JP2004502173A - Method and apparatus configuration for optically ascertaining wavelength-dependent characteristic values of an illuminated sample - Google Patents

Abstract

Disclosed is a characteristic characteristic value of wavelength dependence of an irradiation sample, mainly fluorescence and / or luminescence and / or phosphorescence and / or emission of light activated by an enzyme and / or fluorescence activated by an enzyme. Method for optically grasping characteristic quantities, in particular with respect to radiation and / or absorption, for measuring at least one emphasis and / or maximum value for radiation and / or absorption light to distinguish between different dyes, and / or Or for the measurement of local dye composition at image points in the case of simultaneous use of multiple dyes, and / or for the measurement of local shifts in the emission spectrum that depend on the local surroundings where the dyes are bound, and / or For the measurement of the ion concentration based on the emissivity discriminating dye, the center of gravity and / or the maximum value of the fluorescent chromium emitted light are measured. Method .FIELD 6

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method and a device arrangement for the examination of 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.
Also included is a method based on fluorescence detection for testing active substances (high-throughput screening).
[0002]
By moving from the detection of a small number of broad-spectrum dye bands to the simultaneous recording of complete spectra, the identification of sample properties, mostly from an analytical or functional aspect, related to spatial substructure or kinetic processes, New possibilities open up 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 spatial structure of a thick sample. In addition, it is also possible to detect partial shifts in the emission spectral band of the dye and classify the spatial structure. The data recording rate is not reduced by the configuration of the apparatus.
[0003]
[Prior art]
One of the classical applications of light microscopy for examining biological preparations is fluorescence microscopy (Literature: Pauly "Bioconfocal Microscopy Handbook", Planum Press, 1955). In this case, specific dyes are used for special labeling of the cell parts.
[0004]
The dye molecules are excited from the photon ground state to the excited state by the absorption of one incident photon having a 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, migration under the emission of fluorescent photons is of paramount importance. The wavelength of the emitted photons is in principle shifted to the red compared to the excitation radiation due to the Stokes displacement. That is, it is on the long wavelength side. The Stokes displacement allows separation of the fluorescent light from the excitation light.
[0005]
The fluorescence is separated from the excitation radiation by a suitable dichroic beam splitter in combination with a block filter and observed separately. By doing so, it is possible to depict individual cell parts colored with various dyes. However, in principle, it is also possible to color several parts of the preparation simultaneously with different dyes which have a unique deposition behavior (multiple fluorescence). Again, a special dichroic beam splitter is used to distinguish the fluorescent signals emitted from the individual dyes.
[0006]
In addition to the excitation of dye molecules by one photon with high energy (one-photon absorption), excitation by multiple photons with lower energy is also possible (FIG. 1b). In this case, the sum of the energies of the individual photons is many times higher than the high energy photons. This type of dye excitation is called multiphoton absorption (Literature: Cory, 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, and thus its wavelength is shorter than that of the excitation radiation. Separation of the excitation light from the emitted light is performed in the same manner as in the case of one-photon absorption.
[0007]
The known technique will be described below using an example of a confocal laser scanning microscope (LSM) (FIG. 2 [L1]).
LSM is roughly divided into four modules: (1) light source, scanning module, detection unit, and microscope. These modules are described in more detail below. In addition, DE 19702753A1 is also helpful.
[0008]
Lasers of various 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 inspected. The excitation light is generated in the light source module. In this case, various lasers (argon, argon krypton, TiSa laser) are used. In addition, in the light source module, the selection of the wavelength and the adjustment of the intensity of the necessary excitation wavelength are performed by using, for example, an acousto-optic crystal. Subsequently, the laser beam is directed into the scanning module through a fiber or a suitable mirror device.
[0009]
The laser beam generated by the light source passes through the objective lens (2), and is focused into the preparation through a scanner, a scanning lens system, and a cylindrical lens while suppressing diffraction. A dot is scanned in the xy direction while focusing on the sample. The dwell time on the pixel during sample scanning often ranges from less than a microsecond to several seconds.
[0010]
In the case of fluorescence confocal detection (descanning detection), light emitted from a focal plane (sample) and planes above and below the focal plane reaches a dichroic beam splitter (MDB) through a scanner. MDB separates fluorescence from excitation light. The fluorescence is then focused by a stop (confocal stop / pinhole) exactly in the plane conjugate to the focal plane. This blocks out-of-focus fluorescent components. By varying the size of the stop, it is possible to adjust the optical resolution of the microscope. Behind the stop is another dichroic block filter (EF), which again blocks the excitation beam. After passing through the block filter, the fluorescence is measured by a point image detector (PMT).
[0011]
When multiphoton absorption is used, excitation of dye fluorescence occurs in a small volume portion where the excitation intensity is particularly strong. This area is only slightly larger than the detection area when using a confocal device. Therefore, the use of a confocal stop can be omitted, and detection can be performed immediately after the objective lens (non-descan detection).
[0012]
In another arrangement for detecting dye fluorescence excited by multiphoton absorption, an additional descan detection is performed, but in this case the pupil of the objective lens is imaged into the detection unit (non-confocal). Descan detection).
[0013]
Due to the device configuration of the two detectors connected to the corresponding one-photon absorption or multi-photon absorption, only the plane (optical cross section) existing in the focal plane of the objective lens is reproduced from the three-dimensional illumination image. . Subsequent drawing of several optical cross-sections in the xy plane at various depths z of the sample produces a computer-supported three-dimensional image of the sample.
Therefore, LSM is suitable for testing thick preparations. The excitation wavelength is determined by the intrinsic absorption characteristics of the dye used. The dichroic filter adapted to the emission characteristics of the dye ensures that only the fluorescence emitted from each dye is measured by the point image detector.
[0014]
In biomedical applications, several different cell areas 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 properties or on the basis of emission properties (spectrum). In that case, additional splitting of the fluorescence of the multiple dyes is performed by the sub-beam splitter (DBS), and the individual dye emission detection is performed in separate point image detectors (PMTx).
[0015]
This arrangement does not allow the user to flexibly adapt 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 the device configuration based on DE..., The fluorescence is spectrally divided by one prism. The method differs from the above-described arrangement with a dichroic filter only in that the characteristic curve of the filter used can be adjusted, but also that the emission band of one dye per point image detector is recorded. It is preferable.
[0016]
[Problems to be solved by the invention]
When the emission spectra of the two dyes overlap, conventional detectors have limitations. To avoid overwriting between the two dyes, the spectral detection area must be limited. The area where both dyes overlap is thus simply excised and not detected. This degrades the efficiency of the detection unit. The same signal-to-noise ratio can only be obtained by increasing the pump power, thereby compromising the preparation. Therefore, today up to six dye probes are used simultaneously. Otherwise, dyes cannot be separated because the emission bands are strongly overlapped.
Heretofore, dyes have been modified such that their absorption properties or their emission properties differ from each other.
[0017]
FIG. 3a) shows the emission spectra of various typical dyes. The emitted signal is plotted as a function of wavelength. It can be seen that the positions and shapes of the emission spectra differ between the dyes numbered 1 to 4 and numbered. However, these dyes are often toxic to biological preparations. Therefore, the evolution of syncytia in living preparations cannot be examined. In the late 1990's, so-called fluorescent proteins (GFP, YFP, CFP, TOPAS, GFT, RFP), which are pigments in the natural world, were discovered (Clonetech, USA).
[0018]
These dyes are distinguished by little effect on the sample. Therefore, they are particularly suitable for labeling cellular regions within a biological preparation. However, there is the disadvantage that the emission properties of the dyes are only slightly different.
[0019]
FIG. 3b) shows the wavelength-dependent emission 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.
[0020]
In yet another method for localizing 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 fluoresces. 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, followed by the emission of the second dye.
[0021]
FIG. 3d) shows an energy level diagram of this process, which is referred to in the literature as fluorescence resonance energy transfer (FRET) (Literature: Fan et al .; Biophysics Journal, V76, May 1999, 24122420). ). By detecting the radiation beams of both dyes in this manner and determining the ratio of the two detector signals, the distance between the two molecules can be determined.
It is further 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.
[0022]
FIG. 3c) shows that the emission spectrum of the dye depends on the environment in which the dye is located. In the figure, the radiation signal is depicted as a function of wavelength.
The wavelength shift can be as large as tens of nm. As for the dependence of this wavelength shift on the environment, no more detailed inspection has been known so far. This is because this type of inspection is difficult, if at all possible, with state-of-the-art methods.
[0023]
Certainly, spectrometers are also used today, in combination with LSM. In this case, a conventional, mostly high-resolution spectrometer is used instead of a point image detector (Patent Dixon et al. US Pat. No. 5,192,980). However, they only record the emission spectrum one by one or in an averaged manner over a certain area. That is, this is a kind of spectroscopic analysis.
[0024]
In another arrangement, the lifetime of the dye fluorescence is measured. Again, it can be concluded that the problem is the environment, but recording a complete image requires a long data reception time. Therefore, this method can only be used in a limited manner for testing live preparations.
[0025]
In another application of fluorescence microscopy, ion concentrations (e.g., Ca⁺, K⁺, Mg²⁺, Zn⁺, ...). It uses special dyes or dye formulations whose spectra shift depending on ion concentration (eg, Hula, India, Fluo; Molecular Probes).
[0026]
FIG. 4a) shows the emission spectrum of India 1 depending on the concentration of calcium ions.
FIG. 4b) shows an example of the emission spectrum depending on the calcium ion concentration in the case of the combination of Fluo 3 and the hula red dye. These special dyes are called emissivity discriminating dyes.
By detecting the two fluorescent regions shown in FIG. 4a) and determining the ratio of the two intensities, the corresponding ion concentration can be derived. In many cases, this measurement will analyze the dynamic change in ion concentration in a biological preparation, which requires a time division within 1 millisecond.
[0027]
[Means for Solving the Problems]
Thus, the object of the present invention is a new detection method that is flexible and free of programming. These methods must be usable in imaging systems as well as analytical microscopy systems. Therefore, the data adoption rate must not deteriorate when using these methods.
[0028]
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 of up to 200 nm and a scanning near-field microscope for inspecting the surface with a high resolution of up to 10 nm. There is.
There are also fluorescence correlation microscopes for quantitative measurement of molecular concentration and for measurement of molecular diffusion. Further, 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 setting goals are solved by a method and a device configuration according to the claims.
[0029]
According to the present invention, it is possible to separate even partially or completely overlapping dyes, while avoiding or overcoming the disadvantages and limitations of the prior art. Thereby, multiple fluorescence recording using a fluorescent protein in the natural world is possible. Further, according to this method, the wavelength shift based on the environmental deviation in the preparation to be inspected can be measured very efficiently.
[0030]
BEST MODE FOR CARRYING OUT THE INVENTION
Behind the flexible detection method is spectrally divided fluorescence detection. In that case, the emitted light is split from the excitation light by a main color splitter (MDB) in a scanning module or in a microscope (for multiphoton absorption).
[0031]
FIG. 5 shows a block diagram of the detector unit arranged subsequently.
In the case of confocal detection, light emitted from the sample is focused through the imaging lens system PO and further through the aperture (pinhole) PH, thereby blocking out-of-focus fluorescent light. No aperture is required for non-descan detection.
[0032]
In this case, the light is decomposed by the angular dispersion element DI into its spectral components. Prisms, gratings, and acousto-optic elements are used as the angular dispersion elements. The light split into its spectral components by the dispersive element is subsequently imaged on the line detector DE. That is, the line detector DE measures a wavelength-dependent emission signal and converts it into an electric signal S (). In addition, a linear filter for suppressing the excitation wavelength can be connected in series to the detection unit.
[0033]
FIG. 6 shows a possible embodiment in which the detector unit shown in the block diagram in FIG. 5 is used as the optical beam path.
The structure is essentially based on the Sarny-Turner structure. In the case of confocal detection, light L from a sample is focused by a pinhole lens system PO through a confocal stop PH. In the case of multiphoton absorption in non-descan detection, this aperture is unnecessary.
[0034]
The first imaging mirror S1 collimates the fluorescence. The light then impinges on a linear grating G, for example a grating having 651 lines per millimeter. The grating deflects light in different directions depending on its wavelength. The second imaging mirror S2 focuses the individual wavelength components that have been spectrally split onto the channels of the corresponding line detector DE.
[0035]
It is particularly preferred to use a H7260 line secondary electron multiplier from Hamamatsu. This detector is of a 32-channel type and has high sensitivity. The free spectral range of the above embodiment is about 350 nm. The free spectral range is evenly distributed over the 32 channels of the line detector in this configuration, resulting in an optical resolution of about 10 nm. Therefore, this arrangement is only suitable for spectroscopic measurements. However, it is advantageous to use this in an imaging system. This is because the detection spectrum band is relatively wide and the signal for each detection channel is relatively large. The free spectral range movement can additionally be effected, for example, by twisting the grating.
[0036]
Yet another embodiment involves the use of a matrix detector (eg, CCD,...). In this case, at a certain coordinate, division into various wavelength components is performed by the dispersion element. In the direction that remains on the matrix detector, a complete line (or gap) of the scanned image forms. This embodiment is particularly advantageous when configuring a line scanner (Literature: Cory, Kino; "Confocal Optical Microscopy and Related Imaging Systems"; Academic Press 1996).
[0037]
The structural principle essentially conforms to the LSM shown in FIG. However, an image is formed not at a point focus but at a single line at the focus, and the inspection sample is scanned only in one direction. In such a structure, a slit stop is used as a confocal stop instead of a hole stop. Non-descanning detection using multi-grating absorption is also performed by this device configuration. Again, the confocal stop can be omitted.
[0038]
Hereinafter, a measurement algorithm in the apparatus configuration shown in FIG. 6, that is, in the point scanner will be described. However, this algorithm can be applied to the device configuration of the line scanner without limitation.
As is clear from FIGS. 3 and 4, the individual dyes differ in the position and shape of the emission spectrum. The algorithm (FIG. 7) determines for each image point the position of the center of gravity or the maximum value of the radiation signal detected at the image point.
[0039]
In the following, advantageous and practicable methods of determining the center of gravity will be described in more detail. Other methods of determining the center of gravity or maximum, such as, for example, interpolation fitting, are unconditionally part of the present invention. In that case, the signal for each channel (left diagram) detected by the line detector is multiplied by a calibration function (right diagram). That is, every channel has a certain weight.
[0040]
The left diagram in FIG. 7 shows the measured emission signal for each channel number where the signal was detected. In the diagram on the right, examples of the weighting function are listed for individual channels by channel number.
The weighted individual signals per channel are summed and divided by the sum of the unweighted individual signals (total signal). As a result, a signal is obtained as a feature quantity representing the emission spectrum and thus the center of gravity of the excitation dye (FIG. 8A). This signal is hereinafter referred to as a position signal.
[0041]
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 the position and type of their emission spectrum. Furthermore, for example, if a certain dye is used, it is possible to measure the wavelength shift of the emission spectrum which depends on the environment.
[0042]
If several dyes are present at an image point at the same time, a linear connection of the center of gravity expressed by the following equation is established depending on the relative concentrations of one dye and another dye:
Position =ⁿΣ_kPos_kC_k
Where Pos_kIs the characteristic center of gravity of the pigment, C_kIs the dye concentration, and n is the number of dyes that are simultaneously excited at the image point. Therefore, the present algorithm can also determine the ion concentration and can be used to detect FRET signals. In addition, it is also possible to analyze the partial overlap of two or more dyes (colocalization measurement).
[0043]
FIG. 8b shows a signal that depends on ion concentration when using two dyes (eg, Fluo 3 and Hula Red, Molecular Probes) or one dye having two characteristic emission bands (eg, Molecular Probes, India). It is drawn. It represents a position signal dependent on the ion concentration.
As already mentioned, the position signal is a position measure of the position of the center of gravity of the emission spectrum. That is, it can be used as a mask for calculating a color-coded intensity image.
[0044]
This algorithm is illustrated in FIG. In that case, as a first step, the mask (ie, the position signal) is multiplied with the corresponding look-up data. The look-up data includes a customized color classification of the emission spectral centroid location. Subsequently, the result of the multiplication is multiplied by the intensity value (total signal), that is, the lightness of the color is adapted to the actual fluorescence intensity. Depending on the choice of the look-up data, respectively, a color-masked intensity image (discrete color distribution) can be produced, ie an intensity image with one dye per pixel, or a mixed color by combining individual image points. Can be created as one image.
[0045]
The decisive advantage of this method is that any dye can be detected with the overall fluorescence (total signal) independent of the overlap of the emission spectra, and nevertheless the dye can still be displayed in a separate state (by position signal) It is in the point. Thus, strongly overlapping dyes (FIG. 3c) can be detected particularly effectively.
[0046]
The implementation of the algorithm according to the configuration of FIG. 6 can be performed digitally or analogously. Both device configurations are described in detail below. An arrangement 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 multi-channel PMT is converted into a voltage by the first amplifier A (connected as a current / voltage converter) and amplified. The voltage is directed to an integrator I, where the signal is integrated for that time (eg, the dwell time on the pixel).
[0047]
In order to determine the value quickly, after the integrator I, a comparator K having a switching threshold as follows, which is a simple comparator, that is to say 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 switching thresholds or if the input signal is outside (below or above) the switching threshold. Can be done. The configuration of the comparator or window comparator can be before or after the integrator. A connection arrangement without an integrator (so-called amplification mode) is likewise conceivable.
[0048]
In the case of an arrangement in the amplifier mode, the comparator K is adapted to the appropriate standards. The output of the comparator K is used as a control signal for a switch register Reg that connects directly to the active channel (online), or for individually selecting an active channel (offline), the state at that time is determined by the associated coupler V Through to the computer. The output signal of the integrator I is led directly to another level-matching amplifier A1 for the subsequent A / D conversion. The A / D converted values are 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.
[0049]
FIG. 11 illustrates 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 by the dwell time on the pixel.
After the integrator, a comparator K for comparing the integrated signal with the reference signal is provided.
[0050]
If the integrated signal is smaller than the comparator threshold, the fluorescence signal is not being 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 contributes only to the noise signal to the total signal. The comparator operates the switch S through Reg in such a case, and the individual channel is shut off for the pixel being measured. That is, the spectral region important for the actually measured image point is automatically selected by the combination of the comparator and the switch.
[0051]
Subsequently, the integrated voltage signal is again converted to a current via the resistor R. Therefore, any individual channel generates a current depending on the intensity of the fluorescence arriving at the individual channel. In subsequent positions, all the individual channels adjacent to each other are connected to another resistor R1 between them.
[0052]
The total current generated at the top and bottom of the detector row is also converted to a voltage by the current / voltage converter A1. The voltages Eo and Eu at the top and bottom correspond to the sum of the individual channel signals weighted by the straight lines in the opposite direction. Both signals at the top and bottom are subsequently 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 guided to an analog divider and output a position signal.
[0053]
The integrated signal and the position signal are subsequently converted into digital signals by an analog-to-digital converter, respectively, and further processed by a computer or DSP. However, in that case, the upper and lower signals can be converted without any restrictions and can also be computer processed. In this case, the computer will determine the total signal and the position signal. In both cases, the algorithm of FIG. 9 is executed by a computer.
[0054]
However, the algorithm of FIG. 9 can also be implemented with the connection of FIG. In this regard, three possibilities are described in more detail below.
In the first device configuration, the resistance (R1) existing between the adjacent detection channels is changed, and the multiplication is performed using the lookup data. Remaining connections are left as originally set.
[0055]
In the second configuration, the data is multiplied by the lookup data in the amplifier A1. In that case, the amplifier A1 is operated with a variable nonlinear characteristic curve. In the third arrangement (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 the modification of the amplification in (A), the modification of the integration time in (I), the additional offset input before the integrator and / or the imposition of digital effects by the photons counted by the photon counting device. All three methods can also be combined arbitrarily with one another.
[0056]
In order to avoid artifacts, it is necessary to suppress or at least attenuate the excitation light backscattered from the sample during the fluorescence measurement so that it is smaller than the emission maximum or of the same sequence. For this purpose, an additional linear filter as described above 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 channels, the backscattered or reflected excitation beam can also be blocked by the switch of FIG. it can.
[0057]
The device configuration of FIG. 11 has several advantages over the device configuration of FIG. The most noticeable advantage is that only two channels need to be converted to digital data and sent to the computer. This minimizes the data rate to be processed by the computer. This is especially true when the method is applied to real-time microscopy, for example, in which more than 50 images are detected at 512 × 512 pixels and 12-bit pixel tones, so that very fast-moving dynamic processes can be recorded. is important. Also, with this method, there is no limit on the number of individual channels of the line detector (matrix detector) used, and thus on the size of the detectable spectral region and / or the spectral resolution of the spectral sensor.
[0058]
Further, in the device shown in FIG. 10, the signal level that can be converted is much lower. Thereby, the assumed value of the signal-to-noise ratio becomes small.
In both arrangements for the above evaluation algorithm conversion, integrator switching was used to detect individual channel signals. Also, photon counting can be performed without restriction in individual channels. However, the device configuration shown in FIG. 10 has the advantage that it can even provide complete spectral information in addition to the position signal for additional image processing. The present invention therefore also includes a combination of both device configurations.
[0059]
FIG. 12 shows a measurement result by the device configuration shown in FIGS. 10 and 11.
FIG. 12a depicts the emission spectra of the used dyes GFP, CFP and DI measured with a spectrometer. Excitation of the dye was performed with an argon laser having a wavelength of 488 nm. In the following, these dyes were introduced, in particular, into specific areas of biological preparations.
[0060]
FIG. 12b shows a histogram of the position signal when scanning over a sample piece where all three dyes are present. Three maxima where the three dyes have their characteristic position signals are clearly visible in the histogram. The positional relationship among the three dyes is as shown in the following table.
Pigment CFP GFP DI
Position signal (relative wavelength shift) 14 30 80
Thus, the dyes should be easily separated by the device configuration according to the present invention. In addition, there are local wavelength shifts due to various local environments within the dye. This appears in the maximum value width of the individual dye in the histogram.
[0061]
FIG. 13a shows an intensity image formed from the total signal.
FIG. 13b depicts the corresponding image formed from the position signal.
This image represents the corresponding centroid of the emission spectrum. The differently stained cell nuclei (partially colored with GFP and partly with CFP) and the cellular framework colored with DI are clearly distinguishable.
FIG. 13c shows a calculated and color-coded intensity image corresponding to the algorithm of FIG. Individual regions to which various dyes have been added are separated by color coding. Separation is clarified by displaying the image in three RGB channels.
For comparison, an image measured by detection according to 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 that the fluorescent signals of the different dyes were overwritten on each other.
[0062]
Due to such a very narrow detection band, only a part of the fluorescence signal emitted from the sample could be measured. In order to keep the image at the same signal-to-noise ratio as much as possible, the excitation power must be multiplied.
From this, it is clear that the device configuration based on the present invention has higher efficiency than the device configuration based on the state of the art. 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 area being colored yellow or a double phenomenon in the area within the two image channels (R and G).
[Brief description of the drawings]
FIG. 1 a) one-photon absorption, b) multi-photon absorption
FIG. 2 Known LSM construction
FIG. 3. Typical spectra a) dye, b) fluorescent protein, c) wavelength shift / environment dependent, d) ΔFRET
FIG. 4: Typical spectra for ratio measurement, a) 1 dye and emissivity, b) 2 dye and ion dependent signal
FIG. 5 is a block diagram of a detector unit / optical system.
FIG. 6 shows a configuration example of a detector unit / optical system.
FIG. 7: Algorithm for determining the position of the radiation spectrum
FIG. 8 shows a typical course of the position signal, a) depending on the position of the emission spectrum, b) depending on the ion concentration.
FIG. 9: Algorithm for creating color-coded intensity images when using several dyes
FIG. 10 is an example of an electronic configuration for digital measurement.
FIG. 11 is an example of an electronic configuration for measuring an analog signal.
FIG. 12. Histogram of a) dye spectrum, b) emission spectrum shift of a)
FIG. 13 shows an experiment for separating a dye, a) an overall intensity image, b) an image of a wavelength shift, c) an expanded intensity image, and d) an intensity image with a conventional detector based on the state of the art.
[Explanation of symbols]
PO imaging lens system
DI Angular dispersion element
DE line detector
S () electric signal
PH confocal aperture
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 lattice
S2 Second imaging mirror

Claims (87)

Characteristic values of the wavelength dependence of the irradiated sample, mainly fluorescence and / or luminescence and / or phosphorescence and / or emission of enzyme-activated light and / or especially emission of enzyme-activated fluorescence. And / or in the method of optically grasping characteristic values relating to absorption,
A method for optically grasping characteristic values of wavelength-dependent characteristic of an irradiation sample, wherein at least one emphasis and / or maximum value is measured for the emitted light and / or the absorbed light. Local shifts in the emission spectrum that are dependent on the local surroundings of the dye to distinguish between the various dyes and / or to determine the local dye composition at image points in the case of simultaneous use of multiple dyes, and / or 2. The method according to claim 1, wherein the measurement of the center of gravity and / or the maximum value of the fluorescent chromium radiation is carried out for the determination of and / or for the determination of the ion concentration based on the emissivity discriminating dye. Local shifts in the absorption spectrum depending on the local surroundings of the dye to distinguish between the various dyes and / or to measure the local dye composition at image points in the case of simultaneous use of multiple dyes, and / or The method according to claim 1, wherein the measurement of the center of gravity and / or the maximum of the reflected or transmitted fluorescent chromium excitation light is carried out for the determination of the chromium excitation light and / or the ion concentration measurement based on the dye absorption ratio. The device configuration according to at least one of the preceding claims and splitting of the fluorescent light beam, wherein the device divides the light beam of the sample by a dispersive element and performs detection with spatial resolution in at least one direction. Item 2. The method according to item 1. The method according to at least one of the preceding claims, wherein for the measurement of the absorbance, the light rays reflected from or transmitted through the sample are split by a dispersive element and detection with spatial resolution in at least one direction is performed. . The method according to at least one of the preceding claims, comprising performing a spectral weight calculation between the plurality of detection channels, calculating the sum of the detection channel signals in the weight calculation channels, and calculating the sum of the detection channels. Weighting the signal of the detection channel by multiplying the detection channel by the weighting curve, calculating the total signal by calculating the sum of the target channels, and calculating the position signal by dividing the sum of the weighted signals by the total signal; A method according to at least one of the claims. A method according to at least one of the preceding claims, wherein the weighting curve is a straight line. A method according to at least one of the preceding claims, wherein the signals of the detection channels are converted and read out digitally, and the weighting and summation are performed digitally by a computer. The method according to at least one of the preceding claims, wherein the weighting calculation and the sum calculation by processing the analog data are performed by resistance cascade. A method according to at least one of the preceding claims, wherein the adjustment of the resistance is possible. A method according to at least one of the preceding claims, wherein the weighting curve is adjustable. Method according to at least one of the preceding claims, wherein the signal of the detection channel is affected by non-linear distortion of the input signal. A method according to at least one of the preceding claims, wherein the method affects an integration parameter. A method according to at least one of the preceding claims, wherein the method influences a characteristic line of an amplifier. Method according to at least one of the preceding claims, wherein the position signal and the integrated signal are determined in analog, converted and read out digitally. The method according to at least one of the preceding claims, wherein the upper and lower signals corresponding to the sum of the signals of the individual channels weighted by the inverse weighting curve are read out, digitized and sent to a computer. A method according to at least one of the preceding claims, wherein the position signal and the integrated signal are used to form an image. The method according to at least one of the preceding claims, wherein a color-coded fluorescent image is formed. A method according to at least one of the preceding claims, wherein the overlap is performed by another image. A method according to at least one of the preceding claims, wherein the position signal and the total signal are combined with the lookup data. A method according to at least one of the preceding claims, wherein the look-up data provides an indication of various dyes and / or an expanded indication of the formed image. Method according to at least one of the preceding claims, wherein the comparison of the measurement signal with the reference signal is performed via a comparator in the detection channel, and the activation indicator is changed if the reference signal is below and / or exceeded. . The method according to at least one of the preceding claims, wherein the blocking and / or non-response of each detection channel is performed as required. Method according to at least one of the preceding claims, whereby the spectral width of the spectral region is narrowed. Method according to at least one of the preceding claims, wherein the signal of the detection channel is generated by the connection of at least one integrator, respectively. A method according to at least one of the preceding claims, wherein the signal of the detection channel is generated by photon counting and subsequent digital / analog conversion. A method according to at least one of the preceding claims, wherein the photon counting is performed as a function of time. A method according to at least one of the preceding claims for ascertaining single and / or multiple photon fluorescence and / or fluorescence excited by complex photons. The method according to at least one of the preceding claims, wherein the sample is preferably a microtiter plate, in which irradiation and detection are performed in parallel, in particular for testing active substances. A method according to at least one of the preceding claims for detection with a scanning near-field microscope. A method according to at least one of the preceding claims for single-photon and / or multi-photon dye fluorescence detection in a spectrometer correlated with fluorescence. A method according to at least one of the preceding claims, by confocal detection. A method according to at least one of the preceding claims, comprising a scanning device. A method according to at least one of the preceding claims, wherein an X / Y scanner is used in the irradiation. A method according to at least one of the preceding claims, comprising an X / Y scan table. A method according to at least one of the preceding claims, by non-confocal detection. A method according to at least one of the preceding claims, comprising a scanning device. Method according to at least one of the preceding claims, by descan detection. Method according to at least one of the preceding claims, by bright field imaging. Method according to at least one of the preceding claims, by point imaging. A method according to at least one of the preceding claims, by non-descanning detection. Method according to at least one of the preceding claims, by bright field imaging. Method according to at least one of the preceding claims, by non-scanning, confocal or non-confocal detection and by point or bright field imaging. Method according to at least one of the preceding claims, by means of an X / Y scanning table. Characteristic values of the wavelength dependence of the irradiated sample, mainly fluorescence and / or luminescence and / or phosphorescence and / or emission of enzyme-activated light and / or especially emission of enzyme-activated fluorescence. And / or in an apparatus configuration for optically grasping characteristic values related to absorption,
Apparatus for optically ascertaining the characteristic value of the wavelength-dependent characteristic of an illuminated sample, comprising means for measuring at least one emphasis and / or maximum value for the emitted light and / or the absorbed light. Constitution. 47. The apparatus configuration according to claim 46, wherein the radiation emitted from the sample is split by a dispersive element, and detection with spatial resolution is performed in at least one direction. Apparatus configuration according to at least one of the preceding claims, wherein the splitting of fluorescent light is performed. Apparatus according to at least one of the preceding claims, wherein the light reflected from or transmitted through the sample is split by a dispersive element for absorption measurement and detection with spatial resolution in at least one direction. Constitution. The apparatus configuration according to at least one of the preceding claims, wherein the apparatus performs spectrum weight calculation among a plurality of detection channels, sum calculation of detection channel signals in the weight calculation channel, and sum calculation of the detection channels. Weighting the signal of the detection channel by multiplying the detection channel by the weighting curve, calculating the total signal by calculating the sum of the target channels, and calculating the position signal by dividing the sum of the weighted signals by the total signal; An apparatus configuration according to at least one of the claims. Apparatus arrangement according to at least one of the preceding claims, wherein the weighting curve is a straight line. The apparatus configuration according to at least one of the preceding claims, wherein the signal of the detection channel is converted and read out digitally, and the weighting and sum calculation are performed digitally by a computer. The apparatus configuration according to at least one of the preceding claims, wherein the weight calculation and the sum calculation by processing the analog data are performed by a resistance cascade. Device arrangement according to at least one of the preceding claims, wherein the resistance can be adjusted. Device arrangement according to at least one of the preceding claims, wherein the weighting curve is adjustable. The device configuration according to at least one of the preceding claims, wherein the position signal and the integrated signal are obtained in an analog form, converted and read out digitally. The apparatus configuration according to at least one of the preceding claims, wherein an upper signal and a lower signal corresponding to the signal sum of the individual channels weighted and calculated by the inverse weighting curve are read out, digitally converted, and sent to a computer. Apparatus arrangement according to at least one of the preceding claims, wherein the position signal and the integrated signal are used for forming an image. Apparatus arrangement according to at least one of the preceding claims, wherein a color-coded fluorescent image is formed. Apparatus arrangement according to at least one of the preceding claims, wherein the overlap is effected by another image. Apparatus arrangement according to at least one of the preceding claims, wherein the position signal and the total signal are combined with the lookup data. Apparatus configuration according to at least one of the preceding claims, wherein the look-up data provides an indication of various dyes and / or an expanded indication of the formed image. Apparatus configuration according to at least one of the preceding claims, wherein the comparison of the measurement signal with the reference signal is performed via a comparator in the detection channel, and the activation indicator is changed if the reference signal is below and / or over the reference signal. . The device configuration according to at least one of the preceding claims, wherein blocking and / or non-response of each detection channel is performed as necessary. Apparatus configuration according to at least one of the preceding claims, by narrowing the spectral width of the spectral region to be analyzed. Apparatus arrangement according to at least one of the preceding claims, wherein the signals of the detection channels are each generated by the connection of at least one integrator. Device arrangement according to at least one of the preceding claims, wherein the signal of the detection channel is generated by photon counting and subsequent digital / analog conversion. Apparatus arrangement according to at least one of the preceding claims, wherein the photon counting is performed as a function of time. Apparatus arrangement according to at least one of the preceding claims for ascertaining single and / or multiple photon fluorescence and / or fluorescence excited by complex photons. Apparatus arrangement according to at least one of the preceding claims, wherein the sample is preferably a microtiter plate, in which irradiation and detection are performed in parallel, in particular for testing active substances. Apparatus arrangement according to at least one of the preceding claims, in a microscope. Apparatus arrangement according to at least one of the preceding claims for detection with a scanning near-field microscope. Apparatus arrangement according to at least one of the preceding claims for single-photon and / or multi-photon dye fluorescence detection in a spectrometer correlated with fluorescence. Apparatus configuration according to at least one of the preceding claims, by confocal detection. Apparatus configuration according to at least one of the preceding claims, comprising a scanning device. Apparatus configuration according to at least one of the preceding claims, wherein an X / Y scanner is used in the irradiation. Apparatus configuration according to at least one of the preceding claims, using an X / Y scanning table. Apparatus configuration according to at least one of the preceding claims, by non-confocal detection. Apparatus configuration according to at least one of the preceding claims, comprising a scanning device. Apparatus configuration according to at least one of the preceding claims, based on descan detection. Apparatus configuration according to at least one of the preceding claims, by bright field imaging. Apparatus configuration according to at least one of the preceding claims, by point imaging. The device configuration according to at least one of the preceding claims, wherein the device configuration is based on non-descanning detection. Apparatus configuration according to at least one of the preceding claims, by bright field imaging. Apparatus arrangement according to at least one of the preceding claims, by non-scanning, confocal or non-confocal detection and point or bright field imaging. Apparatus configuration according to at least one of the preceding claims, using an X / Y scanning table.

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