EP1266197A2 - Method and arrangement for optical detection of characteristic variables of the wavelength-dependent behaviour of an illuminated sample - Google Patents
Method and arrangement for optical detection of characteristic variables of the wavelength-dependent behaviour of an illuminated sampleInfo
- Publication number
- EP1266197A2 EP1266197A2 EP01947389A EP01947389A EP1266197A2 EP 1266197 A2 EP1266197 A2 EP 1266197A2 EP 01947389 A EP01947389 A EP 01947389A EP 01947389 A EP01947389 A EP 01947389A EP 1266197 A2 EP1266197 A2 EP 1266197A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- detection
- arrangement according
- fluorescence
- arrangement
- channels
- 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- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/0016—Technical microscopes, e.g. for inspection or measuring in industrial production processes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
- G01Q60/18—SNOM [Scanning Near-Field Optical Microscopy] or apparatus therefor, e.g. SNOM probes
- G01Q60/20—Fluorescence
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/008—Details of detection or image processing, including general computer control
Definitions
- the invention relates to a method and an arrangement in fluorescence microscopy, in particular laser scanning microscopy, fluorescence correlation spectroscopy and scanning near-field microscopy, for examining predominantly biological samples, preparations and associated components. Also included are methods for screening active substances based on fluorescence detection (high throughput screening).
- fluorescence detection high throughput screening.
- the transition from the detection of a few broad spectral dye bands to the simultaneous recording of complete spectra opens up new possibilities for the identification, separation and assignment of the mostly analytical or functional sample properties spatial substructures or dynamic processes. Simultaneous examinations of samples with multiple fluorophores are thus possible with overlapping fluorescence spectra even in spatial structures of thick samples.
- the arrangement does not reduce the data acquisition rate.
- the irradiated photons of a certain energy excite the dye molecules through the absorption of a photon from the ground state into an excited state.
- This excitation is usually referred to as single-photon absorption (Fig. 1a).
- the dye molecules excited in this way can return to the ground state in various ways.
- fluorescence microscopy the transition with the emission of a fluorescence photon is most important.
- the wavelength of the emitted photon is generally red-shifted due to the Stokes shift compared to the excitation radiation, so it has a longer wavelength. The Stokes shift enables the fluorescence radiation to be separated from the excitation radiation.
- the fluorescent light is split off from the excitation radiation with suitable dichroic beam splitters in combination with block filters and observed separately. This makes it possible to display individual cell parts colored with different dyes. In principle, however, several parts of a preparation can also be colored simultaneously using different specific dyes (multiple fluorescence). To distinguish between the fluorescence signals emitted by the individual dyes, special dichroic beam splitters are used.
- the dye emission is not influenced by this type of excitation, ie that The emission spectrum experiences a negative Stokes shift with the multi-photon absorption option, ie it has a shorter wavelength compared to the excitation radiation.
- the separation of the excitation radiation from the emission radiation takes place in the same way as with the single-photon absorption.
- LSM confocal laser scanning microscope
- An LSM is essentially divided into 4 modules: light source, scan module, detection unit and microscope. These modules are described in more detail below. Reference is also made to DE19702753A1.
- lasers with different wavelengths are used in an LSM. The choice of the excitation wavelength depends on the absorption properties of the dyes to be examined.
- the excitation radiation is generated in the light source module.
- Various lasers are used here (argon, argon krypton, TiSa laser).
- the wavelengths are selected and the intensity of the required excitation wavelength is selected in the light source module, for example by using an acousto-optical crystal.
- the laser radiation then arrives in the scan module via a fiber or a suitable mirror arrangement.
- the laser radiation generated in the light source is focused into the specimen with the aid of the objective (2) with limited diffraction via the scanner, the scanning optics and the tube lens.
- the focus raster scans the sample in the xy direction.
- the pixel dwell times when scanning over the sample are usually in the range of less than a microsecond to a few seconds.
- the light which is emitted from the focal plane (specimen) and from the planes above and below it passes via the scanner to a dichroic beam splitter (MDB).
- MDB dichroic beam splitter
- the fluorescent light is then focused on a diaphragm (confocal diaphragm / pinhole) which is located exactly in a plane conjugate to the focal plane. This suppresses fluorescent light components outside of the focus.
- the optical resolution of the microscope can be adjusted by varying the aperture size. Behind the aperture is another dirchroic block filter (EF) which suppresses the excitation radiation again. After passing the block filter, the fluorescent light is measured using a point detector (PMT).
- PMT point detector
- the excitation of the dye fluorescence takes place in a small volume at which the Excitation intensity is particularly high. This area is only marginally larger than the detected area when using a confocal arrangement.
- the use of a confocal diaphragm can thus be omitted and the detection can take place directly after the lens (non-descanned detection).
- a descanned detection still takes place, but this time the pupil of the objective is imaged in the detection unit (non-confocal descanned detection).
- the LSM is therefore suitable for examining thick specimens.
- the excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic filters matched to the emission properties of the dye ensure that only the fluorescent light emitted by the respective dye is measured by the point detector.
- the fluorescent light is switched off using a
- Prism spectrally split The method differs from the arrangement described above with dichroic filters only in that the filter used can be adjusted in its characteristics. However, the emission band of a dye is preferably recorded for each point detector.
- the previous detection devices reach their limits.
- the spectral detection range must be restricted. The area in which the two dyes overlap is simply cut out and not detected. The efficiency of the detection unit thus deteriorates.
- the same signal-to-noise ratio can only be achieved by increasing the excitation power, which can damage the specimen. For this reason, up to 6 different dye probes are used simultaneously at the same time, otherwise the dyes cannot be separated due to the strongly overlapping emission bands.
- dyes have been modified so that they differ from one another either in their absorption properties or in their emission properties.
- 3a shows the emission spectra of various typical dyes. The emission signal is plotted as a function of the wavelength. It can be seen that the dyes labeled 1 to 4 differ in the position and shape of their emission spectra. In most cases, however, these dyes are toxic to live preparations. This means that studies on the evolution of cell aggregates in living preparations are impossible.
- natural dyes the so-called fluorescent proteins (GFP, YFP, CFP, TOPAS, GFT, RFP) were discovered (company: Clonetech. USA). These dyes are characterized by a low sample influence.
- 3b) shows the emission signals as a function of the wavelength for the dyes GFP, Topas, GFT and Cyan-FP.
- both proteins are labeled with different dyes, the emission spectrum of the first dye being superimposed on the absorption spectrum of the second dye.
- the first dye is then excited with a suitable wavelength to fluoresce. If the two molecules are very close to one another ( ⁇ 10 nm), the emission radiation of the first dye can be absorbed by the second, whereby the second dye and not the first dye subsequently emits.
- 3d) shows the energy term scheme for this process, which is called Fluorescence Resonant Energy Transfer (FRET) in the literature (Lit .: Fan et al .; Biophysical Journal, V 76, May 1999, P 2412-2420).
- FRET Fluorescence Resonant Energy Transfer
- the distance between the two molecules can be determined.
- the emission spectrum of a dye which is in a biological preparation can differ from the emission spectrum measured in a dye cell.
- 3c) shows the emission spectra of a dye as a function of the environment in which the dye is located.
- the emission signal is plotted as a function of the wavelength.
- the wavelength shift can be up to several 10 nm.
- the ion concentration (eg: Ca + , K + , Mg 2+ , ZN + , ...) is determined, in particular in biological preparations.
- special dyes or dye combinations eg Fura, Indo, Fluo; Molecular Probes, Inc.
- Fura, Indo, Fluo; Molecular Probes, Inc. which have a spectral shift depending on the ion concentration, have ion concentration.
- Fig. 4a) shows the emission spectra of lndo-1 depending on the concentration of calcium ions.
- Fig. 4b) shows an example of the emission spectra as a function of the calcium ion concentration when using the combination of FLuo-3 and Fura Red dyes.
- These special dyes are called emission ratio dyes.
- the invention therefore relates to new methods for flexible and freely programmable detection. These methods should be used in imaging as well as in analytical microscopy systems. Therefore may the data acquisition rate will not deteriorate when using these methods.
- the microscope systems are imaging systems such as laser scanning microscopes for three-dimensional examination of biological specimens with an optical resolution of up to 200 nm, scanning near-field microscopes for high-resolution examination of surfaces with a resolution of up to 10 nm. Fluorescence correlation microscopes for quantitative Determination of molecular concentrations and for the measurement of molecular diffusions. Furthermore, methods for screening dyes based on fluorescence detection are included. In all of the above systems, fluorescent dyes are used for the specific labeling of the preparations.
- the background of the method for flexible detection is a spectrally split detection of fluorescence.
- the emission light is split off from the excitation light in the scan module or in the microscope (in the case of multi-photon absorption) using the main color splitter (MDB).
- MDB main color splitter
- a block diagram of the following detector unit is shown in Fig. 5.
- the light of the sample is now focused by means of an imaging optics PO with confocal detection through an aperture (pinhole) PH, whereby fluorescence that has arisen out of focus is suppressed.
- the aperture is omitted for undescanned detection.
- the light is now broken down into its spectral components using an angle-dispersive element DI.
- Prisms, gratings and acousto-optical elements come into question as angle-dispersive elements.
- the light split by the dispersive element into its spectral components is subsequently imaged on a line detector DE.
- This line detector DE therefore measures the emission signal as a function of the wavelength and converts this into electrical signals S ().
- a line filter for suppressing the excitation wavelengths can be connected upstream of the detection unit.
- FIG. 6 A possible embodiment of the optical beam path of the detector unit shown in Fig. 5 in the block diagram is shown in Fig. 6.
- the structure essentially describes a Cerny Turner structure.
- the light L of the sample is focused with the pinhole optics PO through the confocal aperture PH. This aperture can be omitted in the case of undescanned detection in the case of multiphoton absorption.
- the first imaging mirror S1 collimates the fluorescent light. Then the light hits a line grating G, for example a grating with a number of lines of 651 lines per mm. The grating bends the light in different directions according to its wavelength.
- the second imaging mirror S2 focuses the individual spectrally split wavelength components on the corresponding channels of the line detector DE.
- the use of a line secondary electron multiplier from Hamamatsu H7260 is particularly advantageous.
- the detector has 32 channels and high sensitivity.
- the free spectral range of the embodiment described above is approximately 350 nm. In this arrangement, the free spectral range is evenly distributed over the 32 channels of the line detector, resulting in an optical resolution of approximately 10 nm. This arrangement is therefore only conditionally suitable for spectroscopy. However, their use in an imaging system is advantageous since the signal per detection channel is still relatively large due to the relatively broad spectral band detected.
- the free spectral range can also be shifted by rotating the grating, for example.
- Another possible embodiment could involve the use of a matrix detector (e.g. a CCD, ).
- the dispersive element splits into different wavelength components in a coordinate.
- a complete line (or column) of the scanned image is imaged in the remaining direction on the matrix detector.
- This embodiment is particularly advantageous when building a line scanner (Lit: Corle, Kino; "Confocal Scanning Optical Microscopy and Related Imaging Systems”; Academic Press 1996).
- the basic structure corresponds essentially to that of an LSM according to Fig. 2. However, instead of one Point focus, a line is mapped into the focus and the sample to be examined is only scanned in one direction.
- a slit diaphragm is used as the confocal diaphragm instead of a pinhole diaphragm. Detection using a multi-photon absorption can also be carried out with this arrangement again the confocal aperture can be omitted.
- the algorithm determines the position of the center of gravity or maxima of the emission signal detected in the pixel for each pixel.
- the following is a advantageous possible way of determining the focus is described in more detail.
- Other types of determining the center of gravity or maxima, such as interpolation fits, etc. are part of the invention without restriction.
- the signals per channel (left diagram) detected by the line detector are multiplied by a calibration function (right diagram), ie each channel is given a specific weighting.
- the diagram on the left in Fig. 7 shows an example of a measured emission signal depending on the channel number in which the signal was detected.
- the diagram on the right shows an example of a weighting function for the corresponding individual channels depending on the channel number.
- Fig. 8a shows the position signal depending on the position of the center of gravity or maximum of the detected emission spectrum.
- the position signal By measuring the position signal, different dyes can be distinguished based on their position and type of emission spectra. Furthermore, when using a dye, for example, the wavelength shift of the emission spectrum, which is dependent on the environment, can be measured.
- Position /, Po k • C k , k
- Pos k is the characteristic position of the center of gravity of a dye
- C k is the concentration of a dye
- n is the number of dyes simultaneously excited in the pixel.
- the algorithm can thus also determine ion concentrations and n for the detection of an FRET signal be used.
- an analysis of the local overlay of 2 or more dyes is possible (colocalization measurement)
- the signal dependent on the ion concentration when using 2 dyes e.g. Fluo-3 and Fura Red, Molecular Probes, Inc.
- a dye with 2 characteristic emission bands e.g. Indo, Molecular Probes, Inc.
- the position signal is plotted as a function of the ion concentration.
- the position signal is a measure of the position of the center of gravity of the emission spectrum. It can therefore serve as a mask for calculating a color-coded intensity image.
- the algorithm is shown schematically in Fig. 9.
- the mask i.e. the position signal
- the lookup table contains the corresponding color assignment depending on the position of the center of gravity of the emission spectrum.
- the result of this multiplication is then multiplied by the intensity value (the sum signal), that is to say the brightness of the color is adapted to the actual fluorescence intensity.
- the intensity value the sum signal
- color-masked intensity images discretrete color distribution
- intensity images with mixed colors can be created by combining the individual pixels into one image.
- the decisive advantage of the method is that the entire fluorescence of each dye (the sum signal) can be detected regardless of the degree of overlap of the emission spectra, and the dyes can still be displayed separately (by the position signal). Highly overlapping dyes (Fig. 3c) can thus be detected particularly efficiently.
- Fig. 10 An arrangement for digital calculation of the sum and position signal is shown schematically in Fig. 10.
- the current flowing at the anodes of a multi-channel PMT is converted into one by the first amplifier A (switched as a current-voltage converter) Tension changed and strengthened.
- the voltage is fed to an integrator I which integrates the signal over a corresponding time (eg pixel dwell time).
- the integrator I can be followed by a comparator K which, as a simple comparator, has a switching threshold which generates a digital output signal when exceeded or which is designed as a window comparator and then forms a digital output signal when the input signal is between the upper and lower ones Switching threshold or if the input signal is outside (below or above) the switching thresholds.
- the comparator or the window comparator can be arranged both before and after the integrator. Circuit arrangements without an integrator (so-called amplifier mode) are also conceivable. When arranged in the amplifier mode, the comparator K is also arranged after corresponding level adjustment.
- the output of the comparator K serves as a control signal for a switch register Reg, which switches the active channels directly (online) or the status is communicated to the computer via an additional connection V in order to make an individual selection of the active channels (off-line ).
- the output signal of the integrator I is directly another ampl. A1 for level adjustment, for the subsequent A / D conversion AD.
- the AD converted values are transmitted via suitable data transmission to a computer (PC or digital signal processor DSP), which carries out the calculation of the position signal and the sum signal according to Figs. 7 and 9.
- Fig. 10 is shown in Fig. 11.
- the signals of the individual channels are in turn transformed with an amplifier into voltage signals.
- the individual voltage signals are then integrated in an integrator I during the pixel dwell time.
- a comparator K which compares the integrated signal with a reference signal, is connected downstream of the integrator.
- the comparator actuates a switch S via Reg and the individual channel is switched off for the pixel just measured. With the aid of the comparators in combination with the switches, the spectral range relevant for the pixel currently being measured is automatically selected.
- the integrated voltage signal is then converted into a current again using a resistor R. Each individual channel thus generates a current which is dependent on the fluorescence intensity impinging on the individual channel. All adjacent individual channels are then connected to another resistor R1 located between them.
- the resulting total current at the upper and lower end of the detector line is in turn converted into a voltage using a current-voltage converter A1.
- the voltage at the upper and lower ends Eo and Eu correspond to the sum of the signals of the individual channels weighted with opposite straight lines.
- the two signals at the top and at the bottom are summed in the following with a summation amplifier SV.
- the resulting signal corresponds to the sum signal of the entire measured fluorescence.
- This sum signal and the signal from the upper end or the signal from the lower end (shown in broken lines) are fed to an analog divider, which forms the position signal at the output.
- the sum and position signals are then converted into digital signals with an analog-digital converter and processed by the computer or DSP.
- the upper and lower signals can also be converted without restriction and processed by the computer.
- the computer would determine the sum signal and the position signal.
- the algorithm according to Fig. 9 is carried out on the computer.
- the algorithm according to Fig. 9 can also be implemented in the circuit according to Fig. 11. Three options are explained in more detail below.
- the multiplication with the lookup table is carried out by changing the Resistors (R1) located between the adjacent single detection channels. The rest of the circuit remains as described above.
- the multiplication is done with the lookup table in the amplifier (A1).
- the amplifier A1 is operated with a variable non-linear characteristic.
- a third arrangement digital (according to Fig. 10) and analog detection (according to Fig.
- the input signals of the individual detection channels are manipulated or distorted by: a change in the gain of (A), a change in the integration times of (I ), by feeding an additional offset in front of the integrator and / or by digitally influencing the counted photons in a photon counting arrangement. All 3 methods can also be combined with one another as desired.
- the excitation light backscattered from the sample or at least to attenuate it so much that it is smaller than or in the same order of magnitude as the emission maximum in a fluorescence measurement.
- the additional line filter described above or a correspondingly optimized main color splitter (MDB) can be used for optical attenuation. Since the spectral width of the excitation laser radiation is much smaller than the bandwidth detected by the individual channel, the backscattered or reflected excitation radiation can also be achieved by specifically switching off the corresponding individual channel using the switch shown in Fig. 11.
- the arrangement according to Fig. 11 has several advantages over the arrangement according to Fig. 10.
- the most noticeable advantage is that only 2 channels need to be converted to digital data and sent to the computer. This minimizes the data rates to be processed by the computer. This is particularly important when using the method in real-time microscopy, in which, for example, more than 50 images with 512x512 pixels and 12 bit pixel depth have to be detected in order to be able to register the extremely fast-running dynamic processes.
- this method is furthermore not limited to the number of individual channels of the line detector (matrix detector) used and thus to the size of the detectable spectral range and / or the spectral resolution of the spectral sensor.
- the signal levels to be converted are significantly smaller. This means that the signal to noise ratio to be expected is lower.
- Fig. 12 shows measurements made with the arrangements shown in Figs. 10 and 11.
- Fig. 12a shows the emission spectra of the dyes GFP, CFP and DI measured with a spectrometer. The dyes were excited with an argon laser with a wavelength of 488 nm. These dyes were subsequently brought specifically to specific regions in a biological preparation.
- Fig. 12b shows a histogram of the position signal when scanning over a sample section where all 3 dyes are located. The 3 maxima in the histogram can be clearly recognized by the 3 dyes having their characteristic position signal. The positions for the 3 dyes are listed in the following table:
- the dyes should therefore be easily separated using the arrangements according to the invention.
- local wavelength shifts are visible due to the different local environments within a dye. This manifests itself in the width of the maxima for the individual dyes in the histogram.
- Fig. 13a shows the intensity image formed from the sum signals.
- Fig. 13b is the corresponding image formed from the position signals. This image embodies the corresponding focus of the emission spectra.
- the differently stained cell nuclei (partly stained with GFP, partly stained with CFP) and cell stains stained with DI are clearly distinguishable.
- Fig. 13c shows the color-coded intensity image calculated according to the algorithm in Fig. 9. The individual regions to which different dyes have accumulated are now separated by the color coding. The separation is illustrated by the representation of an image in its 3 RGB channels. For comparison, an image measured by means of a detection according to the prior art is also shown in Fig. 13d.
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10033180A DE10033180B4 (en) | 2000-06-29 | 2000-06-29 | Method for the detection of dyes in fluorescence microscopy |
DE10033180 | 2000-06-29 | ||
PCT/EP2001/007101 WO2002001222A2 (en) | 2000-06-29 | 2001-06-22 | Method and device for detecting dyes in the field of fluorescence microscopy |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1266197A2 true EP1266197A2 (en) | 2002-12-18 |
Family
ID=7648207
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP01947389A Withdrawn EP1266197A2 (en) | 2000-06-29 | 2001-06-22 | Method and arrangement for optical detection of characteristic variables of the wavelength-dependent behaviour of an illuminated sample |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1266197A2 (en) |
JP (2) | JP3836426B2 (en) |
DE (1) | DE10033180B4 (en) |
WO (1) | WO2002001222A2 (en) |
Families Citing this family (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10222779A1 (en) * | 2002-05-16 | 2004-03-04 | Carl Zeiss Jena Gmbh | Method and arrangement for examining samples |
DE10327382A1 (en) * | 2003-06-16 | 2005-01-05 | Carl Zeiss Jena Gmbh | Method for fluorescence microscopy |
DE102005020543A1 (en) | 2005-05-03 | 2006-11-09 | Carl Zeiss Jena Gmbh | Method and device for adjustable change of light |
DE102005058184A1 (en) * | 2005-12-01 | 2007-06-14 | Friedrich-Schiller-Universität Jena | Motion object e.g. eye-ground, fluorescence and/or reflection spectra detecting method for use during e.g. analysis of sample, involves accumulating local fluorescence and/or reflection signals and spectrometrically evaluating signals |
DE102005058185A1 (en) * | 2005-12-01 | 2007-06-14 | Friedrich-Schiller-Universität Jena | Object e.g. fundus of eye, region`s fluorescence and/or reflection spectra detecting method, involves forming difference signal from accumulated signals for spectral evaluation and presentation of fluorescence and/or reflection spectra |
DE102006034905B4 (en) | 2006-07-28 | 2015-07-30 | Carl Zeiss Microscopy Gmbh | Arrangement for signal processing at the output of a multi-channel detector |
US7532128B2 (en) * | 2006-10-25 | 2009-05-12 | Alverix, Inc. | Position sensitive indicator detection |
DE102007040238A1 (en) | 2007-08-25 | 2009-03-05 | Carl Zeiss Microimaging Gmbh | Method for laser scanning microscopy and beam distributor |
DE102007054602A1 (en) * | 2007-11-15 | 2009-05-28 | Deutsches Zentrum für Luft- und Raumfahrt e.V. | Condition parameter's distribution detecting method for use in e.g. medical diagnosis, involves registering signals with different wavelengths from probes by using camera, and separately detecting signals based on output signals of camera |
JP5244633B2 (en) * | 2008-02-04 | 2013-07-24 | オプテックスエフエー株式会社 | Light intensity monitor sensor and image processing system having the same |
ATE500530T1 (en) | 2008-10-10 | 2011-03-15 | Zeiss Carl Microimaging Gmbh | METHOD FOR IMAGING A SAMPLE USING A MICROSCOPE, MICROSCOPE AND DATA STORAGE |
WO2010041560A1 (en) * | 2008-10-10 | 2010-04-15 | 学校法人 慶應義塾 | QUANTITATIVE EVALUATION METHOD AND DEVICE FOR ZETA POTENTIAL, pH OR TEMPERATURE DISTRIBUTION AT WALL SURFACE, AND QUANTITATIVE VISUALIZATION METHOD AND DEVICE FOR SURFACE MODIFICATION |
DE102009006728A1 (en) | 2009-01-29 | 2010-08-05 | Carl Zeiss Microimaging Gmbh | Optical arrangement for use in deflection beam path of laser-scanning-microscope, has polarization device provided upstream to dispersive element in deflection beam path and adjusting polarization direction for light detection |
DE102009038028A1 (en) | 2009-08-18 | 2011-02-24 | Carl Zeiss Microlmaging Gmbh | Detector arrangement with increased sensitivity |
DE102009043745A1 (en) | 2009-09-30 | 2011-04-07 | Carl Zeiss Microlmaging Gmbh | Variable filtering spectral detector by spatial color separation and laser scanning microscope |
DE102011104379B4 (en) | 2011-06-18 | 2021-11-25 | Carl Zeiss Microscopy Gmbh | Scanning confocal microscope and use, control method and programmable control unit for such a microscope |
JP5867073B2 (en) * | 2011-12-28 | 2016-02-24 | 富士通株式会社 | Resin cure state monitoring device and resin cure state monitoring method |
DE102012203736A1 (en) | 2012-03-09 | 2013-09-12 | Carl Zeiss Microscopy Gmbh | Scanning microscope with spectral detection |
US9201000B2 (en) * | 2013-12-27 | 2015-12-01 | Palo Alto Research Center Incorporated | Sensor apparatus and method based on wavelength centroid detection |
US10317390B2 (en) * | 2014-09-08 | 2019-06-11 | University Of Vienna | Recording dynamics of cellular processes |
JP7424286B2 (en) | 2018-05-30 | 2024-01-30 | ソニーグループ株式会社 | Fluorescence observation device and fluorescence observation method |
JP2023539435A (en) * | 2020-08-05 | 2023-09-14 | 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 | Illumination adjustment method in fluorescence microscope and corresponding fluorescence microscope |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE69115575T2 (en) * | 1990-06-22 | 1996-10-24 | Matsushita Electric Ind Co Ltd | Spectral measurement method |
US5309486A (en) * | 1992-11-12 | 1994-05-03 | Westinghouse Electric Corp. | Non-contact flaw detection for cylindrical nuclear fuel pellets |
DE4331570C2 (en) * | 1993-08-17 | 1996-10-24 | Hell Stefan | Process for the optical excitation of a sample |
DE4416558C2 (en) * | 1994-02-01 | 1997-09-04 | Hell Stefan | Method for optically measuring a sample point of a sample and device for carrying out the method |
US5723288A (en) * | 1994-05-06 | 1998-03-03 | The University Of North Carolina At Chapel Hill | Method of fluorescent detection of nucleic acids and cytoskeleton elements using bis-dicationic aryl furans, and kits useful therefor |
DE19510102C1 (en) * | 1995-03-20 | 1996-10-02 | Rainer Dr Uhl | Confocal fluorescence microscope |
US5612540A (en) * | 1995-03-31 | 1997-03-18 | Board Of Regents, The University Of Texas Systems | Optical method for the detection of cervical neoplasias using fluorescence spectroscopy |
JPH09119897A (en) * | 1995-10-24 | 1997-05-06 | Nippon Soken Inc | Apparatus for detecting adsorptive species |
US5859700A (en) * | 1995-11-22 | 1999-01-12 | Kairos Scientific, Inc. | High resolution imaging microscope (HIRIM) and uses thereof |
DE19702914C2 (en) * | 1997-01-28 | 1998-12-24 | Max Planck Gesellschaft | Method and arrangement for determining predetermined properties of target particles of a sample medium |
DE19701703A1 (en) * | 1997-01-21 | 1998-07-23 | Zeiss Carl Jena Gmbh | Microscope system for determining emission distribution of light emitting samples e.g. in semiconductor ICs |
DE19722790B4 (en) * | 1997-05-30 | 2006-01-05 | Carl Zeiss Jena Gmbh | Arrangement and method for time-resolved measurement according to the scanner principle |
US6483581B1 (en) * | 1997-07-02 | 2002-11-19 | Spectra Code, Inc. | Raman system for rapid sample indentification |
DE19915137C2 (en) * | 1999-03-26 | 2001-10-18 | Michael Schaefer | Fluorescence microscopy method for quantifying multiple fluorochromes in a multi-colored sample and uses of the method |
-
2000
- 2000-06-29 DE DE10033180A patent/DE10033180B4/en not_active Expired - Fee Related
-
2001
- 2001-06-22 JP JP2002506104A patent/JP3836426B2/en not_active Expired - Fee Related
- 2001-06-22 WO PCT/EP2001/007101 patent/WO2002001222A2/en active Application Filing
- 2001-06-22 EP EP01947389A patent/EP1266197A2/en not_active Withdrawn
-
2006
- 2006-02-09 JP JP2006032476A patent/JP4452850B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
See references of WO0201222A2 * |
Also Published As
Publication number | Publication date |
---|---|
WO2002001222A2 (en) | 2002-01-03 |
JP4452850B2 (en) | 2010-04-21 |
WO2002001222A3 (en) | 2002-10-03 |
DE10033180A1 (en) | 2002-05-29 |
JP2004502173A (en) | 2004-01-22 |
JP3836426B2 (en) | 2006-10-25 |
DE10033180B4 (en) | 2006-08-31 |
JP2006138875A (en) | 2006-06-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE10033180B4 (en) | Method for the detection of dyes in fluorescence microscopy | |
EP1504300B1 (en) | Method and arrangement for analyzing samples | |
DE10038526B4 (en) | Method and arrangement for recording the wavelength-dependent behavior of an illuminated sample | |
EP1264169B1 (en) | Improvement of spectral and/or spatial resolution in a laser scanning microscope | |
DE10151217B4 (en) | Method for operating a laser scanning microscope | |
EP1396739B1 (en) | Method and arrangement for adjustable modulation of spectral composition and/or intensity of pump light and/or probe light | |
DE19829981C2 (en) | Method and arrangement for confocal microscopy | |
US6958811B2 (en) | Method for the detection of dyes in fluorescence microscopy | |
DE10257237B4 (en) | Arrangement for the optical detection of light radiation excited and / or backscattered in a sample | |
DE10151216A1 (en) | Method for the optical detection of characteristic quantities of an illuminated sample | |
DE102015107367A1 (en) | Evaluation of Fluorescence Scanning Microscopy Signals Using a Confocal Laser Scanning Microscope | |
DE102009060793A1 (en) | High-resolution microscope and method for two- or three-dimensional position determination of objects | |
DE10339311B4 (en) | System and method for setting a fluorescence spectral measurement system for microscopy | |
WO2006003178A1 (en) | Raster microscope and method for the analysis of biological samples by means of a raster microscope | |
DE10227111A1 (en) | Spectral microscope and method for data acquisition with a spectral microscope | |
DE10118463A1 (en) | Depth-resolved optical imaging method for use in biological scanning microscopy, uses phase or frequency modulation of the imaging light | |
EP4189358A1 (en) | Method for detecting emission light, detection device and laser scanning microscope | |
EP1636624A1 (en) | Method to be used in fluorescence microscopy | |
WO2002014811A1 (en) | Light detector unit and method for detecting luminescence light and confocal microscope for luminescence microscopy | |
WO2013050467A1 (en) | Microscope system and method for recording multi-channel images | |
DE202018102134U1 (en) | Microscope for improved measurement of number and bonds of molecules |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20020323 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: WOLLESCHENSKY, RALF Inventor name: MOEHLER, GUNTER |
|
17Q | First examination report despatched |
Effective date: 20050113 |
|
17Q | First examination report despatched |
Effective date: 20050113 |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: CARL ZEISS MICROSCOPY GMBH |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20150106 |