WO2003098200A1 - Systeme et procede pour analyser des echantillons chimiques et/ou biologiques - Google Patents

Systeme et procede pour analyser des echantillons chimiques et/ou biologiques Download PDF

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Publication number
WO2003098200A1
WO2003098200A1 PCT/EP2003/005107 EP0305107W WO03098200A1 WO 2003098200 A1 WO2003098200 A1 WO 2003098200A1 EP 0305107 W EP0305107 W EP 0305107W WO 03098200 A1 WO03098200 A1 WO 03098200A1
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WIPO (PCT)
Prior art keywords
sample
line
illumination
detector
detection device
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Application number
PCT/EP2003/005107
Other languages
German (de)
English (en)
Inventor
Roland Stange
Jürgen Müller
Achim Kirsch
Original Assignee
Evotec Oai Ag
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 Evotec Oai Ag filed Critical Evotec Oai Ag
Priority to AU2003232778A priority Critical patent/AU2003232778A1/en
Priority to EP03752743A priority patent/EP1504251A1/fr
Publication of WO2003098200A1 publication Critical patent/WO2003098200A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • 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
    • G02B21/08—Condensers
    • G02B21/082—Condensers for incident illumination only
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/063—Illuminating optical parts
    • G01N2201/0631—Homogeneising elements

Definitions

  • the invention relates to a device and a method for examining chemical and / or biological samples, in particular in suspension, with the aid of luminescence spectroscopy.
  • samples for example cells, beads, vesicles, aggregates, DNA strands and the like. contain, excited with electromagnetic radiation, in particular light, and the radiation emitted due to luminescence is detected.
  • the particles are particularly marked with luminescent dye markers.
  • Dye markers of this type emit radiation of different wavelength ranges as a function of their binding to a particle and as a function of the type of particle to which they are connected. For example, these are receptor-ligand compounds.
  • ligands labeled with a dye marker are used here.
  • Luminescence spectroscopy is used in particular in pharmaceutical drug research. This is preferably done in high-throughput.
  • Medium-throughput screening ⁇ plants where a large number of samples are examined in a short time. Often, there are over 1000 per hour Samples examined.
  • titer plates with a large number of wells are used for this purpose. Typical titer plates have 1536 or 2080 wells.
  • the samples are small samples of sometimes only a few microliters per sample. In particular, the sample amounts are less than 50 ⁇ l, preferably less than 10 ⁇ l per well.
  • a suitable method for measuring the luminescence intensity distribution is described, for example, in WO 98/16814.
  • characteristic features such as existing bonds between molecules, concentration ratios and the number of specific particles in the solution can be determined from a heterogeneous brightness distribution of a sample.
  • a confocal microscope is used to carry out this method, which creates a point-like focus within the sample.
  • the focus is generated by an illumination device, in particular a laser.
  • Particles within the point focus, in particular dye markers are excited to luminescence by the radiation.
  • the luminescence radiation emitted by the sample is detected by a detection device.
  • the number of luminescence photons is detected in a time interval of a defined length.
  • a function of the specific brightness distribution is then calculated from this by the number of photons per time interval.
  • good measurement results with regard to the reaction of fluorescence-marked particles, for example molecules can be achieved.
  • this measuring method it is only possible with long measuring times to obtain meaningful statistics, for example on the respective number of individual different particles in the sample. Since the shortest possible measurement times have to be realized in particular in the case of high-throughput screening methods for operating such a system, such statistical analyzes are not economically possible with the method described in WO98 / 16814 and the device described.
  • a device for generating an image of a sample is known from US Pat. No. 6,025,601.
  • the sample To generate the image, the sample must be immobilized on a sample holder, for example a conventional glass slide. ' The sample holder is clamped in a sample table. The sample is illuminated line by line with the aid of an illumination device. The individual lines of the line illumination are recorded one after the other by a detection device and are combined to form an image with the aid of a computer. Since this is to be used to generate an image of a sample, an autofocus mechanism is also required in order to maintain a correct scan height.
  • the method which can be carried out with the aid of the device described in US Pat. No. 6,025,601 is particularly disadvantageous since time-consuming and cost-intensive immobilization of the sample is absolutely necessary. Since the image is built up line by line, the examination of a sample is extremely time-consuming. Such an examination method is not suitable for screening, especially high-throughput screening.
  • EP 0 501 008 describes a so-called imaging flow cytometer.
  • This device has a flow cell through which the sample liquid is moved at a high flow rate. The flowing sample is exposed to pulsed laser light or strobe lamps. To avoid smearing or blurring the image, very short exposure times are required.
  • flow cytometers it is in particular only possible with complex image processing to examine samples which have particles of different sizes.
  • Flow cytometers also have the disadvantage that due to the large-area detection, only a slight suppression of the background radiation is possible.
  • this device is not suitable for examining small particles such as micro and nanoparticles, since it is not possible to distinguish between individual small particles. Furthermore, small particles that are inside or on larger particles cannot be detected with this device.
  • Imaging flow cytometers usually have a point-measuring detector, so that the detector detects and integrates the entire fluorescence of the radiation emitted by the detected particles by this one detector device.
  • the object of the invention is to provide a method and a device for examining chemical and / or biological samples with the aid of luminescence spectroscopy, with which or with which samples having particles of different sizes and / or brightness can be examined. In particular, short examination times and meaningful statistics should be achievable.
  • the object is achieved according to the invention by a method according to claim 1 or by a device according to claim 7.
  • the device according to the invention has a sample holder, which, for example, holds a liquid sample.
  • the sample is particularly in suspension.
  • the sample is illuminated with the aid of an illuminating device, the illuminating device being designed such that it excites the particles contained in the sample to luminescence.
  • the device has an optical device.
  • the optical device is designed such that linear illumination is generated in the sample.
  • the radiation emitted by the sample along this line is detected with the aid of a detection device.
  • the detection device has a plurality of individual detectors. The detection device is connected to an evaluation device for the static evaluation of the radiation detected by the detection device.
  • the detection device is preferably a CCD camera, the individual pixels or pixel groups of the CCD camera being one Form single detector.
  • the radiation perceived by the individual detectors is converted into an electrical signal and transmitted to the evaluation device.
  • the device according to the invention thus performs a highly parallelized processing or evaluation of the radiation perceived by a large number of individual detectors.
  • linear illumination of a liquid sample enables highly parallelized detection of particles in the sample.
  • a considerably larger part of particles is excited to luminescence by a line-shaped illumination. Since this is recorded according to the invention by a detection device with a plurality of individual detectors which carries out a linear detection, a large amount of information can be detected in short examination periods.
  • a detection device with a plurality of individual detectors which carries out a linear detection, a large amount of information can be detected in short examination periods.
  • CCD sensors in which the individual pixel or pixel areas form the individual detectors
  • a very high number of parallelized detections can be carried out.
  • more than 200, preferably more than 500 and particularly preferably more than 1000 measurements can be carried out simultaneously.
  • the individual exposure time is preferably 5 to 100 ms. This means that such an individual detector can be re-detected after only 5 to 100 ms.
  • samples that have particles of different sizes can each be examined in short examination times, wherein particles of different sizes can be examined at the same time.
  • the particles can range from individual molecules to particles with a diameter of a few 100 ⁇ m. Because of the linear illumination, the particles can also diffuse at very low speeds and / or Sample solutions with high viscosity good measurement results can be achieved with short measurement times.
  • CCD detectors with 576 pixels per line are preferably used, each pixel serving in particular as an individual detector and being read out individually. 576 parallel measurements are thus carried out simultaneously.
  • CCD detectors that have 2048 or even 4096 pixels per line are also possible.
  • the individual pixel lengths or widths in the case of preferably used CCD detectors are at most 30 ⁇ m, preferably at most 20 ⁇ m.
  • the device according to the invention makes it possible to obtain structural information about the particles.
  • the linear illumination makes it possible, for example, to obtain information about individual cell areas.
  • the device according to the invention has a high sensitivity, so that even weak luminescence signals can be detected. This is particularly advantageous when analyzing specific cell information.
  • Another advantage of the device according to the invention is that immobilization of the particles on a glass carrier or the like is not necessary, since the device according to the invention is used for statistical evaluation of the detected luminescence signals from particles in suspension and no image of the sample is generated. Due to the linear lighting, a sufficient number of particles is obtained through the measurement volume, i.e. the area of the sample excited by the linear illumination.
  • Good statistics can be achieved by using the device according to the invention.
  • Good, ie meaningful statistics are available, for example, in comparison to confocal measurement methods.
  • confocal Measurement methods have a single focus that has very small dimensions, so that typically only a single particle can be observed in focus at the same time. Typical measuring times are 100 ⁇ s to 1 ms per particle. This results in total measuring times of 1 to 100 s.
  • a line is illuminated, which is preferably imaged on a line of a CCD detector with, for example, 576, 1280 or more pixels.
  • a linear illumination has the advantage over a spot illumination that the particles in the sample are excited to luminescence in a larger area and in particular at the same time.
  • Linear illumination is therefore a highly parallelized measurement focus.
  • the device according to the invention has the particular advantage that it is a simply constructed device. This leads to greater operational reliability than with known devices. In particular when measuring suspensions, the device has the advantage that a smaller amount of data has to be processed.
  • the optical device of the device according to the invention preferably has at least one cylindrical lens for generating the linear illumination. Because the intensity distribution of the radiation, in particular in a laser beam If a single cylindrical lens is provided, the intensity distribution in the longitudinal direction of the illumination line is also Gaussian. It is therefore preferred to use only the middle region of a line generated, in which a high radiation intensity is guaranteed. According to the invention, the device therefore preferably has a limiting device which limits the length of the illumination line. The edge areas on the generated lighting line, which have a low intensity, are thus cut off. For example, an aperture is suitable as a limiting device. As a result, the illumination line generated in the sample is homogenized in its longitudinal direction.
  • Such homogenization can also be achieved with a so-called line generator lens or a power lens.
  • the illumination line it is also possible to provide a plurality of cylindrical lenses in the optical device. These cylindrical lenses are preferably arranged next to one another so that they produce a common line of illumination. The distance between the individual cylindrical lenses is preferably selected such that regions generated by the individual cylindrical lenses overlap with a low intensity. As a result, an illumination line can be generated which has a homogenized high intensity in the longitudinal direction.
  • an illumination line that is as homogeneous as possible
  • a homogeneous lighting line is thus generated on the side of the slit diaphragm facing away from the lighting device.
  • optical fibers are provided, the fiber ends of which are arranged along a line.
  • several rows of optical fibers can be arranged side by side. This increases the width of the lighting line. However, this can be reduced again to the required width by means of downstream lenses. Radiation of the desired wavelength is coupled into the optical fibers with the aid of the illumination device.
  • the detection device is designed as a confocal detection device.
  • the detection device preferably has a slit diaphragm in the direction of the beam path in front of a detector. The position and dimensions of the slit diaphragm are adapted to the illumination line in the sample.
  • detectors that can count individual photons. This is, for example, an APD line array.
  • a line detector (APD line array, CCD line camera) can be provided.
  • a confocal structure of the detection device is realized by a CCD line camera.
  • a CCD line scan camera a CCD area scan camera can also be used, the pixels of which can be read out individually or line by line. This makes it possible to read out only one line of the CCD area camera, so that a confocal construction of the detection device is also realized by the CCD area camera. It is also possible to combine individual lines, for example two lines, into one line. This is possible through software as well as hardware.
  • the illumination device is preferably designed in such a way that it generates radiation for luminescence excitation in several wavelength ranges, typically in the range of 300-900 nm.
  • the individual luminescence wavelength ranges are preferably divided in the detection device.
  • the detection device can have an optical device which is independent of the optical device, by means of which linear illumination of the sample is caused.
  • at least parts of the optical device provided for generating the line illumination are preferably also used for the optical device to be assigned to the detection device.
  • the detection device preferably has a spectrograph. This breaks down the radiation emitted by the sample into spectral components, so that the luminescence radiation can be recorded independently of each spectral component. This has the advantage that a large number of measurement results can be achieved by a single excitation of the sample. It is therefore not necessary to illuminate the sample with radiation of different wavelength ranges at intervals. This significantly increases throughput, particularly in high-throughput screening plants.
  • a CCD area camera is preferably provided for the detection of the different luminescence wavelength ranges.
  • the individual wavelength ranges are split up by suitable optical elements, so that different, in particular parallel lines are generated for each wavelength range on the CCD area scan camera.
  • the individual lines of the CCD area camera, on each of which a spectral line is shown, can be read independently of one another. As a result, the detection of measurement data in different wavelength ranges is implemented in a simple manner.
  • the detection device preferably has at least one dichroic beam splitter or partially transparent mirror.
  • Another possibility for spectrally decomposing the radiation emitted by the sample is to provide a curved grating.
  • the at least one dichroic beam splitter and / or the curved grating is then assigned either a detector per wavelength range or, as described above, a CCD area scan camera, in which several lines can be read independently of one another.
  • the detection device preferably has two polarizing filters and a beam splitter, by means of which the differently polarized radiation emitted by the sample is divided into different polarization ranges.
  • a polarization beam splitter is preferably provided in the beam path, which splits the luminescence radiation into the two polarization directions.
  • a CCD area detector can be used to detect the radiation.
  • the evaluation device connected to the detection device evaluates the one emitted by the sample and detected by the detection device Radiation statistically.
  • the sample is preferably illuminated continuously.
  • the detectors that are preferably used, in particular CCD detectors, have a short exposure time.
  • the line exposure is preferably in the range from 16 ⁇ s to 100 ⁇ s.
  • slower line scan cameras can also be used, since these cameras can be read out after exposure if there are dead times.
  • a surface camera or a surface detector is used in special evaluation methods such as FCS (fluorescence correlation spectroscopy).
  • the evaluation device preferably distributes the intensity of different radiations emitted by the sample. It can thus be determined, for example, which particles occur how often in the sample and / or which bonds of the particles occur how often.
  • correlation methods and histograms can also be carried out in a preferred embodiment of the evaluation device. It is thus possible with the aid of the device according to the invention to obtain a large amount of information about the particles present in the sample in a short time. For example, the number or concentration of specific particles and their binding relationships can be examined with the aid of intensity distributions, anisotropy analyzes or moment analyzes (for example FIDA, FIMDA, C-Mafid).
  • the temporal correlation of the particles can be evaluated across the illumination line, for example.
  • a temporal correlation of the particles along the line can also be measured.
  • the diffusion rate of individual particles can be determined from this.
  • the spatial correlation can take place along a data line, for example in order to obtain structural data information about these particles.
  • the evaluation of the spatial correlation provides, for example, the correlation length as a result.
  • Additional information about the particles can be obtained via fluorescence lifetime measurements.
  • the fluorescence lifetime measurement can be carried out with a further preferred embodiment of the device according to the invention.
  • the sample is excited with pulsed laser light.
  • the detector is synchronized with the laser pulse.
  • the use of avalanche photodiode (APD) arrays is particularly advantageous since the synchronization with these can be carried out directly.
  • the device according to the invention has a movement device in order to realize a relative movement between the particles present in the sample and the illumination line.
  • a movement device makes it possible to obtain good measurement data even with particles with very little or no diffusion speed. It is not necessary to extend the measuring times. This prevents destruction of the particles or dye markers due to long exposure times.
  • the relative movement between sample particles and the illumination line can be realized, for example, by a tilting mirror provided in the optics device, which serves as a movement device for moving the illumination line. It is also possible to or the like the sample carrier with the help of a scanning table. in addition to or instead of moving the lighting line. Flow cells in which the sample flows can also be used. Another option is to use treadmills to move the sample holder.
  • the treadmills are made, for example, of transparent plastic with an optical quality.
  • the sample is preferably introduced into the treadmills in a previous work step, for example by vapor deposition. It is also possible for indentations, for example wells of a titer plate, to be introduced, for example punched, into the plastic band and then sealed with a film. If, for example, the wells have a volume of less than 10 ⁇ l, a drop can no longer because of the surface tension run out of the well even if the treadmill is wound up before the measurement.
  • stirring devices can be provided, for example, which cause the sample liquid to move.
  • the particles can also be moved within the sample holder by applying electrostatic or magnetic fields from the outside and by providing micro stirrers.
  • the device according to the invention and the measurement method that can be carried out with it have the advantage that the flow velocities in the sample or the relative movement between the illumination line and the particles of the sample do not have to be synchronized with the data acquisition.
  • the decisive factor for obtaining good statistics is rather the short measuring time in the method according to the invention.
  • the device according to the invention preferably has a control device for controlling exposure cycles.
  • the exposure cycles can take place at regular or irregular intervals.
  • the exposure time can be controlled by the control device.
  • the control unit preferably only specifies the exposure time of the detector and the read cycle. If, as described above, several lines are exposed in succession and only then read out, the control unit additionally specifies the vertical dock speed. Possibly. the control unit controls the scanning table and / or a stirring device and / or a galvo scanner.
  • the device according to the invention is suitable for high-throughput screening systems.
  • the samples are preferably arranged in titter plates with a large number of wells. Such titter plates have, for example, 1536 or 2080 depressions.
  • the amount of sample provided in each well is in particular less than 10 ⁇ l, preferably less than 5 ⁇ l.
  • the illumination line arranged in such a well preferably has a length of 500-1000 ⁇ m and a width of approximately 1-2 ⁇ m.
  • the invention further relates to a method for examining chemical and / or biological samples, in particular in suspensions, with the aid of luminescence spectroscopy.
  • a linear illumination of the sample which is preferably in suspension, takes place by means of an illumination device.
  • the radiation emitted by the sample along a line is detected in the next step, in particular by means of a detection device which has a plurality of individual detectors.
  • spatially separated subregions of the linear radiation are thus recorded by individual detectors.
  • the radiation received is converted into electrical signals by the individual detectors.
  • the signals emitted by the individual detectors are then processed or evaluated in parallel. This creates a highly parallelized evaluation method.
  • a large number of measurements are thus carried out in parallel or simultaneously. This is done, for example, by taking several measurements side by side in a row. respectively.
  • the linear illumination of the sample corresponds to a large number of individual measuring points arranged next to one another. In particular when using the device described above, each of these measuring points is in one focus.
  • a confocal aperture in the detection beam path in particular one Slit diaphragm or a slit-shaped detector, also in the direction of the optical axis, a spatial limitation of the measurement volumes achieved.
  • the invention it is also possible to carry out evaluations between a plurality of measurement value sequences, which are recorded, for example, in spatially adjacent measurement volumes. This is particularly advantageous for fluctuation measurements and allows, for example, the analysis of spatial correlations within the examined samples.
  • the duration of a measurement is preferably less than 10 ms and particularly preferably less than 1 ms.
  • the individual measurements preferably follow one another directly without a time interval. There may be a time interval between the individual measurements. In this case, poorer statistics of the measurement signals are accepted for a given total measurement period; however, the technical implementation can be simplified for certain types of detectors. This can be done in particular by using the device described above, in particular using CCD detectors with very short readout times.
  • the method according to the invention is, in particular, advantageously further developed, as described above with reference to the device according to the invention.
  • the individual detectors are read out rapidly, with individual pixels or pixel groups of a CCD detector, in particular an avalanche photodiode, preferably being used as individual detectors.
  • FIG. 1 shows a schematic basic illustration of a first embodiment of the device according to the invention
  • FIG. 2 shows a schematic basic illustration of a second embodiment of the device according to the invention
  • FIG. 3 shows a schematic illustration of a further embodiment of the detection device according to the invention
  • FIG. 4 shows a schematic basic illustration in side view of a lighting device according to the invention.
  • Fig. 5 is a schematic basic illustration of the lighting device shown in Fig. 4 in plan view.
  • FIG. 1 The basic arrangement of the individual elements of the device according to the invention shown in FIG. 1 is greatly simplified and contains only that essential components of the device.
  • a line 14 is generated in an image plane 12 on the illumination side.
  • Line 14 is directed in the direction of an optical device 18 via a dichroic beam splitter or a partially transparent mirror 16.
  • a linear illumination 20 is generated in a sample 22 with the aid of the optical device 18.
  • the sample 22 is arranged in a titer plate 26 having a plurality of wells 24.
  • the illumination line is arranged within the sample 22 such that the edges of the wells 24 are preferably not touched.
  • the illumination line is arranged opposite a transparent base plate 28, for example made of glass, in such a way that the base plate 28 is not touched by the illumination line 20 either. In this way, negative influences of the base plate 28 and / or the walls of the wells 24 'are excluded.
  • the optical device 18 serves not only to image the radiation generated by the illumination device 10 in the sample 22, but also to direct the luminescent radiation emitted by the particles present in the sample in the direction of a detection device 30 ,
  • the radiation emitted by the sample is not deflected by the dichroic beam splitter.
  • a line 32 is imaged on the detector device 30, which is, for example, a CCD line camera. If the detector device 30 is not a CCD line camera, a slit diaphragm can be provided in the beam path in front of the detector 30 in order to image the line 32.
  • the camera line itself represents the aperture of a confocal structure.
  • the measurement signals occurring in the individual pixels of the CCD line camera are transmitted from the detection device 30 to the evaluation device 36 via a line 34 or more lines.
  • the evaluation device 36 is used for a static evaluation, for example with regard to the intensity distribution.
  • the expansion device 36 which is preferably a computer, can also be used to control the lighting device 10 and possibly other components to be controlled provided in the device according to the invention.
  • an optical device 18 which is used both for imaging the illumination line 20 generated by the lighting device 10 in the sample 22 and for imaging the luminescent radiation onto the detection device 30
  • two separate optical devices can also be provided.
  • the imaging of the illumination line 20 into the sample 22 is realized with the aid of one optical device and the imaging of the luminescent radiation onto the detection device 30 with the other illumination device.
  • the illumination of the sample 22 can take place from the opposite side of the sample carrier 26 with respect to FIG .
  • the protrusion of two separate optical devices has the advantage that they can be better matched to the illumination wavelength and the luminescence wavelength, for example. However, this is a more expensive device because two separate optical units are required.
  • Laser excitation is particularly suitable for excitation of the particles or luminescence markers in the sample 22.
  • a diffraction-limited illumination line 20 can be realized within the sample 22. This enables high resolution and good suppression of the background radiation to be achieved.
  • Optical devices with a numerical aperture which is higher than 0.7, in particular higher than 0.9, are preferably used in the invention. For example, very good measurement results are achieved with a 20x lens with a numerical aperture of 0.7 or a 40x lens with a numerical aperture of 0.95.
  • the imaging optical device is, for example, a typical microscope structure in which a lens and a tube lens are combined.
  • the lighting device has fiber-coupled lasers 15.
  • the structure of the lighting device in connection with the optics 38, which have lenses 70-76, corresponds to the structure described with reference to FIG. 4.
  • the beams are bundled with the aid of an optical system 38 and in turn generate a line 14 in the image plane 12 on the illumination side - shown as a dot in FIG. 2.
  • the optical system 38 is here a component of the optical device 18, which in the exemplary embodiment shown is an objective 40 and a tube lens 42 in order to image the illumination line 20 in the sample 22.
  • the lighting device is also constructed such that it generates laser light in different wavelength ranges in lasers, not shown, which then emerges divergently from the fiber end 15.
  • different dye markers contained in the sample 22 are simultaneously excited to luminescence.
  • the radiation emitted by the particles present in the sample 22 is transmitted through the dichroic beam splitter.
  • the dichroic beam splitter 16 in accordance with the beam splitter 16 shown in FIG. 1, in turn has the task of directing the radiation coming from the illumination device 10 in the direction of the sample 22 and of transmitting radiation emitted by the sample.
  • a further optical device 46 is provided behind an image plane 44 on the detection side, which corresponds to the surface of the CCD line camera 30 in FIG. 1.
  • the optics device 46 has a mirror 48, which of the Sample 22 incoming radiation is deflected in the direction of a curved grating 50.
  • the curved grating 50 spectrally decomposes the luminescence emitted by the sample into the individual luminescence wavelength ranges.
  • the individual wavelength ranges are imaged in a second detection-side image plane 52 by the curved grating 50.
  • a line 54, 56 shown as a dot in FIG. 2 is depicted in the image plane 52 for each wavelength range. Since the sample is only excited by the illumination device 10 with electromagnetic radiation of certain wavelength ranges and the sample also only has dye markers of certain colors, the curved grating 50 does not result in a continuous spectral division, but rather a division into individual lines.
  • a CCD area camera can be arranged in the image plane 52 in order to detect the radiation emitted by the sample 22 in different wavelength ranges.
  • the individual lines 54, 56 imaged on the CCD area camera are spaced apart from one another since the entire spectrum is not imaged. It is thus possible in a simple manner to obtain information about the individual spectral ranges by reading out individual pixels or individual lines of the CCD area camera. By adding the pixels of one or more adjacent lines, the intensity of the luminescence in the corresponding wavelength range can be increased.
  • the individual spectral lines (54, 56) can then be used to form a histogram or a correlation calculation.
  • the embodiment shown in FIG. 3 corresponds to the embodiment described with reference to FIG. 2 with regard to the illumination device 10 and the excitation of the sample 12. Again, the sample is excited simultaneously in different wavelength ranges.
  • the detection device has a prism 60 for the spectral separation of the luminescence radiation emitted by the sample in the different wavelength ranges. With the help of a lens 62 upstream of the prism and The individual spectral lines 54, 56 are formed in the second image plane 52 of the detection device by a lens 64 connected downstream of the prism.
  • a CCD area camera can in turn be provided as a detector at the level of the image plane 52.
  • a preferred embodiment of the lighting device for generating line 14 is shown.
  • the imaging of the illumination line 14 generated by the illumination device into the sample and the detection of the luminescence radiation emitted by the sample can be carried out as described above with the aid of FIGS. 1 to 3 described.
  • the lighting device has lasers, not shown, which couple light into a fiber, which is divergently scattered by the fiber end 15.
  • a Powell lens 70 is provided in front of the image plane 12 on the illumination side.
  • the Powell lens 70 homogenizes the beam, i.e. homogenization over the length of the lighting line. With known Powell lenses, homogenization down to 5% can be achieved.
  • the Powell lens 70 is followed by a cylindrical lens 72. Since the Powell lens is an aspherical cylindrical lens, the divergence is greatly increased in one direction and not influenced in the other direction.
  • the divergent part of the beam can be collimated by the cylindrical lens 72.
  • a very sharp illumination line 14 can be realized with the aid of a second cylindrical lens 74, which is rotated through 90 ° with respect to the first cylindrical lens 72.
  • the resolution of the device according to the invention can be improved by increasing the sharpness of the illumination line 14.
  • the beam path from the fiber 15 is better collimated before it enters the Powell lens 70.

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Abstract

L'invention concerne un système servant à analyser des échantillons chimiques et/ou biologiques, en particulier en suspension, par spectroscopie de luminescence. Ce système comprend un porte-échantillon (26) destiné à recevoir l'échantillon. Cet échantillon est excité au moyen d'un dispositif d'éclairage (10) destiné à stimuler la luminescence des particules contenues dans l'échantillon. Un dispositif optique (18) permet de projeter un éclairage linéaire dans l'échantillon (22). Comparé à un éclairage ponctuel, cet éclairage linéaire permet d'exciter une partie sensiblement plus importante de l'échantillon avec une intensité élevée constante par point d'éclairage. Le système selon l'invention comprend par ailleurs un dispositif de détection (30) servant à détecter le rayonnement émis par l'échantillon (22) le long d'une ligne. Un dispositif d'analyse (36) est relié au dispositif de détection (30) en vue de l'analyse statistique du rayonnement détecté par ledit dispositif de détection (30).
PCT/EP2003/005107 2002-05-15 2003-05-15 Systeme et procede pour analyser des echantillons chimiques et/ou biologiques WO2003098200A1 (fr)

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AU2003232778A AU2003232778A1 (en) 2002-05-15 2003-05-15 Device and method for analyzing chemical and/or biological samples
EP03752743A EP1504251A1 (fr) 2002-05-15 2003-05-15 Systeme et procede pour analyser des echantillons chimiques et/ou biologiques

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DE10221564.2 2002-05-15
DE2002121564 DE10221564A1 (de) 2002-05-15 2002-05-15 Vorrichtung und Verfahren zur Untersuchung chemischer und/oder biologischer Proben

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EP1617205A1 (fr) * 2004-07-16 2006-01-18 Carl-Zeiss Jena GmbH Microscope à balayage de lumière et son utilisation
EP1959250A1 (fr) * 2006-01-20 2008-08-20 Sumitomo Electric Industries, Ltd. Analyseur, dispositif de verification d'authenticite, procede de verification d' authenticite, et procede de recherche souterraine
WO2008141919A2 (fr) * 2007-05-22 2008-11-27 Leica Microsystems Cms Gmbh Dispositif de détection de lumière dans un microscope à balayage
EP2881458A1 (fr) * 2005-10-26 2015-06-10 Silicon Biosystems S.p.A. Procédé et appareil permettant de caractériser et de compter les particules, notamment de particules biologiques
JP2020509391A (ja) * 2017-01-10 2020-03-26 フォトスイッチ・バイオサイエンシズ・インコーポレイテッド 検出のためのシステムおよび方法

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DE102016105798A1 (de) * 2016-03-30 2017-10-05 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts, Universitätsmedizin Verfahren zum Erzeugen eines Mikroskopbildes

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EP1959250A4 (fr) * 2006-01-20 2011-10-12 Sumitomo Electric Industries Analyseur, dispositif de verification d'authenticite, procede de verification d' authenticite, et procede de recherche souterraine
EP1959250A1 (fr) * 2006-01-20 2008-08-20 Sumitomo Electric Industries, Ltd. Analyseur, dispositif de verification d'authenticite, procede de verification d' authenticite, et procede de recherche souterraine
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AU2003232778A1 (en) 2003-12-02
EP1504251A1 (fr) 2005-02-09

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