HK1057100A - Method for the optical determination of characteristical parameters of a sample - Google Patents

Description

The invention relates to a method and arrangement in fluorescence microscopy, in particular laser scanning microscopy, fluorescence correlation spectroscopy and scanning near field microscopy, for the examination of predominantly biological samples, preparations and associated components, including high-throughput scanning and flow cytometers. The transition from detecting a few broad spectral bands of dye to simultaneous absorption of complete spectra opens up new possibilities for identifying, seeding and allocating the mostly analytical or functional structures into partial or dynamic spectral units. This will also increase the number of samples that can be detected simultaneously through the use of fluorescence.

State of the art

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The state of the art is illustrated below by an example of a confocal laser scanning microscope (LSM) (Fig. 2).

An LSM is essentially divided into 4 modules: light source, scanning module, detection unit and microscope.

The excitation radiation is generated in the light source module. Various lasers (argon, argon krypton, TiSa-laser) are used. Furthermore, the light source module selects the wavelengths and sets the intensity of the required excitation wavelength, e.g. by using an acoustic optical crystal. The laser radiation produced in the light source is focused into the preparation by means of the lens (2) with a deflection limit via the scanners, the scanning optics and the tubular lens.The focus is point-like, scanning the sample in the x-y direction. In a decanated detection of fluorescent light, the light emitted from the focus plane (specimens) and from the levels above and below reaches the scanner to a dichroic beam divider (MDB), which separates the fluorescent light from the excitation light. The fluorescent light is then focused on a aperture (confocal aperture/pinhole) located precisely in a plane conjugated to the focus plane. This suppresses fluorescent light fractions outside the focus.Behind the aperture is another dichroic block filter (EF) which again suppresses the excitation radiation. When using a multiphoton absorption, the excitation of the dye fluorescence occurs in a small volume where the excitation intensity is particularly high. This range is only slightly larger than the detected range when using a confocal arrangement.

In another arrangement for detecting a dye fluorescence induced by multiphoton absorption, a descended detection is continued, but this time the lens pupil is imaged into the detection unit (nonconfocal descended detection).

The two detection devices, in conjunction with the corresponding single-photon or multiphoton absorption, only reproduce the plane (optical cross-section) in the focal plane of the lens in a three-dimensional illuminated image. The LSM is therefore suitable for the examination of thick preparations. The excitation wavelengths are determined by the dye used with its specific absorption properties. Dichroic filters, adjusted to the emission properties of the dye, ensure that only the fluorescent light emitted by the dye is measured by the spot detector.

In biomedical applications, several different cell regions are currently marked with different dyes at the same time (multi-fluorescence). The individual dyes can be detected separately, with the state of the art, either by different absorption properties or emission properties (spectrums) (Fig. 3a). For example, emission signals are applied depending on the wavelength for different dyes (1-4). For separate detection, additional splitting of the fluorescent substance from several dyes with the secondary beam splitters (DBS) and separate detection of individual dye emissions in different point detectors (PMT x) are performed. A flexible approach to detection and to creating corresponding emission properties by adjusting the new dyes is possible with the new colour block (s) described above and does not require a new colour filter.

In a known arrangement, the fluorescent light is split spectrally by means of a prism. The method differs from the dichroic filter arrangement described above only in that the filter used is adjustable in its characteristics.

A rapid local measurement of the emission spectrum is only possible with both arrangements, since the setting of the emission range is based on mechanical movements of the dichroic filter or apertures and thus the maximum spectral resolution is limited to some 5 nm. A high spectral resolution is required, for example, when the emission spectra overlap as shown in Figure 3b. Figure 3b shows such a behavior of two naturally occurring dyes CFP and GFP. These dyes are particularly suitable for the examination of living preparations as they do not exert toxic effects on the samples to be examined.

If the position of the emission spectrum of the dyes used is unknown or if a shift in the emission spectrum (Fig. 3c) is due to the environment, high-resolution detection of the dye fluorescence is necessary. The wavelength shift can be up to several 10 nm. Spectrometers are now also used in conjunction with an LSM to measure the emission spectrum in the sample. Instead of a point detector, a traditional, mostly high-resolution spectrometer (patent Dixon, et al. US 5,192,980) is used.

The purpose of the invention is therefore to create a new method for efficient spectral resolution detection of fluorescent dyes with a line detector. The spectral resolution is determined by the number of individual channels in the optical arrangements shown above. If not all individual channels of the detector can be read out simultaneously, a sequential reading of the individual channels is performed according to the state, called multiplex. The multiplexing can be done for one pixel while scanning a sample. This has the disadvantage that the integration time per single channel in which a signal can be detected is reduced by the number of multiplex adjustments.In another method of multiplexing, the signals of the individual channels can be interspersed. The individual channels are then read out one after the other. However, no new data can be taken during the read-out time. Therefore, in this type of multiplexing, the read-out speed of the line detector would decrease. The method or arrangement according to the invention thus has the following tasks: The method is intended to be used in both imaging and analytical microscopy systems. Microscopic systems are imaging systems such as laser scanning microscopes for the three-dimensional examination of biological preparations with an optical, optical, and optical detection system.The following are included: spatial resolution up to 200 nm, scanning near-field microscopes for high-resolution examination of surfaces up to 10 nm, fluorescence correlation microscopes for the quantitative determination of molecular concentrations and for the measurement of molecular diffusions, and fluorescence-based methods for the screening of dyes.

In all the above systems fluorescent dyes are used for the specific labelling of the preparations. Preferential training is the subject of the dependent claims.

Description of the invention

The present invention is based on a spectrally split detection of fluorescence, summing up the different spectral components. The emission light in the scanning module or microscope (in the case of multiphoton absorption) is separated from the detected radiation by means of an element for separating the excitation radiation from the detected radiation, such as the main color divider (MDB) or an AOTF according to 7346DE or according to 7323DE.The light is then split into its spectral components by means of a line detector DE, which measures the emission signal in relation to the wavelength and converts it into electrical signals. The method described below is used to obtain a summation of individual channels, i.e. a summation of the channels sent through a single channel of the line. In addition, a sub-unit of the line detector can be proposed for the regulation of the wavelength of the light.

A possible embodiment of the optical beam path of the detector unit shown in Fig. 4 in the block diagram is shown in Fig. 5. The structure essentially describes a Cerny Turner structure. In a confocal detection, the light L of the sample is focused by the pinhole optics PO through the confocal aperture PH. In a non-descanned detection, in the case of multiphoton absorption, this aperture may be lost. The first imaging mirror M2 collimates the fluorescent light.The detector has 32 channels and a high sensitivity. The free spectral range of the above embodiment is about 350 nm. The free spectral range is distributed evenly in this arrangement among the 32 channels of the line detector, resulting in an optical resolution of about 10 nm. The use of the unit in an imaging system is advantageous because the signal per detection channel is still relatively large due to the relatively wide detected spectral band.

The spectral resolution is determined by the number of individual channels in the optical arrangements shown above. In the embodiments described above, each individual channel detects a spectral band of the emission spectrum with a spectral width of approximately 10 nm. If all individual channels of the detector cannot be read simultaneously, a sequential reading of the individual channels (multiplexes) is performed according to the state. The invention is based on the addition of individual channels with different patterns.

For example, if 8 channels can be read out simultaneously, then using the 32 channel detector described above, a summation is performed on 4 channels each. The summation of the entire N=32 channels is then performed in n=4 steps, with the summation window moving by one single channel (L/n=4/4=1) each. The measured single channel signals are denoted by ck,j (in Figure 6 as

If the signal at the detector edge does not decrease, the last single channel of the detector can be covered (hidden) so that only a width of L/n is available for measurement, as shown in Figure 6. This is necessary to avoid artifacts in the calculation. To calculate the N times n spectral values Sm, differences of sums over individual channels are now calculated using the following algorithm: The following The following The following The following The following

The spectral values S (intermediate values) thus calculated can then be graphically represented on the displayed image, e.g. during a spectral scan.

The summation over different individual channels and thus the measurement of ck,j is shown schematically in Figure 7. The signals of the individual channels are in turn transformed into voltage signals by an amplifier A. The individual voltage signals are then integrated into an integrator 1 during the pixel residence time. The integrator is connected to a comparator K which compares the up-integrated signal with a reference signal. If the split signal is smaller than the comparator threshold, then the corresponding single channel would not measure any or too small a fluorescence signal. In such a case, the single channel signal should not be further processed, as this channel contributes only a noise component to the overall signal. In such a case, the comparator operates a switch S via SR and the single channel is turned off for the pixel just measured.

The integrated voltage signal of the individual channels can then be switched to different sum points by a demultiplexer MPX connected to the switch register SR via the register Reg1.

The sum of the signals ck,j is then converted into digital signals by an analogue-to-digital converter and further processed by the computer or DSP. The sum of the signals SV can also be operated with a variable nonlinear characteristic.

The above arrangement used an integrated circuit to detect the single channel signals, but also allows for full photon counting in the single channels.

The MPX's switching speed requirements depend on the type of setting. For example, if a point setting is used, the scan must be performed within the integration time for that point (i.e. within a few microseconds). If the setting is used, the scan must be performed within a few milliseconds to seconds. The calculation of the single channel signals is carried out using the above algorithm using the ck,j.

In both of the above arrangements, an integrated circuit was preferably used for detecting the single channel signals.

However, a photon counting can be performed in the individual channels and the photon numbers added. The advantage of the invention is that the wavelength distribution (λ-stack) captured is allocated to the respective image point coordinates x, y or z and/or an additional time allocation is made in memory for measured time-varying processes.

Claims (12)

1. a method for optical recording of characteristic dimensions of an illuminated sample, where the signal reflected, reflected and/or fluoresced and/or transmitted by the sample is detected by a location-resolving detector in several channels by imaging the radiation coming from the sample as a spectral breakdown on the detector, whereby a linkage of detection channels is made so that the number of read and processed readings is less than the number of detection channels
2. Method according to claim 1, where the link is a summation
3. Method according to claim 1 or 2, shifting the linked measurements and/or changing the number of linked detector channels
4. procedure according to one of the preceding claims, a length of not more than 30 mm,
5. procedure according to one of the preceding claims, with a change in displacement
6. procedure according to one of the preceding claims, whereby an algorithm is used to determine intermediate values from the readings for the efficient reading of a detector line.
7. The method according to one of the above claims, whereby a multiplexer moves the summation pattern along a line detector.
8. The procedure is based on one of the above claims, whereby a spectrally resolved spectral measurement is performed over a dispersive element pre-specified for the detector
9. procedure according to one of the preceding claims, where the spectrally split radiation is shifted relative to the detector.
10. The following conditions shall apply: the dispersive element is capable of rotating on at least one axis
11. The following conditions shall apply: in the case of a fixed dispersive element in at least one of its axes of oscillation, the spatially altering effect of the oscillation in this axis is caused by a scanning unit and/or a displacement of the detector.
12. The method according to one of the above claims, whereby a memory-based point-by-point mapping of the captured respective wavelength distribution (λ-stack) to the respective point-by-point coordinates x,y or z and/or an additional time mapping is made for measured time-varying flows.