLIP.
- By using a motorized rotatable λ/2-plate before each laser and a polarization beam splitter cube in the combined beam path of all lasers, a variable, loss-free beam splitting into two illumination canals takes place. Thereby, by rotating the
- b. This application-related limitation can however be eliminated, if, in lieu of the rotatable λ/2-plate, fast electrooptic or magnetooptic polarization rotators (for example Pockels cells, Faraday rotators or LC retarders) are used, which have switching periods in the microsecond range or shorter (
FIG. 2 ). - c. A variable, wavelength-specific beam splitting into two illumination canals can be done also with two AOTFs (acousto-optical tunable filter) arranged successively one after the other as in
FIG. 3 , whereby, for instance, the 1st order of diffraction of the first AOTF is used for the coupling in the imaging system, whereas the 0th order of diffraction is coupled in through a second AOTF in the manipulator module (FIG. 3 ).- The imaging should thereby not be impaired by switching over of the bleaching ROI.
- This method has the disadvantage in applications that in case of simultaneous manipulation and imaging, the second manipulator AOTF must be adjusted simultaneously through software control with the switching of the first AOTF (for example switching off of the laser power of the imaging system at the reversal points of the raster scan).
- d. A variant of c. without functional limitations can be realized when an AOTF is exclusively used for variable beam splitting between two illumination canals and the laser power can be adjusted separately in both canals through two other AOTFs (
FIG. 4 ). - e. A simple economical method for beam splitting can be realized with the help of a neutral graduating wheel with different positions or a continuously coated neutral filter wheel or a neutral slider (graduated filter).
- a. Laser-specific, variable beam splitting with a rotatable λ/2-plate and polarization beam splitters (ref.
The present invention relates to a method and apparatus for actuation control of a microscope, in particular of a Laser Scanning Microscope, in which, at least one first illumination light, preferably moving at least in one direction, as well as at least one second illumination light moving at least in one direction, illuminate a sample through a beam combination. A detection of the light coming from the sample takes place. At least one part of the illumination light is generated through the splitting of the light from a common illuminating unit. A common control unit accomplishes a controlled splitting of the illumination light into the first and the second illumination lights. The intensity of the first illuminating light, as specified by a user or specified automatically, is assigned a higher priority (is prioritized) compared to the specified value for the second illumination light, and an adjustment for the second illumination light takes place until a maximum value is obtained, which is determined by the value specified for the first illumination light.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
In describing preferred embodiments of the present invention illustrated in the drawings, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected, and it is to be understood that each specific element includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.
If the manipulating as well as imaging systems compete for the power of the laser line in such a manner that it is as high as possible in the simultaneous operation in this type of microscope system, it is an advantage if the power requirement of the imaging system has a higher priority compared to the manipulator module. In commercial laser scanning microscope systems with only one scan module, typically the laser power for the manipulation process and the subsequent imaging can in each case be adjusted through the operating interface of the control software. This takes place, for example, using the corresponding software slider. In contrast to that, in the methods for simultaneous, variably tunable division of a laser line between two independent scan systems, shown in
According to the invention, the beam-splitting ratio as well as the subsequent intensity modulation are so optimally adjusted that, on one hand, the laser power requirement of the imaging system is fulfilled (higher priority) and, on the other hand, the manipulating system also receives laser power that is as high as possible at the same time. This makes it necessary to provide a method for optimal management of the laser power that is as automatic as possible, in which the user of the device only needs to define the laser powers necessary for imaging and manipulation in the customary manner (as in LSM systems with only one scan module) and, against that, the control software takes care on its own of the optimal tuning of the components shown in
Implementation of this principle of the actuation control, shown in the flow charts in
This principle is explained as follows on the basis of the variable splitting of the beam by means of a rotatable λ/2-plate and intensity modulation of the two split partial beams by means of an AOM (acousto-optic modulator).
The AOMs correspond, for instance, to the attenuators in the beam paths to the manipulator or the line scanner shown in
The principle of the controlling actuation shown generally in
As already explained above, in most of the applications, the power for the light required by the imaging system has the first priority. The imaging system (for example the line scanner in
The software slider in the operating software represents (analogous to the software interface of “stand alone” LSM systems) the total power for the light demanded by the respective scan module (image forming as well as manipulating systems). Screenshots of a user interface for the user are shown in
whereby Rλ/2 and TAOM represent the part of the light reflected by the polarization beam splitter cube and the part of the light transmitted by the AOM. Thereby the designations “Master” and “Slave” stand for the “imaging” or the “manipulating” scan system. The “Master” part of the imaging system after the polarization beam splitter (Rλ/2) is obtained here from the angular position Θ of the λ/2-plate
Rλ/2=cos2(2θ)
In the present invention, the strategy for the control is so arranged as in
Pideal is the value specified by the user, on response yes to the comparison in the first box, it goes to the next query, on response no, the attenuator (AOM) of the master part must be adjusted.
In the next comparison, on no, the lambda half plate of the master system is adjusted, on yes, the attenuator (AOM) of the master system.
However, in the control, the power demanded by the manipulating system (“Slave”) comes to an expression as in
To illustrate the actuation control processes shown in
The examples 1)-5) follow successively one after the other, whereby the reaction without the manipulating system is described first (ref.
1) Imaging 100%, manipulation 0%, →Rλ/2=1, TAOM,Master=1, Tλ/2=0
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- the λ/2-plate is set to Rλ/2=1, that is, the master (imaging) receives the entire laser energy when the transmission of the corresponding attenuator is maximum (TAOM,Master=1), the attenuator is arranged in sequence after the λ/2-plate;
2) Imaging 50%, manipulation 40%
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- The imaging demands 50% of the available energy, thus a maximum of 50% remains for the manipulation
- However the manipulation asks for only 40%, so that the manipulation can also actually receive its 40%
- For that the λ/2-plate must be regulated, because at that moment all the energy flows in the direction of the imaging system R□/2=1, the λ/2-plate is thereby regulated as little as possible and hence moves according to T□/2=0.4→Rλ/2=0.6 (the total is 1).
- But now the imaging system receives too much energy (60% because R□/2/2=0.6 and TAOM,Master=1), that is, it must now be slightly attenuated: TAOM,Master=0.
8 3
Final result: Tλ/2/2=0.4→Rλ/2=0.6→TAOM,Master=0.
3) Imaging 50%, manipulation 70%
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- The manipulation demands 70%, but can have only 50%, because the power requirement of 50% for the imaging system has a higher priority, that is, increase by 10% from 40% to 50% is possible, for that the λ/2-plate must be moved slightly, from Rλ/2=0.6 to Rλ/2=0.5; after that the attenuators of both systems are each adjusted to give 100% transmission.
Final result: Tλ/2=0.5→Rλ/2=0.5→TAOM,Master=1.0, TAOM,Slave=1.0, PSlave=0.5 (insted of 0.7)
4) Imaging 10%, manipulation 40%
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- The λ/2-plate can remain as it is, only the attenuators must be readjusted, this is done fast: TAOM,Master=0.2, TAOM,Slave=0.8
Final result: Tλ/2=0.5→Rλ/2=0.5→TAOM,Master=0.2, TAOM,Slave=0.8, PSlave=0.4
5) Imaging 10%, manipulation 70%
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- The imaging (master) demands 10% of the laser power, that is, the manipulation can receive 70%; for that the λ/2-plate must be moved: Tλ/2=0.7→R□/2=0.3
- After that the attenuators are adjusted so as to yield the total values of 10% and 70% respectively
Final result: Tλ/2=0.7→Rλ/2=0.3→TAOM,Master=0.
The generalized principle of the control shown in
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- at least one source of light can be divided with a variably adjustable ratio of RST/TST between two scanning systems by means of a beam splitting element ST;
- the power requirement of one scanning system (“Master”) is assigned higher priority than that of the other scanning system (“Slave”);
- suitable intensity modulators are provided for, if necessary, reducing the intensity of the transmitted light distributed between the two partial branches TMaster and TSlave;
- the user of the devices defines only the power required by the two scanning systems through the interface of the operating SW, and the control SW determines on its own the optimal settings for the variable beam splitting and for the intensity modulators of the master and the slave scan modules.
Neutral combiners (for example T20/R80) can be employed universally as beam combiners for most diverse varieties of applications and, in addition to that, enable applications in a simple manner, in which the same laser wavelengths can be used in simultaneous operation, both of the imaging system as well as of the manipulation system (in particular photobleaching, FRET, FRAP, FLIP). On the other hand, neutral combiners often represent a compromise, especially when the same laser line is used simultaneously for the manipulation as well as for the imaging, between the branching ratio for the respective laser wavelength, on one hand, and maximizing the signal efficiency in the range of the detection wavelength, on the other hand. Therefore, this demands an optimal design for the beam combiner, which is explicitly optimized for simultaneous operation of a manipulating and an imaging system for the same laser wavelength.
It is evident from
The remaining laser power (1−PNV,imag) of the common source of light of wavelength λ is thus available to the manipulating “Slave” system according to the actuation control schema in
PNV, mani, sample=(1−PNV,imag)*(1−RNV)
The optimal reflectivity RNV of the neutral beam combiner is obtained by maximizing the resulting manipulating laser power in the object plane PNV, mani, sample for the same fluorescence signal intensity as in the layout in
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- Example: P0,imag=0.08 (8% excitation power for the embodiment 8a.) RNV=0.4939 PNV, mani, sample=0.3401
PSV,imag=P0,imag/(RSV*RFL)
The remaining power (1−PSV,imag) of the common light source of wavelength λ is thus available to the “slave” manipulation system according to the actuation control principle shown in
PSV, mani, sample=(1−PSV,imag)*(1−RSV)
The reflectivity RSV of the beam combiner for the excitation and manipulation wavelength λ is now to be so optimized that for a given fluorescence reflectivity RFL (in the ideal case as nearly equal to 1 as possible) and the same fluorescence signal intensity as in the embodiment 8a, a highest possible manipulation laser power PSV, mani, sample in the object plane is obtained. Analytically one obtains the optimum for:
[RSV]opt=(P0,imag/RFL)1/2
In
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- Example: P0,imag=0.08 (8% excitation power for the embodiment 8a.), RFL=0.85 RSV=0.3068 and PSV, mani, sample=0.4805
For the same fluorescence signal intensity in the imaging system, one thus obtains, using this beam combiner, about 30% higher manipulation laser power in the sample—compared to the optimized neutral combiner of the embodiment 8b.
If in contrast to the devices shown in
To generalize, an optimized beam combiner design for the superposition of the optical axes of two independent scanning systems is required, in which both the modules are operated with at least one common laser wavelength λ. Thereby, at least one of the two scanning systems is designed as an imaging system and its power requirement is assigned higher priority compared to the other scanning system in such a manner that the detected fluorescence signal intensity is comparable with the corresponding “stand alone” system. For the wavelength(s) λ commonly used by both the systems, the branching ratio of this beam combiner is so selected that for a given fluorescence signal intensity, which would correspond to the typical intensity in a “stand alone” scanning system for free passage of the beam without a beam combiner, laser power that is as high as possible in the sample plane is obtained for one scanning system. Outside the common wavelength(s) λ used by the two scanning systems, the beam combiner is so designed that it is either only reflecting or transmitting as far as possible. The optimized spectral design of this beam combiner corresponds therefore to a “bad” bandpass filter in transmission or reflection.
In other words, as the control variables for the method according to the invention serve the grade of the reflectivity (Rsv, Rfl) or the transmission of the corresponding beam combiner for the excitation beam and fluorescence beam in the imaging system with respect to the proportion of the manipulation system or if specific power is given, the selection of a suitable beam combiner is optimized as the control variable.
In
Outside the bandpass range of 488 nm, the beam combiner is as reflecting as possible as in
The described invention relates in a general sense to any type of imaging and manipulating system. Besides the (confocal and partially confocal) point and line scanners, it can also be of relevance in particular in multifocal laser scanning systems (for example, those based on lens arrays, diode laser arrays, with any type of beam splitting arrangement) and spinning disk systems/Nipkow systems. Further, in the present invention, the sample can be scanned with a scanning method according to current state-of-the-art. Thereby, one of the following can be the underlying scanning principle of the device for the deflection of the beam in the imaging or the manipulating system:
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- Galvo mirror or
- guidable, in particular rotatable and tiltable mirrors, for example step motor driven deflecting mirrors
- polygon mirrors
- acousto-optical deflecting devices, in particular acousto-optical deflectors (AODs)
- movable aperture masks, in particular in the form of a Nipkow disk
- movable (monomode) fibers
- movable objectives or objective parts
- mechanical x- and y-adjustment of a suitable component or of the entire scanning system, for example by means of acousto-optical modulators
However, since both the scanning systems must be independent of each other in the sense of this invention, a mechanical x- and y-adjustment of the sample is not admissible.
Besides the use of microscope systems with coherent light sources (lasers) and confocal or partially confocal scan modules, an advantageous application of the invention in analogous manner is conceivable also in the simultaneous manipulation of the sample and/or the imaging with the help of (structured) wide-field illumination systems with incoherent light sources.
Modifications and variations of the above-described embodiments of the present invention are possible, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims and their equivalents, the invention may be practiced otherwise than as specifically described.
Claims
1-34. (canceled)
35. Method for actuation control of a microscope in which, a first illumination light moves at least in a first direction, and a second illumination light moves at least in a second direction, the first and second illumination lights illuminating a sample through a beam combination, the method comprising the steps of:
- generating at least one part of the first and second illuminating lights by splitting the light from a common illuminating unit, in which the intensity of the first illuminating light, specified by a user or specified automatically, is assigned a higher priority compared to the specified value for the second illumination light, and an adjustment for the second illumination light takes place until a maximum value is obtained, which is determined by the value specified for the first illumination light; and detecting the light coming from the illuminated sample.
36. The method for actuation control of a microscope according to claim 35, further comprising the steps of moving the first and second illumination lights through the sample.
37. The method for actuation control of a microscope according to claim 35, wherein before the adjustment of the second illumination light, a change in the distribution ratio of the light from the illumination unit takes place.
38. The method for the actuation control of a microscope claim 35, wherein, besides the splitting, intensity modulation takes place.
39. The method for actuation control of a microscope according to claim 35, further comprising the steps of providing a first imaging system and a second manipulating system.
40. The method for actuation control of a microscope according to claim 39, whereby the imaging system is chosen from the group consisting of a wide-field microscope, a point scanning, a line scanning microscope, a microscope scanning with point-distribution; and a Nipkow microscope.
41. The method for actuation control of a microscope according to claim 35, wherein the first and second illuminating lights have same wavelengths and are divided.
42. The method for the actuation control of a microscope according to claim 35, wherein common illumination from the illumination lights takes place in the same or different regions of the sample.
43. A light raster microscope comprising:
- a beam combiner for bringing together at least one first illumination light moving in a first direction as well as at least one second illumination light moving in a second direction, for the illumination of a sample;
- at least one detection unit for the detection of the light coming from the sample;
- an illumination unit for the generation of at least one part of the illumination light by means of splitting the light into the first and the second illumination lights;
- an actuation control unit for controlling the splitting of the illumination light of the illumination unit by assigning priority to the intensity of the first illumination light specified by a user or automatically compared to the value specified for the second illumination light and making adjustment for the second illumination light until a maximum value is obtained, the maximum value being determined by the value specified for the first illumination light.
44. The light raster microscope according to claim 43, further comprising means for the adjustment of the intensity of at least the first or the second illumination unit.
45. The light raster microscope according to claim 43, further comprising an imaging first system and a manipulating second system.
46. The light raster microscope according to claim 45, wherein the imaging system is selected from the group consisting essentially of a wide-field microscope, a point-scanning, a line-scanning microscope, a microscope scanning with point-distribution, and a Nipkow microscope.
47. The light raster microscope according to claim 45, whereby the manipulating system is a point-scanner.
48. The light raster microscope according to claim 43, wherein a tunable laser is split into at least two channels.
49. The light raster microscope according to claim 43, wherein before the splitting, combining with at least one or more lasers takes place.
50. The light raster microscope according to claim 43, further comprising means for adjusting the intensity and/or the wavelength and/or the polarization of at least one of the first and second illumination lights.
51. The light raster microscope according to claim 43, further comprising means for optically coupling one of the split illumination canals with another light raster microscope and/or an optical manipulation unit.
52. A light raster microscope system comprising:
- a first light raster microscope; and
- a second light raster microscope and/or an optical manipulation unit, each of the raster microscopes and/or the manipulation unit illuminating a sample simultaneously and/or alternately, wherein the illumination unit from the first and/or the second light raster microscope and/or the manipulation unit is optically split and serves in each case the purpose of illuminating the other light raster microscope and/or the manipulation unit.
53. The light raster microscope system of claim 52 wherein at least one of the first and second raster microscopes and the manipulation unit comprises:
- a beam combiner for bringing together at least one first illumination light moving in a first direction as well as at least one second illumination light moving in a second direction, for the illumination of a sample;
- at least one detection unit for the detection of the light coming from the sample;
- an illumination unit for the generation of at least one part of the illumination light by means of splitting the light into the first and the second illumination lights; and
- an actuation control unit for controlling the splitting of the illumination light of the illumination unit by assigning priority to the intensity of the first illumination light specified by a user or automatically compared to the value specified for the second illumination light and making adjustment for the second illumination light until a maximum value is obtained, the maximum value being determined by the value specified for the first illumination light.
54. Light raster microscope according to claim 52, further comprising optical fibers for optically coupling with the respective second system.
Type: Application
Filed: Apr 6, 2007
Publication Date: Mar 20, 2008
Inventors: Bernhard Zimmermann (Jena), Ralf Netz (Jena), Frank Hecht (Weimar), Joerg-Michael Funk (Jena), Ralf Engelmann (Jena)
Application Number: 11/783,290
International Classification: G02B 21/06 (20060101);