microscope and a method for examining a sample with a microscope

Abstract

Microscope, in particular confocal microscope, with one or more lasers for generating pulsed illumination light for a sample and a detection device for detection signals from the sample, wherein the detection device has several spectral detection channels (Ch.1, Ch.2) adjustable for detecting predefinable different wavelength ranges (1, 2), and wherein each of the spectral detection channels (Ch.1, Ch.2) is assigned several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3), characterized in that the several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) assigned to each of the spectral detection channels (Ch.1, Ch.2) are adjustable during a measurement, that the The temporal detection windows each have a different size, and the multiple temporal detection windows (Ch.1.1, Ch.1.2, Ch. 1.3, Ch.2.1, Ch.2.2, Ch.2.3) are each arranged between two immediately consecutive pulses of illumination light.

Description

[0001] The present invention relates to a microscope, in particular a confocal microscope, comprising one or more lasers for generating illumination for a sample and a detection device for detection signals from the sample, wherein the detection device has several spectral detection channels adjustable for detecting predefinable different wavelength ranges. Furthermore, the present invention relates to a method for examining a sample with a microscope, in particular a confocal microscope, wherein the microscope comprises one or more lasers for generating illumination for a sample and a detection device for detection signals from the sample, and wherein the detection device has several spectral detection channels adjustable for detecting predefinable different wavelength ranges.

[0002] Microscopes of the type mentioned above and methods for examining a sample with such a microscope are well-known in practice and exist in a wide variety of configurations. For example, in confocal laser microscopy, biological samples are typically labeled with dyes. Often, different organelles are labeled with different dyes. These dyes are excited to fluorescence by illumination generated by one or more lasers. If several dyes are present in the sample, they are usually displayed on different image channels by wavelength separation in the detection unit. The corresponding detection signals pass through detection channels that are adjustable or configured to detect predefined different wavelength ranges.

[0003] The emission spectra of dyes often overlap. Furthermore, the reflected excitation light usually falls within the emission spectra of the dyes. Phenomena such as autofluorescence, second harmonic generation, or resonant energy transfer cannot be distinguished from "normal" fluorescence photons by wavelength separation alone. Since these phenomena either provide additional information about the sample or interfere with the pure fluorescence pattern, it is desirable to be able to separate these phenomena from the actual fluorescence signal during detection.

[0004] In confocal laser microscopes, for example, the fluorescence light is often chromatically split before detection. This is achieved either via a cascade of optical beam splitters, a prism, or an arrangement with a grating. After spectral splitting, the light encounters different detectors to form different detection channels. Each detector or detection channel is thus assigned a specific wavelength range.

[0005] Furthermore, two methods are known for measuring fluorescence lifetimes. The first method is single-photon counting, where the time between excitation and detection signal is measured for each photon. This generates statistics based on a very large number of individual measurements. The aim here is to determine the decay curve and thus the lifetime of dye molecules. Due to the large amount of data required, the evaluation is usually performed offline. However, a rough estimate of the lifetimes is also possible online by averaging the measured individual values.

[0006] A second well-known method for measuring fluorescence lifetimes is the so-called gating method, in which the photons are sorted into time windows defined before the measurement. As with single-photon measurements, statistics are generated to calculate the lifetime of the dyes after the measurement. The known gating methods perform one measurement per gating window.

[0007] A problem with known microscopes and methods for examining a sample is that many phenomena that carry information about the sample cannot be detected. Often, the detection signals of these phenomena are suppressed or at least reduced, preventing them from interfering with the measurement of other phenomena. Ultimately, this results in a waste of potentially usable detection signals.

[0008] The present invention is therefore based on the objective of providing a microscope and a method for examining a sample with a microscope of the type mentioned at the outset, whereby a particularly versatile application is made possible taking into account a wide variety of phenomena with particularly good separation of the phenomena during the examination.

[0009] According to the invention, the foregoing problem is solved by a microscope having the features of claim 1 and by a method for examining a sample with a microscope having the features of claim 6. The microscope is characterized in that several temporal detection windows can be set for each of the spectral detection channels, and the method is characterized in that several temporal detection windows can be set for each of the spectral detection channels, and thus detection signals are recorded in the temporal detection windows.

[0010] In accordance with the invention, it has first been recognized that, through the clever design and implementation of a measurement method, it is possible to separate and evaluate a wide variety of phenomena during sample analysis. Specifically, several temporal detection windows are adjustable for each of the spectral detection channels, allowing detection signals to be recorded within these adjustable windows. Consequently, the temporal dependence of the detection signals is taken into account, for example, the very rapid onset of reflected light and the typically later occurrence of fluorescence light. By separating these signals using the temporal detection windows, mutual interference between the reflected and fluorescence signals is largely avoided. Both phenomena can thus be investigated and evaluated independently.With the microscope and method according to the invention, a dynamic interplay between wavelength selection and selection of the detected time window is thus possible, enabling, for example, optimization of image contrast. Furthermore, the combination of both selection methods – wavelength and temporal detection window – makes it possible to generate additional information, for example, in confocal imaging. In particular, the user can receive immediate visual feedback via the imaging and, if necessary, optimize the settings of the detection channels and the detection window even during a measurement.

[0011] Consequently, the microscope and the method according to the invention for examining a sample enable a particularly versatile application, taking into account a wide variety of phenomena with particularly good separation of the phenomena during an examination.

[0012] Specifically, the wavelength ranges of the spectral detection channels could be adjustable and / or displayed during a measurement. This allows for optimization of the measurement settings to the specific application. Visual feedback via imaging could be helpful in this regard. Alternatively or additionally, the temporal detection windows could be adjustable and / or displayed during a measurement. This also allows for adaptation to existing measurement situations, for example, to optimize image contrast. A visual representation simplifies the measurement process and makes it more convenient.

[0013] In a further advantageous embodiment, the wavelength ranges could have different sizes. This also allows for optimization of the measurement conditions with regard to the respective application. Depending on the application, the wavelength ranges could also be the same size. Alternatively or additionally, the temporal detection windows could each have different sizes. This allows for adaptation to individual temporal profiles of detection signals.

[0014] For particularly convenient and versatile evaluation of the detection signals, each spectral detection channel could be assigned separate evaluation electronics or a separate evaluation channel. Alternatively, each spectral detection channel could be assigned several separate evaluation channels to allow for particularly flexible adaptation of the evaluation to individual experimental situations. Furthermore, each evaluation channel could be assigned one or more temporal detection windows.

[0015] Depending on the application, the evaluation of the recorded detection signals could be performed in parallel or serially. If several separate evaluation channels are assigned to a single spectral detection channel, parallel evaluation is possible, with each evaluation channel potentially having a predefined number of detection windows. If there is only one evaluation channel, serial evaluation of the recorded detection signals is possible, with each detection window being evaluated sequentially.

[0016] In a further advantageous manner, the evaluation in the evaluation electronics, in the evaluation channel(s), could be activated or deactivated by control signals that correlate with the temporal detection windows. If the evaluation is activated by the control signals, evaluation begins upon receipt of the control signal. If it is deactivated, evaluation ceases upon receipt of the control signal. Depending on the application, the appropriate control process can be selected, either according to the activation mode or the deactivation mode.

[0017] Advantageously, two or more temporal detection windows could be combined during or after data acquisition. Preferably, the results of this combination could be displayed, for example, by output on a screen.

[0018] A further advantage would be that different lasers could be assigned to different temporal detection windows. For example, different lasers could be assigned to each temporal detection window or to groups of temporal detection windows. Similarly, multiple lasers could be assigned to a single temporal detection window or a group of temporal detection windows. The specific application must be taken into account in this regard.

[0019] Similarly, in a suitably designed method for examining a sample with a microscope, two or more temporal detection windows during or after data acquisition could advantageously be combined, and the results of the combination could then preferably be presented. Likewise, in a suitable method, different lasers could be assigned to different temporal detection windows.

[0020] In the case of serial processing, an address counter could be incremented each time the control signal is deactivated at the end of a temporal detection window. This allows for separate representation of the individual time periods during image acquisition. The processed image information for each time period or temporal detection window could be stored in a separate memory cell due to the incremented address counter. If multiple time periods or detection windows are to be combined during serial processing, as is easily done with parallel processing, this could also be easily achieved in serial processing by subsequently compiling the contents of the corresponding memory cells.

[0021] Even with such serial evaluation, it would be possible to invert the evaluation process, allowing the targeted suppression of specific time segments of the image signal. For this purpose, each memory cell containing evaluated image information from a temporal detection window could be assigned an individually configurable marker. This marker would then allow the content of that time segment or detection window to be subtracted from the overall image during subsequent evaluation.

[0022] In a further advantageous method of evaluation, the detection signals to be recorded could be evaluated using a track-and-hold circuit, whereby a data accumulation could be started with each start of a gating pulse.

[0023] To obtain meaningful data using the methods described, it is advantageous to acquire data at a high repetition rate and achieve high temporal resolution. Repetition rates of 80 MHz are beneficial in this regard. Both of these requirements can be implemented particularly effectively with a design approach using an FPGA (Field-Programmable Gate Array) or an ASIC (Application-Specific Integrated Circuit), with the combination of an FPGA / ASIC and as few extremely fast individual components as possible for sampling and data acquisition being especially advantageous.

[0024] In summary, it can be stated that confocal laser microscopy imaging has not yet taken into account the temporal dependence of detection signals. Many phenomena that convey information about a sample therefore cannot be detected. Furthermore, existing methods for measuring temporal phenomena in fluorescence microscopy aim solely to determine the lifetime of dye molecules as precisely as possible. Adjusting parameters during the measurement is not provided for. In particular, a dynamic interplay between wavelength selection and selection of the detected time window is not possible with current methods.

[0025] There are now various ways to advantageously elaborate and further develop the teaching of the present invention. Reference is made, on the one hand, to the dependent claims and, on the other hand, to the following explanation of preferred embodiments of the invention with reference to the drawings. In conjunction with the explanation of preferred embodiments of the invention with reference to the drawings, generally preferred embodiments and further developments of the teaching are also explained. The drawings show

[0026] Fig. 1 in a schematic representation a design according to the invention of detection channels and associated temporal detection windows,

[0027] Fig. 2 in a schematic representation with different detection windows and different detection channels with a design according to Fig. 1 recorded image data,

[0028] Fig. Figure 3 in a diagram shows an exemplary embodiment of an arrangement of three evaluation channels for parallel evaluation,

[0029] Fig. 4 in a schematic representation a parallel evaluation,

[0030] Fig. 5 in a diagram the temporal sequence of detection windows in a serial evaluation,

[0031] Fig. 6. A serial evaluation is shown in a schematic representation.

[0032] Fig. Figure 7 shows a schematic representation of a track-and-hold circuit.

[0033] Fig. 8a in a schematic representation the relationship of detection windows of different detectors to a laser,

[0034] Fig. 8b in a schematic representation the relationship of detection windows of different detectors to different lasers,

[0035] Fig. 8c in a schematic representation the relationship of detection windows of a detector to different lasers,

[0036] Fig. 8d in a schematic representation the removal of cross signals by sequential image acquisition on a pixel and

[0037] Fig. 8e in a schematic representation the use of cross signals in sequential image acquisition on one pixel for data evaluation.

[0038] In one embodiment of the present invention, the time or time window between the excitation of a dye and the arrival of the generated photons at the detector is used as an additional property of the detected photons. Since, for example, reflection, autofluorescence, and fluorescence differ in this property, this makes it possible to distinguish them from one another during detection.

[0039] In the present embodiment, the temporal detection window is introduced as a second criterion for the separation of detection signals in confocal laser microscopy imaging, alongside the detection wavelength range. Fig. Figure 1 shows a schematic representation of a setting option for two separation channels, Ch.1 and Ch.2, in a user interface. This includes the number of wavelength ranges. 1 and 2 as well as the number of temporal detection windows Ch.1.1, Ch.1.2 and Ch.1.3 or Ch.2.1, Ch.2.2 and Ch.2.3 for the sake of clarity in Fig. The detection is limited to two wavelength ranges or three temporal detection windows per wavelength range or detection channel. However, significantly more wavelength ranges and temporal detection windows can be configured.

[0040] According to Fig. 2. Imaging could occur during the in Fig. The embodiment described in Figure 1 is represented as follows: Ch.1 receives only green light – dashed borders – and Ch.2 detects only red light – borders with solid lines. The single borders of the symbols in the detection windows represent, in this example, the very early reflected light, as shown in Ch.1.3 and Ch.2.3. The symbols with double borders represent the fluorescence signal. For example, the heart represents a long-lasting fluorescence, while the face represents a short-lasting fluorescence.

[0041] For example, the following practical applications are possible in fluorescence microscopy, although this is not an exhaustive list.

[0042] It would be possible to separate the fluorescence and reflected light. This allows for the simultaneous display of detection channels with overlapping wavelength ranges in two or more channels. On the one hand, reflected light can interfere with the imaging of the fluorescence light. On the other hand, the pure reflected image can provide further information, for example, about the position and location of optical interfaces. Simply suppressing the reflected signal would result in the loss of this information.

[0043] Another application could be the separation of second harmonic generation (SHG) and multimodal fluorescence (MP) signals. The SHG signal, like reflection, is an extremely fast signal and can therefore be easily separated from the fluorescence in time. In contrast, purely spectral separation can be difficult or impossible, especially when the emission wavelength of the fluorescence overlaps with the wavelength of the SHG. Particularly when dealing with samples that are prone to fading or rapidly moving, it is advantageous for the user to be able to detect SHG and fluorescence signals simultaneously, rather than relying on two separate measurement processes.

[0044] Another application is the separation of autofluorescence and fluorescence. Autofluorescence frequently occurs in plant preparations. The lifetime and wavelength of autofluorescence vary considerably. The temporal detection window can serve as an additional criterion to facilitate the separation of fluorescence and autofluorescence.

[0045] In principle, it is conceivable that two or more temporal detection windows or detection channels can be combined during (online) or after (offline) data acquisition, and preferably, that the results of this combination can be displayed. FRET representation could be an example. FRET often shows only a slight decrease in donor fluorescence with a simultaneous reduction in lifetime, while the acceptor shows a slight increase in intensity with a flatter rise slope. Dividing the intensity of the long-lived fluorescence of the donor by the short-lived intensity of the acceptor provides a more sensitive measurement method than pure intensity or lifetime measurements. Other applications where temporal detection windows or detection channels can be subtracted, added, multiplied, and / or combined in any way are conceivable.

[0046] If multiple pulsed lasers are present in the system, they can pulse simultaneously or alternately in a user-selectable sequence, i.e., emit excitation light. The user-adjustable temporal detection windows of the various detectors can all refer to a single laser; see [reference]. Fig. 8a. It is also possible that the temporal detection windows of different detectors refer to different lasers, see Fig. 8b, or that the temporal detection windows of one or more detectors refer alternately to different lasers, see Fig. 8c. This flexible assignment of the temporal relationship between the laser pulse and the temporal detection window enables, among other things, sequential data acquisition at the pixel level, see Fig. 8d. This allows cross-excitation and cross-emission of spectrally similar fluorochromes to be separated at the hardware level. Compared to line-wise or frame-wise sequential scanning, pixel-wise sequential data acquisition eliminates pixel offset between individual detection channels for rapidly moving samples. Furthermore, if cross-excitation and cross-emission channels are also recorded, online data analysis allows differentiation between "normal" cross-emission and emission by FRET (see [reference]). Fig. 8e. In all Fig. 8a– Fig. 8e the time axis runs from left to right, or the horizontal or x-axis forms the time axis.

[0047] Furthermore, one application is the visualization of anisotropy phenomena. Anisotropies can lead to longer or shorter decay times of dyes. While the described method is not intended for quantifying lifetimes, changes in lifetime can be clearly visualized by simply dividing the short-lived intensity by the long-lived intensity.

[0048] Furthermore, one application is gated FCS. With very weak FCS samples, scattered light from water can influence the measurement results. This scattered light can be easily identified and rendered invisible to the detection channel through temporal selection during FCS measurements. Additionally, the influence of interfering autofluorescence can be minimized by appropriately selecting the FCS detection time window.

[0049] The separation of detection based on the temporal arrival of the photons takes place in the electronics downstream of the detector. In principle, both a parallel and a sequential design of the electronics are conceivable.

[0050] In a sequential setup, the pulses emitted by the light source and those arriving from the detection unit are sampled by fast serial-parallel scanning units. The pulses can be sampled by very fast ADCs, and the arrival times of the pulses can be determined using appropriate processing algorithms. Subsequently, the time interval(s) between the pulse from the light source and the pulse(s) from the detection unit are determined. This information about durations can be assigned to the corresponding time intervals or detection windows using suitable methods.

[0051] A parallel setup could be implemented according to Fig. 3. This can be implemented. The time intervals of the sample emissions to be detected can be represented by electrical signals. Each electrical evaluation channel is assigned one or more time windows or detection windows. These evaluation channels can then be used to control further electrical circuit components that perform the evaluation of the image information. Fig. Figure 3 shows three evaluation channels with a total of five time periods or detection windows distributed across these three channels. However, the number of evaluation channels and time periods is not limited to the number shown; this is merely an example.

[0052] Each of the depicted evaluation channels controls its own evaluation electronics. Each time interval starts and stops the evaluation of the detection signal or confocal signal in confocal microscopy. This is shown in the diagram. Fig. Figure 4 is an example of parallel evaluation, activated by the control signals. In the depicted arrangement, a previously defined spectral channel can be divided into any number of time intervals, and these time intervals can be evaluated separately. The control logic and evaluation circuitry shown is implemented for each spectral detection channel.

[0053] Alternatively, an inverted application can be used, which allows the evaluation to be stopped using the control signals and thus selectively hides certain signal components.

[0054] In a serial evaluation, the evaluation can be carried out according to Fig. 5. In this case, there is only one control signal for one evaluation channel. All time intervals to be detected are set within this control signal.

[0055] Such a control signal is used to control evaluation electronics. To enable the separate display of individual time intervals during image acquisition, an address counter is incremented each time the control signal is deactivated at the end of a time interval. The basic structure of the arrangement is shown in Fig. Figure 6 shows the serial evaluation and the incrementing of the address counter particularly well.

[0056] The evaluated image information for each time period is stored in a separate memory cell due to the incrementing of the address counter. If several time periods are to be combined, as is possible with parallel evaluation, this can be achieved by subsequently compiling the contents of the corresponding memory cells.

[0057] Serial evaluation also allows for an inversion of the evaluation process, enabling the targeted suppression of specific time segments of the image signal. This is achieved by marking a memory cell with a label that, during subsequent evaluation, allows the content of that time segment to be subtracted from the overall image.

[0058] Another type of evaluation can be performed according to Fig. 7. Implement this using a track-and-hold circuit. A gating signal is fed as a serial signal to the clock input of a track-and-hold stage (T / H).

[0059] With each gating pulse, the accumulation of image data in the track-and-hold stage begins. At the end of the time window, the charge stored in the track-and-hold stage's memory is sampled by an analog-to-digital converter and further processed. The result of this type of signal processing is a value integrated over the gating interval, proportional to the number of detected photons.

[0060] Regarding further advantageous embodiments of the teaching according to the invention, reference is made to the general part of the description and to the attached claims in order to avoid repetition.

[0061] Finally, it should be expressly pointed out that the exemplary embodiments described above serve only to illustrate the claimed teaching, but do not limit it to these exemplary embodiments. Reference symbol list 1 wavelength range 2 wavelength range Ch.1 Detection Channel Ch.2 Detection Channel Ch.1.1 Detection window Ch.1.2 Detection window Ch.1.3 Detection window Ch.2.1 Detection window Ch.2.2 Detection window Ch.2.3 Detection window

Claims

[1] Microscope, in particular confocal microscope, with one or more lasers for generating illumination light for a sample and a detection device for detection signals from the sample, wherein the detection device has several predefinable different wavelength ranges for detection ( 1 , 2 ) features adjustable spectral detection channels (Ch.1, Ch.2), characterized by that for each of the spectral detection channels (Ch.1, Ch.2) several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) can be set. [2] Microscope according to claim 1, characterized by that the wavelength ranges ( 1 , 2 ) the spectral detection channels (Ch.1, Ch.2) and / or the temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) are adjustable and / or displayable during a measurement. [3] Microscope according to claim 1 or 2, characterized by that the wavelength ranges (1 , 2 ) and / or the temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) each have a different size. [4] Microscope according to any one of claims 1 to 3, characterized by that each spectral detection channel (Ch.1, Ch.2) is assigned separate evaluation electronics or a separate evaluation channel, or several separate evaluation channels are assigned, wherein preferably each evaluation channel is assigned a temporal detection window (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) or several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [5] Microscope according to claim 4, characterized by that the evaluation in the evaluation electronics, in the evaluation channel or in the evaluation channels can be activated or deactivated by control signals that correlate with the temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [6] Microscope according to any one of claims 1 to 5, characterized by that two or more temporal detection windows during or after data acquisition can be combined and preferably the results of the combination can be presented. [7] Microscope according to any one of claims 1 to 6, characterized by that different lasers can be assigned to different temporal detection windows. [8] Method for examining a sample with a microscope, in particular a confocal microscope and in particular a microscope according to any one of claims 1 to 7, wherein the microscope has one or more lasers for generating illumination light for a sample and a detection device for detection signals from the sample and wherein the detection device has several predefinable different wavelength ranges for detecting ( 1 , 2 ) features adjustable spectral detection channels (Ch.1, Ch.2), characterized bythat for each of the spectral detection channels (Ch.1, Ch.2) several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) can be set and thus detection signals are recorded in the temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [9] Method according to claim 8, characterized by that the evaluation of the detection signals to be recorded is carried out in parallel or serially. [10] Method according to claim 8 or 9, characterized by that each spectral detection channel (Ch.1, Ch.2) is assigned separate evaluation electronics or a separate evaluation channel, or several separate evaluation channels are assigned, wherein preferably each evaluation channel is assigned a temporal detection window (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) or several temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [11] Method according to claim 10, characterized bythat the evaluation in the evaluation electronics, in the evaluation channel or in the evaluation channels is activated or deactivated by control signals that correlate with the temporal detection windows (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [12] Method according to any one of claims 9 to 11, characterized by that in a serial evaluation, an address counter is incremented each time the control signal is deactivated at the end of a time detection window (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3). [13] Method according to any one of claims 8 to 12, characterized by that each memory cell containing evaluated image information from a temporal detection window (Ch.1.1, Ch.1.2, Ch.1.3, Ch.2.1, Ch.2.2, Ch.2.3) is provided with an individually predefinable marker. [14] Method according to claim 8, characterized bythat the detection signals to be recorded are evaluated using a track-and-hold circuit, whereby data accumulation is started with each start of a gating pulse. [15] Method according to any one of claims 8 to 14, characterized by that two or more temporal detection windows during or after data acquisition are combined and preferably the results of the combination are displayed. [16] Method according to any one of claims 8 to 15, characterized by that different lasers are assigned to different temporal detection windows.

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