EP1177426A1 - Systemes microscopiques utilises pour le balayage optique d'objets microscopiques - Google Patents

Systemes microscopiques utilises pour le balayage optique d'objets microscopiques

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Publication number
EP1177426A1
EP1177426A1 EP00927164A EP00927164A EP1177426A1 EP 1177426 A1 EP1177426 A1 EP 1177426A1 EP 00927164 A EP00927164 A EP 00927164A EP 00927164 A EP00927164 A EP 00927164A EP 1177426 A1 EP1177426 A1 EP 1177426A1
Authority
EP
European Patent Office
Prior art keywords
detection
microscope system
image
image detection
optical
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.)
Withdrawn
Application number
EP00927164A
Other languages
German (de)
English (en)
Inventor
Thomas LÖRCH
Andreas Plesch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Metasystems Hard and Software GmbH
Original Assignee
Metasystems Hard and Software GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Metasystems Hard and Software GmbH filed Critical Metasystems Hard and Software GmbH
Publication of EP1177426A1 publication Critical patent/EP1177426A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor

Definitions

  • the invention relates to microscope systems for optical scanning of microscopic objects, as described in claims 1 and 8, a corresponding method for optical scanning, as described in claim 9, and the use of the microscope system, as described in claim 1 1 .
  • An automated scanning of an object to be examined (e.g. a slide) is required for a large number of applications in a wide variety of technology areas, particularly in biological and medical research. Conventionally, this is realized with the aid of a motorized microscope in such a way that the slide is moved in the x and y directions, e.g. is meandered under the object and is successively captured by a CCD camera.
  • the speed of the automatic scanning of microscopic specimens is particularly under fluorescence conditions, i.e. using a fluorescence microscope, limited by the following factors:
  • the signal intensities of fluorescence signals from microbiological preparations are sometimes relatively small. In the case of high magnifications in particular, a comparatively long integration or detection time of such signals is necessary for sufficient image quality, which in some cases can be a few seconds.
  • fluorescence signals of different wavelengths must be examined in the same image or detection area of a microscopic object. This is particularly the case with microbiological objects that are prepared with several fluorescent dyes or fluorochromes have been. In such cases, the same detection area must be recorded several times using the microscope filter combination suitable for the respective fluorescent dye. Color cameras that can record three color channels (red R, green G, blue B) at the same time are unsuitable for this, because on the one hand the integrated R, G, B filters do not match the
  • Emission spectra of the fluorescent dyes are adapted, and since it is not possible, on the other hand, to individually control the detection time for the individual color channels. In view of the considerable differences in the fluorescence intensities of, for example, DAPI counterstaining and hybridization signals, this is absolutely necessary in the case of FISH.
  • a motorized object shift table is used for a step-by-step scanning or "scanning" of a microscopic object, then after stopping the object shift table, a settling time which is characteristic of the object shift table must be waited for in order to obtain a motion blur, i.e. to avoid "blurring" of an image to be detected. Such a motion blur would otherwise inevitably result from the conventionally used detection times of CCD cameras.
  • the scanning speed or rate should preferably be limited only by the minimum detection time required for image detection. It is also an object of the invention to provide a corresponding method for optically scanning objects and uses of the device according to the invention.
  • a microscope system for optically scanning objects in particular under fluorescence conditions, comprises at least two preferably electrical, electronic and / or photographic image detection devices which are designed for the simultaneous storage-capable detection of a detection area of the object.
  • the image detection devices preferably share a part of an optical recording system of the microscope.
  • a part of the recording system on the lens side can be used for detection by all image detection devices, whereas parts of the recording system on the side of the image detection devices fall apart.
  • the microscope system according to the invention with the at least two image detection devices has considerable advantages over conventional microscope systems, in particular due to the possible simultaneous and inexpensive detection of the object by the multiple image detection devices.
  • the image detection devices comprise at least one preferably high-resolution CCD camera, in particular so-called megapixel cameras, ie CCD cameras which have more than 10 6 image-resolution pixels.
  • the image detection devices are preferably also designed such that respective detection times can be set independently of one another.
  • the integration or accumulation time of the CCD array can be extended for small intensities to be detected, the readout rate of the CCD being suitably adapted.
  • the detection period, ie the integration or accumulation period, of the CCD can be shortened.
  • This "electronic shutter" of the image detection device usually makes a mechanical diaphragm arrangement for weakening large detection intensities superfluous, which results in a simpler and less expensive solution.
  • a first of the image detection devices detects the detection area in a first object focus plane and a second of the image detection devices the detection area in a second object focus plane, the first and the second object focus plane being offset relative to one another along an optical detection axis of the microscope system.
  • This allows simultaneous detection of the object to be optically scanned in different focal planes, ie different depth layers of the object can be examined.
  • the object or object planes, which are detected in a focused or sharp manner by the at least two image detection devices, are thus spaced apart from one another along the optical detection axis of the microscope system.
  • the storable images or signals obtained by the image detection devices can subsequently be converted into a so-called "extended focus" image or projection image in order to represent the different detected levels of the object, in particular in an image.
  • extended focus image or projection image
  • known algorithms for generating a projection image from images of several depth layers can be used.
  • the first and the second image detection device are spaced from the first object focus plane by different geometric path lengths along the respective optical detection paths.
  • the image detection devices in the beam path of the microscope system can be offset such that the geometric path lengths along the respective detection paths are of different lengths.
  • At least one of the Image detection devices an additional optics for setting the respective object focus level.
  • the additional optics are, in particular, an optical lens or a lens system which is arranged in the detection beam path of the microscope system in such a way that different focus or depth planes of the object are imaged sharply or in a focused manner, in particular even without the displacement of the image detection devices described above.
  • the additional optics are arranged in the optical detection path of only the first image detection device and consequently lie only in the detection beam path between the object to be detected and the first image detection device, while the second image detection device will detect the object without the additional optics.
  • the additional optics are preferably arranged directly in front of at least one of the plurality of image detection devices.
  • a first of the image detection devices is designed for the detection of a first and a second one of the image detection devices for the detection of a second optical wavelength range.
  • This is particularly advantageous for fluorescence applications of a microscope system in which the microscopic object has been prepared with several fluorescent dyes to be detected, which have their emission bands in different optical wavelength ranges.
  • the provision of at least two image detection devices according to the invention thus enables simultaneous detection of the detection range of the object in a plurality of optical wavelength ranges, which results in a noticeable improvement in the scanning speed or rate compared to conventional, sequentially operating microscope systems.
  • the first image detection device comprises a first camera and a dichroic beam splitter and the second image detection device a second camera, the beam splitter being designed and arranged in the detection beam path in such a way that detection beams of the detection range in the first wavelength range are transmitted by the beam splitter onto the first camera and detection beams of the detection range in the second wavelength range by reflection on the beam splitter the second camera fall.
  • This embodiment which is particularly advantageous for fluorescence applications, therefore in principle permits the use of identical cameras, in particular high-resolution CCD cameras, since the wavelength range selection is carried out by the dichroic beam splitter. If emission bands of fluorescent dyes are to be detected in three different wavelength ranges, three cameras with two dichroic beam splitters can be provided. An expansion to more than three cameras is also possible.
  • a microscope system for optically scanning objects comprises at least one object illumination device, at least one image detection device for detecting detection areas of the object within a detection period t detektlon and an object displacement device for displacing the object, the object displacement device is adapted to a displacement of the object at a predetermined speed, so that after a displacement time V ersch i the object eb to a detection area or to a maximum length of the detection area in the direction of displacement is moved, wherein t detektlon a fraction of t strig ⁇ eb is.
  • Such a shortening of the detection period i detect i o ⁇ is particularly advantageous in transmission or transmitted light examinations .
  • Such a microscope system allows accordingly to detect a detection area of the object, even if it should move because any move by the compared to the displacement time t 29 ⁇ eb small detection time t detektlon "frozen" is. In this way, motion blur of a detected image is effectively countered.
  • detection areas of the object can be detected with the maximum mechanically possible displacement rate of the object displacement device (typically about 10 detection areas or positions per second) without having to wait for the settling time of the object or object displacement device.
  • a possibly reduced intensity of the image signal detected by the image detection device can often be increased again by a correspondingly increased illumination level.
  • the microscopic object can be moved continuously (for example uniformly at a constant speed), as a result of which there is no longer any need to wait for a settling time of the object displacement device and for it to accelerate and slow down in comparison to conventional microscope systems.
  • An increased scanning speed of the object can therefore advantageously be achieved.
  • the detection time period t detektlon ⁇ t 29 ⁇ eb / 500 preferably t detektlon ⁇ t 29 ⁇ eb / 1 000.
  • the detection time period t detektl0n is preferably less than 1 msec, preferably less than 0.1 msec.
  • the object displacement devices are designed for at least two-dimensional displacement of the object perpendicular to the optical axis of the microscope system (x and y direction).
  • the object displacement device also enables the object to be displaced along the optical axis (z direction), which is particularly advantageous for the automated scanning of "thick" objects.
  • the object displacement device comprises an object displacement table and an electrical displacement motor which displaces it.
  • the microscope system according to the invention is provided with a control device which is in signal connection with the object illumination device, the image detection device and the object displacement device and controls an automatic (ie unattended) optical scanning of an area of the object comprising several detection areas or image fields.
  • a control device which is in signal connection with the object illumination device, the image detection device and the object displacement device and controls an automatic (ie unattended) optical scanning of an area of the object comprising several detection areas or image fields.
  • a method for optical scanning of objects is preferably provided by means of a microscope system as defined above, wherein a detection area of the object is simultaneously detected with at least two image detection devices in a storable manner.
  • the image detection devices simultaneously detect the detection area in at least two different object focus planes, which are offset along an optical detection axis of the microscope system, and / or the detection area in at least two optical wavelength ranges, which differ at least in certain areas.
  • the microscope system according to the invention and the method according to the invention can preferably be used in all applications in which the automatic scanning or scanning of microscope specimens and the (at least partially) automatic evaluation of objects of a certain type are important. Such requirement profiles can be found particularly in medical research.
  • a preferred use of the device according to the invention relates to the detection of labeled, in particular fluorescence-labeled biological material in a sample.
  • biological material encompasses all biological material and the substances that make up this material, for example viruses, viroids, multicellular and unicellular organisms, in particular cells, for example bacteria, cells in tissue association or culture cells, their components such as cell organelles, for example cell nuclei, mitochondria, plastids , Endoplasmic reticulum (ER), dictyosomes, ribosomes etc., genetic material such as chromosomes and their components, nucleic acids such as DNA and RNA, proteins such as structural proteins, enzymes and antibodies, polysaccharides and lipids.
  • a preferred example of the use defined above relates to the search for "rare” events, such as the search for little fluorescent-labeled biological material to be found in a sample.
  • the term "rare event” means that the ratio of unlabelled to the labeled biological material in the sample is more than 10 4 , in particular more than 10 6 .
  • the ratio of unlabelled to the labeled biological material in the sample can be approximately 10 8 in prenatal diagnosis and approximately 10 6 in tumor diagnosis.
  • the label comprises one, more preferably several, different fluorescent labels.
  • Preferred fluorescent labels are, for example, fluorescent dyes of the Cy family, fluorescein isothiocyanate (FITC), nucleic acid-binding fluorescent dyes such as DAPI (4 ', 6'-diamidine-2'phenylindole dihydrochloride) and Hoechst 33342 etc.
  • the biological material to be detected can be directly involved in this be labeled with a specific dye, for example the DNA in a cell nucleus using DAPI, and / or being labeled indirectly, for example using one or more differently specific fluorescence-coupled nucleic acid probes (fluorescence - // 7-s / Yü hybridization, FISH) and / or by means of fluorescence-coupled antibodies, which can be monoclonal or polyclonal.
  • a specific dye for example the DNA in a cell nucleus using DAPI
  • FISH fluorescence- // 7-s / Yü hybridization
  • the use defined above is the search of a few stained cells of a certain phenotype in a large cell population.
  • the cells to be detected can be selectively labeled, for example with the aid of a fluorescence-coupled antibody.
  • a fluorescence-coupled antibody In general, however, such a label is not absolutely specific, ie cells are also stained that do not have the desired phenotype (false positive cells).
  • several independent antibodies directed against the same cell type and coupled with different fluorescent dyes can be used simultaneously.
  • false positive results can also be obtained through the additional analysis of morphometric parameters, for example the shape and / or area of a counter-staining of the cell nucleus with the aid of a nucleic acid-binding fluorescent dye (eg DAPI) can be recognized and eliminated.
  • morphometric parameters for example the shape and / or area of a counter-staining of the cell nucleus with the aid of a nucleic acid-binding fluorescent dye (eg DAPI) can be recognized and eliminated.
  • Preferred applications of such a search for "rare" events relate, for example, to tumor diagnostics such as the search for micrometastases in blood or bone marrow or the monitoring of the course of tumor diseases, the ratio of unlabeled to marked cells often being in the range of about 1 0 6 , as well as the identification of fetal cells circulating in the mother's blood in the non-invasive genetic prenatal diagnosis, the ratio of unlabelled to labeled cells often being in the range of about 10 8 .
  • Another example of a preferred use of the microscope system according to the invention and the method according to the invention relates to the counting of FISH signals or FISH spots in a large number of cell nuclei. It is necessary here to record images from several focal planes in different fluorescence channels and then to convert them into an “extended focus” image or projection image as described above in order to record the signals from different planes of the three-dimensional nuclei.
  • the projection images obtained in this way are then used to determine the number of fluorescence signals, for example to determine deviations in the number of chromosomes.
  • the distance between FISH signals from different fluorescent dyes can be determined.
  • translocation 9/22 Philadelphia chromosome
  • CML chronic myeloid leukemia
  • FIG. 1 is a schematic view of an embodiment of a microscope system according to the invention.
  • FIG. 2 shows a schematic basic arrangement of an embodiment of a microscope system according to the invention with an afocal relay system designed as an illumination manipulator;
  • FIG. 3 shows a schematic view of an embodiment of a microscope system with three image detection devices, which are controlled via wavelength-sensitive, dichroic beam splitters.
  • the structure of a microscope system 10 is shown schematically in FIG.
  • the microscope system comprises an object displacement table 1 2 which can be displaced in all spatial directions and which is provided with an electrical displacement motor (not shown).
  • the displacement motor and the object displacement table 1 2 form an object displacement device.
  • the microscopic object to be examined is fixed on the object shift table.
  • the microscope system 10 has two object illumination devices, a transmitted light illumination device 14 and a second microscope-side illumination device (EPI illumination) 16 being provided.
  • the detection light is collected by a lens 1 8 and fed via a lens system to a CCD camera 20, which serves as a first image detection device.
  • the detection area of the object to be examined can also be directly viewed visually via an eyepiece 22 by means of a swivel mirror that can be folded into the beam path or a beam splitter.
  • the object lighting devices (14, 1 6), not shown, the displacement motor of the object table 1 2 and the CCD camera 20 are connected to an external control device, not shown.
  • the CCD camera For example, 20 has a chip resolution of 1 280x1 024 pixels and can typically be read out within a few msec become.
  • the possible pulsed illumination by the object illumination device 1 6 can result in a substantial reduction in the integration time or detection time which is effectively required, since the required light energy can be obtained in a much shorter time than can be transferred to the specimen during the continuous output of a conventional fluorescent lighting.
  • the shifting time period t shift is the time during which the object must be shifted in its shifting direction by the object shifting table 12 in order to be shifted in the shifting direction by a detection area or by the maximum length of the detection area.
  • the microscope system 10 for optical scanning of (microscopic) objects or specimens preferably comprises at least one object illumination device 14, 16, at least one image detection device 20 for detecting detection areas of the object and an object displacement device 12 for displacement of the object, the object displacement device 1 2 being designed for displacement of the object at a predetermined speed, so that after a displacement Time period t shifts the object by a detection area or by the maximum detection area length in the direction of displacement, and the object lighting device 14, 16 is designed to generate object lighting pulses of the (respective) time period t pu , s , with t pu , s a fraction of t strig ⁇ eb is.
  • the detecting portion has for example a rectangular shape
  • the object shifter 1 2 is adapted to the object direction within the time ⁇ eb ersch i to the maximum length of the rectangular detection region in shift to shift. If the direction of displacement runs parallel to a side edge of the detection area, for example, this maximum length corresponds to this edge length. Since the time period t pulse of the object illumination pulse is only a fraction of this shifting time period t displ ⁇ eb , a high-quality image detected by the image detection device 20 is obtained even if the object should move during the detection. Accordingly, the pulsed lighting (similar to flash or stroboscopic lighting) effectively prevents the motion blur or a “risk of blurring” of an image to be detected.
  • the duration of the object lighting pulses is preferably t pu
  • S of the object illumination pulses is preferably chosen to be less than 1 msec, preferably less than 0.1 msec.
  • a method for the optical scanning of objects, in particular under fluorescence conditions by means of a (preferably according to the invention) microscope system which comprises at least one object illumination device, at least one image detection device for the detection of detection areas of the object and an electrically driven object displacement device for displacing the object following steps in that order:
  • the continuous displacement of the object by the electrical object displacement device 1 2 is preferably a uniform displacement movement at a constant speed, but the object can also move more slowly at the time of the pulse illumination or be at a standstill. Since the pulse duration t pu , s is only a fraction of t 29 ⁇ eb , a high-quality, time-integrated image signal can also be obtained by the image detection device 20 if the object moves during the detective integration of the image, without any noticeable impairment due to motion blur would result. Consequently, the method advantageously allows optical scanning of objects with an increased sampling rate, since there is no need to react to the problem of settling or settling of the object shifting device 1 2 or other problems of motion blur by disadvantageously waiting for a characteristic settling time.
  • T pulse is preferably less than or equal to t Versch , eb / 500, preferably t Versch ⁇ eb / 1000 .
  • an additional object moving step (d) is preferably carried out, which in particular can have a different object moving speed and direction.
  • Such an additional object moving step can be advantageous if the detection areas of the Object should not directly adjoin one another in the direction of displacement, but should be spaced apart by undetected regions of the object. This is particularly necessary when a "spot-like" detection of distributed detection areas of an object is required.
  • the direction of displacement can be reversed so that larger objects can be meandered scanned.
  • method steps (a) to (c) or (a) to (d) can be repeated a predetermined number of repetitions, in order in this way to automatically recognize a large number of detection areas of the object.
  • the motion blur can also be reduced effectively by shortening the detection period t detetlon .
  • the detection time period t detektlon ie the time period during which the incident light is collected on the CCD chip, can be set to, for example, 0. Shorten 1 msec.
  • a possibly reduced CCD detection signal strength can often be raised again by a correspondingly increased lighting level.
  • the very short detection time allows an image to be taken before the stage 1 2 has swung or settled and is therefore stable, since, as with pulse lighting, any residual movement is "frozen" to a certain extent.
  • the image can be recorded at the maximum shift frequency determined by the object table 12 (typically approximately 10 detection areas or positions per second) without having to wait for the object table 12 to settle.
  • FIG. 2 schematically shows the arrangement of an illumination manipulator 24 in the illumination beam path of the microscope system.
  • control light directed parallel to the optical axis of the microscope system is generated by a light source 28 which is installed in a lamp house 30.
  • the control light strikes a first converging lens 32 of the lighting manipulator 24, which is designed as an afocal relay system.
  • the Collecting lens 32 focuses the parallel light beam into a focal point on the optical axis of the microscope system, which is at a distance f 2 from the main plane of the collecting lens 32.
  • This focal point coincides with the focal point of a second converging lens 34 of the illumination manipulator 24, which in turn subsequently generates a light bundle parallel to the optical axis, which is directed further towards the microscope. Since the focal length i of the converging lens 34 is smaller than the focal length f 2 of the converging lens 32, the original excitation beam diameter D 2 is compressed to the diameter D 1, where 0 ⁇ 0 2 ⁇ .
  • a microscope system 10 for optically scanning objects can include at least one object illumination device 28, at least one first image detection device for detecting a first detection area of the object and a second image detection device for detection of a second detection area, which essentially comprises the first detection area, the object illumination device 28 comprising an illumination manipulator 24 for selectively illuminating the first and / or the second detection area.
  • the lighting manipulator 24 accordingly makes it possible to selectively illuminate the entire second detection area or only the first detection area.
  • the first image detection device can comprise a CCD camera, the second image detection device an eyepiece for direct optical viewing and the lighting manipulator 24 an afocal relay system or a beam compressor.
  • the illumination manipulator 24 accordingly allows the small (first) detection area of the CCD camera to be illuminated selectively without simultaneously having to also illuminate the surrounding or other parts of the (second) detection area visible through the eyepiece of the microscope system.
  • the use of an afocal relay system or a beam compressor thus advantageously enables the excitation lighting to be concentrated on the (first) detection area which is decisive for detection with the CCD camera.
  • this embodiment advantageously avoids unnecessarily rapid bleaching of the fluorescent dyes by an unnecessarily large illuminated image field.
  • Another solution to adapt the camera image to the illuminated surface of the object and thus to optimally use the excitation light is to use a camera adapter that reduces the factor M.
  • the image area captured by the camera is enlarged by the same factor M.
  • the discrepancy between the illuminated eyepiece image field and the camera image field is reduced accordingly.
  • the (larger) eyepiece field remains fully illuminated.
  • FIG. 3 schematically shows part of the beam path of a further embodiment of the microscope system according to the invention.
  • three different and separately controllable image detection devices are used, which are designed as first 40, second 42 and third 44 CCD cameras.
  • An object lighting device 46 which consists of a light source and a lens system, generates incident light illumination on a microscopic object, not shown, by means of a first beam splitter 48.
  • the first beam splitter 48 is designed as a three-band beam splitter (triple band beam splitter), which has a high transmission in the optical wavelength ranges FF 2 and F 3 having.
  • the beam splitter 48 can be used, for example, for the simultaneous excitation of a plurality of fluorescent dyes or fluorochromes in these wavelength ranges.
  • Two further beam splitters 50, 52 are arranged in the detection or detection beam path of the microscope system, which distribute the light to be detected from the detection area of the object to the CCD cameras 40, 42 and 44.
  • the detected light is spectrally split by using dichroic table beam splitters.
  • the beam splitter 50 has a large transmission coefficient in the wavelength range F 1, but effectively reflects the wavelength range F 2 and F 3 .
  • the second dichroic beam splitter 50 has a high transmission coefficient in the wavelength range F 3 and a large reflection coefficient in the wavelength range F 2 .
  • light to be detected in the wavelength range F 2 falls on the second CCD camera 42, while light to be detected in the wavelength range F 3 falls on the third CCD camera 44.
  • the use of a single, spectrally sensitive image detection device is superior to the multi-channel recording according to the invention with different image detection devices.
  • the detection parameters of the image detection devices in particular the integration time, can be set independently of one another. This is particularly advantageous for the detection of individual fluorochromes in the fluorescence microscopy of biological material, since the fluorescence intensities of the individual fluorochromes often differ by orders of magnitude.
  • the optical wavelength ranges that are to be detected can be individually determined depending on the desired task, while they are predefined in a color camera.
  • high-resolution CCD cameras can be used, the number of pixels of which clearly exceeds that of color cameras.
  • the detection described in the different focal planes can be carried out with a microscope system, which is shown schematically in FIG. 3. If no additional simultaneous recording of several color channels is to take place, as was described above, color-neutral beam splitters 50, 52 are used, which suitably distribute the detection light to the CCD cameras 40, 42 and 44 without color splitting. In particular, the beam splitters 50, 52 split the optical input signal into two beams with typically 50% of the input signal strength.
  • the geometrical path lengths along the respective detection paths from the respective CCD cameras 40, 42 and 44 to an object plane can be selected to be of different lengths. In FIG.
  • the CCD camera 40 has a shorter geometric path length to the object than that of the CCD cameras 42 and 44.
  • the geometric distance along the respective detection paths from the beam splitter 50, at which the common detection beam path of the various CCD cameras is divided, to the CCD camera 40 is shorter than to the CCD cameras 42 and 44.
  • the CCD camera 40 sharpens a focal plane of the object or focused, which will differ slightly from the focus plane, which is imaged by the cameras 42 and 44.
  • additional optics (not shown) can also be used in front of at least one of the CCD cameras in order to thus determine the desired focal plane.
  • the analog camera outputs of the CCD cameras 40, 42 and 44 can each be connected to signal inputs of a digitizing device or module (not shown) which is designed for multiplex processing of analog camera signals.
  • a digitizing device or module (not shown) which is designed for multiplex processing of analog camera signals.
  • Such multiplex processing of the individual camera signals advantageously enables the cost-effective use of a single digitizing module for several image detection devices.
  • the signals from the CCD cameras cannot be read out and digitized at the same time, but this does not imply any practical limitation.
  • the readout time of a CCD camera with a value of typically 0.1 seconds is generally shorter than typical detection times.
  • the detection times for different fluorescent dyes are of different lengths, so that the CCD camera signal of the stronger fluorescent dye can already be read out, while the other CCD camera still detects and integrates the weaker signal.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

La présente invention concerne un système microscopique utilisé pour le balayage optique d'objets, notamment dans des conditions de fluorescence. Ce système comprend au moins deux dispositifs de détection d'image (40, 42, 44) qui sont de préférence électriques, électroniques et/ou photographiques et qui sont conçus pour la détection simultanée à capacité de stockage d'une zone de détection de l'objet.
EP00927164A 1999-05-07 2000-05-08 Systemes microscopiques utilises pour le balayage optique d'objets microscopiques Withdrawn EP1177426A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19921127 1999-05-07
DE1999121127 DE19921127A1 (de) 1999-05-07 1999-05-07 Mikroskopsysteme zur optischen Abtastung von mikroskopischen Objekten sowie Verfahren zur optischen Abtastung
PCT/EP2000/004115 WO2000068667A1 (fr) 1999-05-07 2000-05-08 Systemes microscopiques utilises pour le balayage optique d'objets microscopiques

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