JP4452850B2 - Method and apparatus configuration for optically grasping characteristic characteristic values depending on wavelength of illuminated sample - Google Patents

Description

  The present invention relates to a method and apparatus configuration for examining mainly biological samples, preparations and associated components in fluorescence microscopy, in particular laser scanning microscopy, fluorescence correlation spectroscopy and scanning near-field microscopy. It is about.

Along with that, a method based on fluorescence detection (high throughput screening) for examining agents is also included.
Confirmation of sample properties, mostly from the analytical or functional side, in relation to spatial substructures or kinetic processes, by moving from the detection of a small number of broad-spectrum dye bands to the simultaneous recording of complete spectra; Open up new possibilities in separation and classification.

  Thereby, in the case of a fluorescence spectrum having an overlapping portion, simultaneous inspection of a sample containing multiple fluorophores is possible even in a thick sample spatial structure. In addition, it is also possible to classify spatial structures by detecting partial shifts in the emission spectral band of the dye. The data recording rate is not reduced by this apparatus configuration.

  One of the classic fields of application of optical microscopes for examining biological preparations is fluorescence microscopy (reference: Pauly "Biological Confocal Microscopy Handbook", Planum Press 1955). In this case, specific dyes are used for special labeling of cell parts.

  The dye molecule is excited from the photon ground state to the excited state by absorption of one incident photon having constant energy. This excitation is commonly referred to as one-photon absorption (FIG. 1a). The dye molecule can return to the ground state from the excited state in various ways. In fluorescence microscopy, the transfer of fluorescent photons under emission is most important. The wavelength of the emitted photon is essentially shifted to the red side compared to the excitation radiation due to Stokes displacement. That is, on the long wavelength side. Stokes displacement allows separation of fluorescent light from excitation light.

  The fluorescence is separated from the excitation radiation by a suitable dichroic beam splitter in combination with a block filter and separately observed. By doing so, it is possible to depict individual cell parts colored with different dyes. In principle, however, several parts of the preparation can also be colored simultaneously with different dyes that have a unique deposition method (multiple fluorescence). Again, a special dichroic beam splitter is used to distinguish the fluorescence signals emitted from the individual dyes.

  In addition to excitation of dye molecules by one photon having high energy (one-photon absorption), excitation by a plurality of photons having lower energy is also possible (FIG. 1b). In this case, the total energy of individual photons corresponds to many times that of high energy photons. This type of dye excitation is referred to as multiphoton absorption (literature: Collie, Kino; “Confocal scanning optical microscopy and related imaging systems”; Academy Press 1996). However, dye emission is not affected by this type of excitation. That is, in the case of multiphoton absorption, the emission spectrum causes a negative Stokes shift, so its wavelength is short compared to the excitation radiation. The excitation light is separated from the emitted light in the same way as in the case of one-photon absorption.

A known technique will be described below using an example of a confocal laser scanning microscope (LSM) (FIG. 2 [L1]).
The LSM is roughly divided into the following four modules: light source, scanning module, detection unit, and microscope. These modules are described in more detail below. In addition, DE 19702753A1 is also helpful.

  Lasers of different wavelengths are used in the LSM for individual excitation of the various dyes in the preparation. The excitation wavelength is selected according to the absorption characteristics of the dye to be examined. Excitation light is generated in the light source module. In this case, various lasers (argon, argon krypton, TiSa laser) are used. In addition, the light source module performs wavelength selection and necessary excitation wavelength intensity adjustment by using, for example, an acousto-optic crystal. Subsequently, the laser beam is directed into the scanning module through a fiber or suitable mirror device.

  The laser beam generated by the light source passes through the objective lens (2), and is focused into the preparation via the scanner, the scanning lens system, and the cylindrical lens under the suppression of diffraction. Focus on the sample and perform dot scanning in the xy direction. In many cases, the residence time on the pixel in the sample scan is in the range of less than microseconds to several seconds.

  In the case of fluorescence confocal detection (descan detection), light emitted from the focal plane (sample) and the plane above and below it reaches the dichroic beam splitter (MDB) through the scanner. MDB separates fluorescence from excitation light. Subsequently, the fluorescence is focused with a stop (confocal stop / pinhole) that is exactly in the plane conjugate with the focal plane. This blocks out-of-focus fluorescent components. It is possible to adjust the optical resolution of the microscope by varying the dimensions of the stop. There is another dichroic block filter (EF) behind the stop, which again stops the excitation beam. Fluorescence is measured by a point image detector (PMT) after passing through the block filter.

  When multiphoton absorption is utilized, excitation of dye fluorescence occurs in a small volume where the excitation intensity is particularly high. This area is only slightly larger than the detection area when the confocal device is used. Therefore, the use of a confocal stop can be omitted, and detection can be performed immediately after the objective lens (non-descan detection).

  In another apparatus configuration for detecting dye fluorescence excited by multiphoton absorption, an additional descan detection is performed, but in this case the objective pupil is imaged into the detection unit (non-confocal). Descan detection).

  Of the three-dimensional illumination image, only the plane (optical cross section) existing in the focal plane of the objective lens is reproduced by the device configuration of the two detectors connected to the corresponding one-photon absorption or multi-photon absorption. . Subsequent drawing of several optical cross sections in the xy plane at various depths z of the sample produces a computer supported 3D image of the sample.

  Therefore, LSM is suitable for inspecting thick preparations. The excitation wavelength is determined by the intrinsic absorption characteristics of the dye used. With the dichroic filter that matches the radiation characteristics of the dye, only the fluorescence emitted from each dye is reliably measured by the point image detector.

  In biomedical applications, several different cell regions are currently labeled simultaneously with different dyes (multiple fluorescence). In the state of the art, individual dyes are detected separately on the basis of various absorption characteristics or on the basis of radiation characteristics (spectrum). In that case, additional splitting of the fluorescence of multiple dyes is performed by a sub-beam splitter (DBS), and individual dye radiation detection is performed in separate point image detectors (PMTx).

  With this arrangement, it is not possible for the user to adapt flexibly to new dye properties during detection and excitation. Therefore, for each (new) dye, a new dichroic beam splitter and block filter must be provided. In a device configuration based on DE ..., the fluorescence is spectrally divided by a single prism. This method is different from the above apparatus configuration having a dichroic filter only in that the characteristic curve of the filter used can be adjusted, but in addition, the emission band of one dye per point image detector is recorded. This is preferable.

  If the emission spectra of two dyes overlap, there is a limit in conventional detection devices. In order to avoid overwriting between the two dyes, the spectral detection region must be limited. The area where both dyes overlap is thus simply excised and not detected. This deteriorates the efficiency of the detection unit. The same signal-to-noise ratio can only be obtained by increasing the excitation power, thereby compromising the preparation. Therefore, up to six types of dye probes are used at the same time today. Otherwise, the emission bands cannot be separated because the emission bands are strongly overlapped.

In the past, dyes have been modified so that their absorption characteristics or their emission characteristics differ from each other.
FIG. 3a) shows the emission spectra of various typical dyes. The radiation signal is depicted as a function of wavelength. It can be seen that the position and shape of the emission spectrum differ between 1 to 4 and the numbered dyes. However, these dyes are often toxic to biological preparations. Therefore, it is impossible to examine the evolution of syncytia within the biological preparation. At the end of the 90s, so-called fluorescent proteins (GFP, YFP, CFP, TOPAS, GFT, RFP), which are natural pigments, were discovered (Clontech, USA).

  These dyes stand out because they have little effect on the sample. They are therefore particularly suitable for labeling cell regions within biological preparations. However, it has the disadvantage that the emission properties of the dyes differ only slightly.

FIG. 3b) shows the wavelength dependent radiation signal for the dyes GFP, Topaz, GFT and Cyan FP.
In conventional methods, only CFP and RFP could be effectively distinguished from others based on the changed absorption characteristics. That is, this is a case of sequential imaging. Separation of the dyes GFP and GFT would not be possible at all by conventional means.

  In yet another method for locating the two proteins, both proteins are labeled with different dyes. However, the emission spectrum of the first dye overlaps with the absorption spectrum of the second dye. Subsequently, the first dye is excited by the appropriate wavelength and emits fluorescence. If both molecules are very close to each other (<10 nm), the radiation beam of the first dye is absorbed by the second dye, so that the first dye is not emitted and the second dye is subsequently emitted.

  FIG. 3d) shows the energy level diagram of this process, referred to in the literature as Fluorescence Resonance Energy Transfer (FRET) (literature: Fan et al .; Biophysical Journal, V76, May 1999, page 24122420). ). By detecting the radiation beams of both dyes in this way and determining the ratio of both detector signals, the distance between the two molecules can be determined.

Furthermore, it is known that the emission spectrum of the dye present in the biological preparation can be distinguished from the emission spectrum measured in the dye cuvette.
FIG. 3c) shows that the emission spectrum of the dye depends on the environment in which the dye is placed. In the figure, the radiation signal is depicted as a function of wavelength.

  The wavelength shift may be several tens of nm. As for the dependence of this wavelength shift on the environment, no more detailed examination has been known so far. This is because this type of inspection is difficult if possible with the state of the art methods.

  Indeed, today, spectrometers are also used in combination with LSM. In this case, instead of a point image detector, a conventional and mostly high-resolution spectrometer is used (Patent Dixon et al., US Pat. No. 5,192,980). However, they only record the emission spectrum one by one or in an averaged form over a certain area. That is, this is a kind of spectroscopic analysis.

  In another apparatus configuration, the lifetime of dye fluorescence is measured. Again, it can be concluded that the problem is in the environment, but it takes a long data reception time to record a complete image. This method can therefore only be used in a limited way for the examination of raw preparations.

In another application of fluorescence microscopy, it is used in particular for the measurement of ion concentrations in biological preparations (for example Ca ⁺ , K ⁺ , Mg ²⁺ , Zn ^{+ ,.} For this purpose, special dyes or dye combinations with spectral shifts depending on the ion concentration (for example, Hula, India, Fluo; Molecular Probes) are used.

FIG. 4a) shows the emission spectrum of India 1 depending on the concentration of calcium ions.
FIG. 4 b) shows an example of a radiation spectrum depending on the calcium ion concentration in the case of a combination of Fluoro 3 and the Hula red pigment. These special dyes are called emissivity discrimination dyes.

  By detecting the two fluorescent regions shown in FIG. 4a) and calculating the ratio of both intensities, the corresponding ion concentration can be derived. In many cases, this measurement analyzes dynamic changes in ion concentration in the life preparation, which requires a time division within 1 millisecond.

  From the above, the object of the present invention is a new detection method that is flexible and free of programming. These methods should be usable with imaging systems as well as analytical microscopy systems. Therefore, the data adoption rate must not deteriorate when using these methods.

As a microscope system, an image creation system such as a laser scanning microscope for three-dimensional inspection of biological preparations with an optical resolution up to 200 nm and a scanning near-field microscope for inspection of a surface with high resolution up to 10 nm. There is.
There are also fluorescence correlation microscopes for quantitative measurement of molecular concentration and for molecular diffusion measurement. Furthermore, a method of dye inspection based on fluorescence detection is also included.

  In all of the above systems, fluorescent dyes are used to specially label the preparation. These set goals are solved by the method and apparatus configuration according to the claims.

  In accordance with the present invention, it is possible to separate even partially or even overlapping dyes, while avoiding or overcoming the disadvantages and limitations of the prior art. Thereby, multiple fluorescence recording using a fluorescent protein in nature is possible. Furthermore, according to this method, the wavelength shift based on the environmental deviation in the preparation to be inspected can be measured very efficiently.

  The background of flexible detection methods is spectrally divided fluorescence detection. In that case, the emitted light is split from the excitation light by the main color splitter (MDB) in the scanning module or in the microscope (in the case of multiphoton absorption).

A block diagram of the detector unit arranged subsequently is shown in FIG.
In the case of confocal detection, the light emitted from the sample is focused through the imaging lens system PO and further through the aperture (pinhole) PH, thereby blocking the fluorescence generated outside the focus. No aperture is required for non-descan detection.

  In this case, the light is decomposed into its spectral components by the angular dispersive element DI. As the angular dispersion element, a prism, a grating, and an acousto-optic element are used. The light split into its spectral components by the dispersive element is subsequently imaged onto the line detector DE. That is, the line detector DE measures a radiation signal depending on the wavelength and converts it into an electrical signal S (). In addition, a linear filter for suppressing the excitation wavelength can be connected in series to the detection unit.

FIG. 6 shows a possible embodiment when the detector unit shown in the block connection diagram of FIG. 5 is used as an optical beam path.
This structure is essentially based on the Sirnie-Turner structure. In the case of confocal detection, the light L from the sample is focused through the confocal stop PH by the pinhole lens system PO. In the case of non-descan detection and multiphoton absorption, this diaphragm is not necessary.

  The first imaging mirror S1 collimates the fluorescence. The light then strikes a linear grating G, for example a grating having 651 lines per millimeter. The grating deflects light in various directions depending on its wavelength. The second imaging mirror S2 focuses the spectrally divided individual wavelength components onto the channel of the corresponding line detector DE.

  It is particularly preferable to use Hamamatsu H7260 line secondary electron multiplier. This detector is a 32-channel type and has high sensitivity. The free spectral region of the above embodiment is about 350 nm. Since the free spectral range is evenly distributed over the 32 channels of the line detector in this arrangement, the optical resolution is about 10 nm. Therefore, this apparatus configuration is only suitable for spectroscopic measurements. However, it is advantageous to use it in an imaging system. This is because the detection spectrum band is relatively wide and the signal for each detection channel is relatively large. In addition, the movement of the free spectral region can also be performed, for example, by twisting the grating.

  In another embodiment, there is an example involving the use of a matrix detector (eg, CCD,...). In this case, division into various wavelength components is performed by a dispersive element at a certain coordinate. A complete line (or gap) of the scanned image is imaged in the direction that remains on the matrix detector. This embodiment is particularly advantageous when constructing a line scanner (literature: Corey, Kino; “Confocal Optical Microscopy and Related Imaging Systems”; Academic Press 1996).

  The structure principle essentially conforms to the LSM described in FIG. However, an image is formed at a focal point instead of a point focal point, and the inspection sample is scanned only in one direction. In the case of such a structure, a slit diaphragm is used as the confocal diaphragm instead of the hole diaphragm. Non-descanned detection using multi-grating absorption is also performed by this apparatus configuration. Again, the confocal stop can be omitted.

  The measurement algorithm in the apparatus configuration shown in FIG. 6, that is, the point scanner will be described below. However, this algorithm can be applied to the line scanner apparatus configuration without any limitation.

  As is apparent from FIGS. 3 and 4, the individual dyes differ in the position and shape of the emission spectrum. The algorithm (FIG. 7) determines the maximum value of the radiation signal detected at the centroid position or image point for each image point.

  In the following, an advantageous and feasible centroid determination method is described in more detail. Other methods of determining the center of gravity or maximum value, such as the interpolation fitting method, are unconditionally part of the invention. In that case, the signal for each channel (left diagram) detected by the line detector is multiplied by the calibration function (right diagram). That is, every channel has a certain weight.

  The left diagram of FIG. 7 represents the measured radiated signal by the number of the detected channel of the signal. The right diagram shows an example of the weighting function for each channel by channel number.

  The weighted individual signals per channel are added together and divided by the sum of the unweighted individual signals (total signal). As a result, a signal is obtained as a feature amount representing the radiation spectrum and hence the center of gravity of the excitation dye (FIG. 8a). This signal is hereinafter referred to as a position signal.

FIG. 8a shows a position signal depending on the position of the center of gravity or the position of the maximum value of the detected radiation spectrum.
By measuring the position signal, the various dyes can be distinguished based on their radiation spectrum position and type. Further, for example, when a certain dye is used, it is possible to measure the wavelength shift of the emission spectrum depending on the environment.

If several dyes are present at the same time at the image point, a linear connection relationship of the center of gravity expressed by the following equation is established according to the relative density of one dye and another:
Position = ⁿ Σ _k Pos _k C _k
Here, Pos _k is the characteristic barycentric position of the dye, C _k is the dye concentration, and n is the number of dyes simultaneously excited at the image point. Thus, the algorithm can also determine the ion concentration and can be used to detect the FRET signal. In addition, it is possible to analyze the partial overlap of two or more dyes (colocalization measurement).

  FIG. 8b shows a signal that depends on the ion concentration when using two dyes (eg Fluo 3 and Hula Red, Molecular Probes) or one dye with two characteristic emission bands (eg India, Molecular Probes). It is drawn. It represents a position signal that depends on the ion concentration.

  As already mentioned, the position signal is a position measure of the centroid position of the emission spectrum. That is, it can be used as a mask for calculating a color-coded intensity image.

  This algorithm is illustrated in FIG. In that case, as a first step, the mask (ie, the position signal) is multiplied with the corresponding lookup data. The look-up data includes color classifications made separately for the position of the center of gravity of the emission spectrum. Subsequently, the result of this multiplication is multiplied by an intensity value (total signal), that is, the color brightness is adapted to the actual fluorescence intensity. Depending on the choice of look-up data, each color masked intensity image (discrete color distribution) can be created, i.e. an intensity image with one dye per pixel, or a mixed color by combining individual image points It is also possible to create a single intensity image.

  The decisive advantage of this method is that any dye can detect the entire fluorescence (total signal) independent of the degree of overlap of the emission spectra, yet the dye can still be displayed as separated (by position signal). It is in that point. Accordingly, strongly overlapping dyes (FIG. 3c) can be detected particularly effectively.

  Implementation of the algorithm with the configuration of FIG. 6 can be performed either digitally or analogly. Both device configurations are described in detail below. An apparatus configuration for digitally calculating the total signal and the position signal is shown in FIG. In this case, the current flowing through the anode of the multichannel PMT is converted into a voltage and amplified by the first amplifier A (connected as a current / voltage converter). The voltage is guided to an integrator I, where the signal is integrated for that time (for example, the residence time on the pixel).

  In order to obtain the value quickly, after integrator I, a comparator K having the following switching threshold as a simple comparator, ie a digital output signal when the threshold is exceeded, or a window comparator And a comparator K that emits a digital output signal if the input signal is between the upper and lower limits of the switching threshold, or if the input signal is outside (below or above) the switching threshold. Can be made. The configuration of the comparator or window comparator can be before or after the integrator. A connection device configuration without an integrator (so-called amplification mode) can also be considered.

  In the case of the device configuration in the amplifier mode, the comparator K configures the device in conformity with an appropriate standard. The output of the comparator K is used as a control signal for the switch register Reg which is directly connected to the active channel (online), or to select the active channel individually (offline), the current state is the associated coupler V To the computer. The output signal of the integrator I is directly led to another amplifier A1 for level adaptation for subsequent A / D conversion. The A / D converted value is transmitted through a suitable data transmission device to a computer PC (or digital signal processor DSP) which performs the calculation of the position signal and the total signal according to FIGS.

  FIG. 11 shows a device configuration based on analog data processing equivalent to the device configuration of FIG. In this case, the signals of the individual channels are converted into voltage signals by the amplifier. Subsequently, the individual voltage signals are integrated in the integrator I for the residence time on the pixels.

A comparator K for comparing the integrated signal with the reference signal is installed after the integrator.
If the integrated signal is less than the comparator threshold, no fluorescence signal is measured in the individual channel, or the measured signal is too small. In such a case, signal processing of the individual channel should not continue. This is because this channel only contributes to the total signal. In such a case, the comparator operates the switch S through the Reg, and the individual channel is cut off for the actually measured pixel. That is, the spectral region important for the measured image point is automatically selected by the combination of the comparator and the switch.

  Subsequently, the integrated voltage signal is again converted into a current via a resistor R. Therefore, any individual channel generates a current depending on the intensity of fluorescence arriving at the individual channel. In subsequent positions, all individual channels adjacent to each other are connected to another resistor R1 between them.

  The total current generated at the top and bottom of the detector row is also converted to voltage by the current / voltage converter A1. The voltages Eo and Eu at the upper and lower ends correspond to the sum of the signals of the individual channels weighted by the reverse straight line. Both signals at the top and bottom are then summed by the summing amplifier SV. The signal thus generated corresponds to the total signal of all measured fluorescence. The total signal and the signal at the upper end or the signal at the lower end (indicated by a broken line) are led to an analog divider to output a position signal.

  The total signal and the position signal are each subsequently converted into a digital signal by a single analog-to-digital converter and further processed by a computer or DSP. In that case, however, the upper and lower signals can be converted without any limitation and can be processed by a computer. In this case, the computer determines the total signal and the position signal. In either case, the algorithm of FIG. 9 is executed by a computer.

However, the algorithm of FIG. 9 can also be implemented with the connections of FIG. In this regard, the three possibilities are described in more detail below.
In the first device configuration, the resistance (R1) existing between adjacent individual detection channels is changed, and multiplication is performed using the lookup data. The remaining connections are left at their original settings.

In the second device configuration, multiplication is performed by the look-up data in the amplifier A1. In that case, the amplifier A1 is operated using a variable non-linear characteristic curve. In the third device configuration (digital detection (according to FIG. 10) and analog detection (according to FIG. 11)), the input signal of the individual detection channel is manipulated or distorted by:
It is a change in amplification in (A), a change in integration time in (I), an additional offset input in front of the integrator and / or a digital influence by photons counted by the photon counter. All three methods can be arbitrarily combined with each other.

  In order to avoid artifacts, the fluorescence measurement needs to suppress the excitation light backscattered from the sample, or at least weaken it so that it is less than or equal to the maximum of the radiation. For this purpose, the above-described additional linear filter or a suitably optimized main color splitter (MDB) can be used for optical attenuation. Since the spectral bandwidth of the excitation laser beam is much narrower than the bandwidth detected by the individual channel, the backscattered or reflected excitation beam can also block the corresponding individual channel as intended by the switch of FIG. it can.

  The apparatus configuration of FIG. 11 has several advantages over the apparatus configuration of FIG. The most prominent advantage is that only two channels need to be converted into digital data and sent to the computer. This minimizes the data rate to be processed by the computer. This is particularly true when the method is applied to real-time microscopy, which detects, for example, 50 images or more with 512 × 512 pixel and 12-bit pixel tones, so that dynamic processes can be recorded very quickly. is important. In addition, if this method is used, there is no restriction on the number of individual channels of the line detector (matrix detector) used, and therefore on the width of the detectable spectral region and / or the spectral resolution of the spectral sensor.

Furthermore, in the apparatus shown in FIG. 10, the signal level that can be converted is much lower. Thereby, the assumed value of the signal-to-noise ratio is reduced.
In both apparatus configurations for the above evaluation algorithm conversion, integrator switching was used to detect individual channel signals. Also, photon counting can be performed within the individual channel without any restrictions. However, the apparatus configuration shown in FIG. 10 has an advantage that it can provide even complete spectrum information in addition to the position signal for additional image processing. The present invention therefore also includes a combination of both device configurations.

FIG. 12 shows measurement results obtained by the apparatus configuration shown in FIGS. 10 and 11.
FIG. 12a shows the emission spectra of the used dyes GFP, CFP and DI measured with a spectrometer. The dye was excited by an argon laser having a wavelength of 488 nm. In the following, these dyes were brought in particular areas of the biological preparation.

  FIG. 12b shows a histogram of the position signal when scanning over a sample piece where all three dyes are present. Three maxima can clearly be seen in the histogram, with the three dyes having their characteristic position signals. The positional relationship of the three dyes is as shown in the following table.

Dye CFP GFP DI
Position signal (relative wavelength shift) 14 30 80
Therefore, the dye should be easily separated by the apparatus configuration according to the present invention. In addition, local wavelength shifts based on various local environments within the dye are observed. This appears in the histogram in the maximum value width of the individual dye.

FIG. 13a shows an intensity image formed from the total signal.
FIG. 13b shows the corresponding image formed from the position signal.
This image represents the corresponding centroid of the emission spectrum. Differently stained cell nuclei (partially colored with GFP and partly colored with CFP) and cell frameworks colored with DI are clearly distinguishable.

  FIG. 13c shows an intensity image calculated and color-coded corresponding to the algorithm of FIG. The individual areas to which the various dyes are added are separated by color coding. Separation is clarified by displaying the image in three RGB channels.

  For comparison, an image measured by detection based on the state of the art is also shown in FIG. 13d. In this case, the detection was performed only in an extremely narrow spectral band in order to avoid overwriting the fluorescence signals of the different dyes with each other.

  Since the detection band was thus narrowed, only a part of the fluorescence signal emitted from the sample could be measured. To keep the image as close as possible to the same signal-to-noise ratio, the excitation power must be increased many times.

  From this, it is clear that the device configuration based on the present invention has higher efficiency than the device configuration based on the current technology. In addition, the region where CFP or GFP is deposited may not be separated because the emission spectra of both dyes overlap. This is manifested in the cell region being colored yellow or in a double phenomenon in the regions within the two image channels (R and G).

a) One-photon absorption, b) Multiphoton absorption Known LSM construction Typical spectrum a) Dye, b) Fluorescent protein, c) Wavelength shift / environment dependent, d) FRET Typical spectrum for ratio measurement, a) 1 dye and emissivity, b) 2 dye and ion dependent signal Block diagram of detector unit / optical system Example of detector unit / optical system configuration An algorithm for determining the position of the emission spectrum. Typical course of the position signal, a) dependent on the position of the emission spectrum, b) dependent on the ion concentration Algorithm for creating color-coded intensity images when using several dyes Example of electronics configuration for digital measurement Electronics configuration example for measuring analog signals a) Dye spectrum, b) Histogram of emission spectrum shift of a) Experiments for separating dyes, a) total intensity image, b) wavelength shift image, c) developed intensity image, d) intensity image with conventional detector based on current technology

Explanation of symbols

PO Imaging lens system DI Angular dispersion element DE Line detector S () Electrical signal PH Confocal stop A First amplifier I Integrator K Comparator V Attached coupler Reg Switch register DSP Digital signal processor PC Computer S Switch A Current / Voltage converter SV Summing amplifier S1 First imaging mirror G Linear grating S2 Second imaging mirror

Claims (87)

Characteristic wavelength-dependent characteristic values of the irradiated sample, mainly emission and absorption for any one of fluorescence, luminescence, phosphorescence, emission of light activated by enzyme, and fluorescence activated by enzyme In the optical grasping method of the characteristic value regarding any of
Measuring at least one position of the spectral centroid and the maximum value with the one of the radiation beam or absorption light, and at least one position of said spectral centroid and the maximum value among the spectral centroid and a maximum value The irradiation sample has a combination of data including color classification corresponding to at least one of the positions, generates an addition signal of a plurality of detection channels, and multiplies the combination result and the addition signal to generate an intensity image. Optical grasping method for characteristic values of wavelength dependence.   For differentiating different dyes, for measuring the local dye composition at the image point in the case of simultaneous use of multiple dyes, and for measuring the local shift of the emission spectrum depending on the local area where the dye is bound And a centroid and maximum measurement of the fluorescent chromium radiation for any one of the measurements for ion concentration based on the emissivity-identifying dye.   For differentiating different dyes, for measuring the local dye composition at the image point in the case of simultaneous use of multiple dyes, and for measuring the local shift of the absorption spectrum depending on the local periphery to which the dye is bound And measuring the center of gravity and the maximum of the reflected or transmitted fluorescent chromium excitation beam for any one of the measurements for ion concentration based on the dye absorption ratio. .   The method according to claim 3, wherein the radiation beam of the sample is divided by a dispersive element and detection is performed with local decomposition in at least one direction.   The method according to claim 4, wherein the light beam reflected from the sample or transmitted through the sample is divided by a dispersive element, and detection with local decomposition is performed in at least one direction for measuring the absorbance. Wherein the plurality of spectral weighting calculation between detection channels, performs the sum calculation and the summation calculation of detection channels of the detecting channel signals in a plurality detection channels, The method of claim 5.   Positioning is performed by weighting the detection channel signal by multiplying the detection channel and the weighting curve used in the weight calculation, calculating the total signal by the sum calculation of the target channel, and dividing the sum of the weight signal by the total signal. The method of claim 6, wherein the signal is calculated.   The method of claim 7, wherein the weighting curve is a straight line.   9. The method of claim 8, wherein the detection channel signal is converted and read out digitally, and the weighting and summation calculations are performed digitally by a computer.   The method according to claim 9, wherein the weighted calculation and the total calculation by analog data processing are performed by resistance cascade.   The method of claim 10, wherein the resistance can be adjusted.   The method of claim 11, wherein the weighting curve is adjustable.   13. The method of claim 12, wherein the detection channel signal is affected by nonlinear distortion of the input signal.   The method of claim 13, wherein the method affects the integration parameter.   The method of claim 14, wherein the method affects the characteristic line of the amplifier.   The method according to claim 15, wherein the position signal and the total signal are obtained in analog, converted and read out digitally. The method according to claim 16, wherein an upper signal and a lower signal corresponding to a sum of signals of individual channels calculated by weighting with a weighting curve having a reverse weighting direction are read out, digitally converted, and sent to a computer.   The method of claim 17, wherein the position signal and the total signal are used to form an image.   The method of claim 18, wherein a color-coded fluorescent image is formed.   The method of claim 19, wherein the overlap is performed by another image.   21. The method of claim 20, wherein the position signal and the overall signal are combined with lookup data.   The method of claim 21, wherein the look-up data provides either a display of various dyes and a developed display of the formed image.   23. The method according to claim 22, wherein the measurement signal is compared with the reference signal through a comparator in the detection channel, and the operation indicator is changed in any of the cases below the reference signal and when the reference signal is exceeded. .   24. The method according to claim 23, wherein each detection channel is blocked or made unresponsive as required.   25. The method of claim 24, thereby narrowing the analysis width of the spectral region.   26. The method according to claim 25, wherein each detection channel signal is generated by a connection of at least one integrator.   27. The method of claim 26, wherein the detection channel signal is generated by photon counting and subsequent digital / analog conversion.   28. The method of claim 27, wherein the photon counting is performed as a function of time.   29. The method of claim 28, for grasping any of single fluorescence, multiple photon fluorescence, and fluorescence excited by complex photons.   30. A method according to claim 29, wherein the sample is preferably a microtiter plate, wherein irradiation and detection are in parallel, especially in the examination of the active substance.   The method according to claim 30, for detection with a scanning near-field microscope.   32. The method of claim 31 for either single-photon and multi-photon dye fluorescence detection in a spectrometer correlated with fluorescence.   35. The method of claim 32, by confocal detection.   34. A method according to claim 33, comprising a scanning device.   35. The method of claim 34, wherein an X / Y scanner is used for irradiation.   36. The method of claim 35, comprising an X / Y scan table.   35. The method of claim 32, by non-confocal detection.   38. The method of claim 37, comprising a scanning device.   35. The method of claim 32, by descan detection.   40. The method of claim 39, by bright field imaging.   The method of claim 32, by point imaging.   35. The method of claim 32, with non-descan detection.   43. The method of claim 42, by bright field imaging.   33. The method of claim 32, by non-scanning, confocal or non-confocal detection and point imaging or bright field imaging.   45. The method of claim 44, according to an X / Y scan table. Characteristic wavelength-dependent characteristic values of the irradiated sample, mainly emission of at least one of fluorescence, luminescence, phosphorescence, emission of light activated by enzyme, and fluorescence activated by enzyme In the device configuration for optical grasping of the characteristic value related to either
Measuring at least one position of a spectral centroid and a maximum value for either the emitted or absorbed light, and measuring at least one position of the spectral centroid and a maximum value of the spectral centroid and the maximum value. Means are provided for synthesizing data including color classification corresponding to at least one position, generating an addition signal of a plurality of detection channels, and multiplying the synthesis result and the addition signal to generate an intensity image. A device configuration for optically grasping characteristic characteristic values of wavelength dependency of irradiated samples.   The apparatus configuration according to claim 46, wherein the radiation beam of the sample is divided by a dispersive element and detection is performed with local decomposition in at least one direction.   48. The apparatus configuration according to claim 47, wherein fluorescent light is split.   49. The apparatus configuration according to claim 48, wherein the light beam reflected from the sample or transmitted through the sample is divided by a dispersive element to perform detection with local decomposition in at least one direction for the absorbance measurement. The spectral weighting calculation between a plurality of detection channels, performs the sum calculation and the summation calculation of detection channels of the detecting channel signals in a plurality detection channels, device structure of claim 49.   Positioning is performed by weighting the detection channel signal by multiplying the detection channel and the weighting curve used in the weight calculation, calculating the total signal by the sum calculation of the target channel, and dividing the sum of the weight signal by the total signal. The apparatus configuration according to claim 50, wherein the apparatus calculates a signal.   52. The apparatus configuration of claim 51, wherein the weighting curve is a straight line.   53. The apparatus configuration according to claim 52, wherein the signal of the detection channel is converted and read out digitally, and weighting and summation calculation are performed digitally by a computer.   54. The apparatus configuration according to claim 53, wherein the weight calculation and the sum calculation by the processing of analog data are performed by a resistance cascade.   55. The device configuration of claim 54, wherein the resistance can be adjusted.   56. The apparatus configuration of claim 55, wherein the weighting curve is adjustable.   57. The apparatus configuration according to claim 56, wherein the position signal and the total signal are obtained in analog, converted and read out digitally. 58. The apparatus configuration according to claim 57, wherein an upper signal and a lower signal corresponding to a sum of signals of individual channels calculated by weighting with a weighting curve having a reverse weighting direction are read out, digitally converted, and sent to a computer.   59. Apparatus arrangement according to claim 58, wherein position signals and total signals are used to form an image.   60. The apparatus configuration of claim 59, wherein a color-coded fluorescent image is formed.   61. Apparatus arrangement according to claim 60, wherein the overlap is performed by another image.   62. The apparatus configuration of claim 61, wherein the position signal and the overall signal are combined with the lookup data.   64. The apparatus configuration according to claim 62, wherein at least one of display of various dyes and development display of a formed image is made by look-up data.   64. The apparatus configuration according to claim 63, wherein the measurement signal and the reference signal are compared through a comparator in the detection channel, and the operation indicator is changed when the reference signal is equal to or lower than the reference signal or when the reference signal is exceeded.   The apparatus configuration according to claim 64, wherein each of the detection channels is blocked or made unresponsive as necessary.   The apparatus configuration according to claim 65, wherein the analysis target width in the spectral region is narrowed.   68. The apparatus arrangement of claim 66, wherein each detection channel signal is generated by a connection of at least one integrator.   68. Apparatus arrangement according to claim 67, wherein the detection channel signal is generated by photon counting and subsequent digital / analog conversion.   69. The apparatus configuration of claim 68, wherein photon counting is performed as a function of time.   70. The apparatus configuration according to claim 69, for grasping at least one of single photon fluorescence, multiple photon fluorescence, and fluorescence excited by complex photons.   71. Apparatus arrangement according to claim 70, wherein the sample is preferably a microtiter plate, and irradiation and detection are performed in parallel, especially in the examination of the active substance.   72. Apparatus configuration according to claim 71 used in a microscope.   73. Apparatus arrangement according to claim 72 for detection with a scanning near-field microscope.   74. The apparatus configuration of claim 73 for detecting dye fluorescence of at least one of single photons and multiphotons in a spectrometer correlated with fluorescence.   75. A device configuration according to claim 74 by confocal detection.   76. A device configuration according to claim 75, according to a scanning device.   77. The apparatus configuration according to claim 76, wherein an X / Y scanner is used for irradiation.   78. Apparatus configuration according to claim 77, based on an X / Y scan table.   75. The device configuration of claim 74, with non-confocal detection.   80. Apparatus configuration according to claim 79, according to a scanning device.   75. Apparatus configuration according to claim 74, based on descan detection.   82. Apparatus configuration according to claim 81, by bright field imaging.   75. A device configuration according to claim 74, by point imaging.   75. The apparatus configuration of claim 74, with non-descan detection.   85. Apparatus arrangement according to claim 84, by bright field imaging.   75. Apparatus arrangement according to claim 74 by non-scanning, confocal or non-confocal detection and point imaging or bright field imaging.   90. The apparatus configuration according to claim 86, according to an X / Y scan table.

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