HU231357B1 - Method and apparatus for analysing fluorescence of a sample - Google Patents

Description

Procedure and equipment for fluorescence testing of a sample

The invention relates to a method and equipment for the fluorescence examination of a sample. The test sample according to the invention is preferably transparent and liquid, and the invention is used to identify fluorescent objects in the sample.

Conventional flow cytometers are capable of high-speed counting of objects with fluorescent properties (autofluorescent (e.g. chlorophyll) or fluorescently dyed, labeled) in a flowing liquid sample. However, they are typically not able to create a sufficiently high-resolution image of the passing measured objects.

In addition to counting the flowing bodies, determining their shape and spatial form can also be important, for this purpose, liquid samples containing objects with fluorescent properties are also subjected to holographic examination.

Several solutions are known in which holographic and fluorescence (displaying fluorescent properties) images of the liquid sample are taken one after the other in time with a single sensor. Such a solution is described in US 2004/0156098 A1, US 7,362,449 B2 and US 2012/0148140 A1. A major disadvantage of these solutions is that between the two shots, the examined objects can move (even as a result of their natural movement in still liquid). Due to their sensitivity to movement, these solutions are not suitable for testing flowing media. Another big disadvantage of such solutions is that since the holographic and fluorescence recordings are made with the same sensor, this sensor cannot be selected according to either fluorescence or holographic recordings, i.e. the parameters of the sensor cannot be chosen optimally for either type of recording.

- 2 Fluorescence recording and holographic recording are also used in document US 2015/0056607 A1, but with these they examine a sample located in a different part of space, i.e. the two measurements can only be combined statistically.

Document US 2014/0376816 A1 describes a device suitable for analyzing and sorting objects in a flowing liquid. The document describes a solution in which a holographic and fluorescent image is recorded from a given area of space without the use of imaging optics on adjacent areas of a single sensor. In order for the image belonging to the holographic sensor part and the image belonging to the fluorescence sensor part to be projected onto different areas of the sensor, the sensor must be placed relatively far from the cell containing the sample to be tested. However, in the absence of a reducing optic, an extremely weak fluorescent signal can be recorded and the resolution of the holographic image will be very poor. In addition, the selection and operating parameters of the sensor cannot be specifically optimized for fluorescence or holographic measurement, because it must serve both functions simultaneously.

US 2014/0376816 A1 also describes an embodiment in which an imaging zone is divided into two parts along a flow channel. One of these two parts is suitable for holographic recording, the other for fluorescence recording. These recordings are therefore made along a flow channel. In both of the above-described embodiments of document US 2014/0376816 A1, the holographic and fluorescence recordings are made in two different areas of the same sensor.

In certain known solutions, both fluorescent and holographic images are recorded with a single camera, however, the brightness to be detected (which determines the exposure time and amplification and sensitivity required for their detection) is quite different, so the resolution required for recording should also differ in the optimal case, but this common not possible because of the camera. In these known solutions, it is also very difficult (or impossible) to measure several different types of fluorescent signals at the same time.

- 3 Because of the common sensor, it is absolutely impossible to measure synchronously, which makes the registration of the fluorescent and holographic image almost impossible in the case of a flowing sample (in the case of extremely slow flow, it is only possible with a large error).

In the light of the known solutions, the need arose for a procedure and equipment with which fluorescent objects in a sample can be reconstructed in great detail and can be separated from non-fluorescent objects.

The primary goal of the invention is to create a process and equipment that are as free as possible from the disadvantages of the state-of-the-art solutions.

Another goal of the invention is to create a method and device that can be used to produce a holographic image of fluorescent objects with good resolution and to separate fluorescent objects from non-fluorescent objects.

Another goal of the invention is to create a method and equipment in which, in addition to achieving the above goal, the resource requirements of holographic reconstruction can be significantly reduced compared to known solutions.

The purpose of the invention is to create a method and equipment in which the parameters of the fluorescence sensor and holographic sensor used can be selected independently of each other in accordance with the requirements of such devices.

The objectives set in relation to the invention were achieved by the method according to claim 1 and the device according to claim 8. Advantageous embodiments of the invention are defined in the subclaims.

Hereinafter, exemplary preferred embodiments of the invention are described with drawings, where

Figure 1 is a block diagram illustrating an embodiment of the device according to the invention, a

- Figures 4 2A-2C show schematic graphs illustrating the recording order in one embodiment of the method according to the invention, the

Figure 3A is an exemplary fluorescence recording, a

Figure 3B is the holographic image corresponding to Figure 3A, a

Figure 3C is a reconstruction of the part framed in Figure 3B, and a

Figure 4 is another exemplary fluorescence recording.

The method according to the invention is used for the fluorescence examination of a sample. As will be shown below, the method according to the invention can be particularly advantageously applied to a liquid, transparent sample. In such a sample, water, as a liquid translucent medium, typically carries fluorescent objects that emit fluorescent light upon appropriate fluorescent excitation, i.e. give a fluorescent response. The wavelength of the fluorescence response differs from the wavelength of the excitation light, and typically the response has a longer wavelength than the excitation.

The sample examined by the method according to the invention is preferably flowed through the sample holder at a suitable (suitably slow) speed.

In addition, the method according to the invention is naturally also suitable for examining a stationary sample. The method according to the invention is also suitable for examining a solid sample in which it is thin enough to excite the fluorescent objects in it and detect their emitted light. The holographic recording required for the implementation of the method according to the invention can typically be made of such thin samples. With sufficiently short exposure times, the method and equipment according to the invention are also suitable for testing gaseous samples.

For example, the method according to the invention can preferably be carried out with the device according to the invention, an embodiment of which is illustrated in Figure 1.

During the method according to the invention, a specific examined sample volume of the sample (i.e. a sample volume currently under examination) is illuminated with fluorescent excitation light and the fluorescent excitation light of the given sample volume examined

- 5 by mapping the emitted fluorescent light, we make a fluorescence recording. The fluorescent emitted light can be imaged directly (without the interposition of an imager element) on the plane of the sensor, or - as we will see below - preferably by the interposition of an imager lens. In the embodiment illustrated in Fig. 1, the given sample volume under investigation is the part of the sample that is located in the 10 sample holder spaces during the excitation.

By fluorescence recording, we mean a recording that is made by mapping the emitted fluorescent light emitted by fluorescent objects onto a sensor, i.e. it is used to display the mapping of light from a fluorescent source. By holographic recording we mean a recording on which holographic information is recorded; the holographic recording contains information that can be holographically reconstructed.

In the method according to the invention, a holographic recording is made of the sample volume under investigation in a time-overlapping manner with the fluorescence recording. In the method according to the invention, therefore, a fluorescence recording and a holographic recording are made in a time-overlapping manner on the same sample volume determined as above.

By fluorescence recording, we mean that we make a recording of the fluorescent emitted light (i.e. the fluorescent response) emitted as a result of excitation, i.e. the fluorescent response is recorded on the recording; in the fluorescence recording, as much as possible, only information about objects that fluoresce (give a fluorescent response to the corresponding fluorescent excitation) is visible.

And the holographic recording contains the hologram of the given examined sample volume, which is made with a suitable holographic arrangement of the given examined sample volume. Such an arrangement can be, for example, the in-line holographic arrangement illustrated in the embodiment according to Figure 1, but other holographic arrangements can also be used by definition. The holographic recording is preferably a digital holographic recording and the sensor used is a digital holographic sensor. Digital holographic layout

- In case of application of 6, the advantages associated with digital holography can be applied in the invention.

The fluorescence recording and the holographic recording are both taken from the given sample volume under investigation, and the excitation light also arrives in this sample volume.

Due to the nature of the excitation light, it does not necessarily illuminate the entire given sample volume uniformly, however, the excitation light is preferably used in such a way that it illuminates a slightly larger volume than the given sample volume. In the latter case, all of the fluorescent objects in the given sample volume can be advantageously excited.

The fluorescence recording is therefore made from the given sample volume under investigation. By taking a picture of the given sample volume, we mean that the given sample volume, i.e. a two-dimensional projection of it on the sensor, will appear as much as possible on the fluorescence recording (it will be added to the fluorescence recording), and in addition, the area outside the given sample volume may appear information about parts. The making of a holographic recording should also be understood in this way (since the holographic beam also typically overhangs laterally over the given sample volume). In the recordings made in this way, you can search for the common part, which will essentially be a mapping of the given sample volume.

In addition, during the method according to the invention, a fluorescent object identification step is performed on the fluorescence recording, and if at least one fluorescent object is identified in the given sample volume in the fluorescent object identification step, then at least one object on the fluorescence recording is corresponding to at least one fluorescent object we assign a position, and by reconstructing the holographic recording, we create a reconstructed holographic recording, and we identify the reconstructed model (reconstructed image, three-dimensional model) of at least one fluorescent object based on the position of at least one object in the reconstructed holographic recording. By identifying the reconstructed image, we mean determining which can be separated from the reconstruction of the entire holographic recording

- 7 reconstructed models belong to the fluorescent object. The fluorescent object identification step is discussed in detail below, in this step we examine the fluorescence recording and determine whether, based on the recorded information, there is a part of the recording (even just one pixel) that belongs to a fluorescent object, i.e. the fluorescent object light emitted by was formed in that part of the fluorescence recording.

These additional steps of the method are therefore only performed if at least one fluorescent object is identified based on the fluorescence recording in the fluorescent object identification step. If a fluorescent object is currently present in the given sample volume under investigation, the mapping of its emitted light, i.e. its image (hereinafter: object image) is displayed on the fluorescence recording. With the help of this object image, the fluorescent objects can be identified in the fluorescence recording. Therefore, in the fluorescent object identification step, it is necessary to check whether there is an object image of a fluorescent object in the fluorescence recording and, if there is such an object image at all, then to identify all such object images. With identification, we assign an object position to each fluorescent object based on the location of the object image. If the currently examined sample volume does not contain a fluorescent object of which an (evaluable) object image is mapped on the fluorescence sensor, then the current steps of the procedure are not carried out (in the case of a flowing sample, in this case we proceed to the examination of an additional sample volume).

The object position can by definition be determined for small, point-like object images, if an object image is larger, then an object position must be assigned to it in some way - for example, by selecting the geometric center of the object image.

If we have identified (found) at least one fluorescent object in the given sample volume, we reconstruct the holographic recording also belonging to that sample volume. This reconstructed holographic recording will contain a reconstructed image, i.e. a three-dimensional model, of the identified fluorescent object. The reconstructed holographic recording

- 8, however, will typically also contain the reconstructed image (3D model) of additional objects that have not been identified as fluorescent objects (we cannot perform the hologram reconstruction selectively in this regard). Among the typically large number of reconstructed objects, at least one fluorescent object can be very advantageously found easily with the method according to the invention. Since the fluorescence recording and the holographic recording were taken from the given sample volume as described above, it can be determined which position on the holographic recording belongs to the position of the object identified on the basis of the fluorescence recording. Based on the position determined on the holographic recording, it is possible to specify which reconstructed image (3D model) belongs to the fluorescent object. Of course, in the case of the presence of several fluorescent objects, several 3D models are selected in this way based on the positions of several objects.

During its operation, the device according to the invention preferably searches for fluorescently emitting objects in the flowing sample. When it detects such a thing in the fluorescence recording, it reconstructs the image of the given fluorescent object based on the parallel recorded holographic image. To do this, he takes advantage of the fact that the two recordings are made of essentially the same sample volume and overlap in time, so that the position identified on the fluorescence recording can be found on the holographic recording. For the search, according to the above, the two recordings are preferably registered to each other in space and time, by which we mean that the region (window) that appears in both recordings is identified on the two recordings, and the assignment is determined between them, i.e. that on one what position on the other side belongs to a specific position.

In summary, the position of the fluorescent (fluorescent) object determined on the basis of the fluorescence recording shows where the object is located in the cell (provides x, y coordinates), while the exact depth (z) of the fluorescent object is determined by the holographic reconstruction (the given object in the holographic recording however, knowing the x, y coordinates is sufficient to find it).

- 9 According to the above, the invention also covers a device suitable for the fluorescence examination of a sample, on which the method according to the invention can be carried out as an example. An embodiment of the device according to the invention is illustrated in figure 1.

The apparatus according to the figure contains - 10 sample holder spaces suitable for receiving a given sample volume of the sample subjected to the test, one or more, in the present embodiment two, 12a, 12b excitation light sources suitable for illuminating the sample holder space 10 with fluorescent excitation light, the 10 14 fluorescence sensors suitable for recording fluorescence by mapping the fluorescent emitted light of a given sample volume in the sample holder space during the test, and a holographic arrangement containing 16 holographic light sources and 18 holographic sensors suitable for making a holographic recording of the given sample volume. The wall of the sample holding space part 10 is typically transparent, i.e. the excitation light, the emitted light and the light of the holographic light source can penetrate it (preferably without refraction or reduction in intensity).

By fluorescence sensor we mean a sensor that is used to record emitted fluorescent light emitted by fluorescent objects, i.e. on which light from a fluorescent source is imaged. By holographic sensor we mean a sensor that is used to record holographic information. Both sensors (imaging devices) are light sensors (typically with different resolution and sensitivity), only their function differs, i.e. what kind of light (emitted fluorescent light or light from a holographic light source) we use these sensors to capture. The fluorescence and holographic indicators in their names are therefore not used to display any pre-existing property, but merely to distinguish the sensors from each other; so this marker can even be omitted. The same is true for fluorescence and holographic recordings, in which names the indicators show what kind of light mapping we want to record in the given recording; thus, the adjectives in these names also serve to differentiate, they can even be omitted from it. The above also applies to the 22 fluorescence reducing lens described below.

- 10 In the present embodiment of the device according to the invention, the fluorescence sensor 14 and the holographic sensor 18 are arranged opposite each other in relation to the sample holder space part 10, together with the sample holder space part 10, on a common optical axis. In the case of such an arrangement, the fluorescence and holographic recording of the sample holder space part can be made very advantageously, since in this case both sensors see the sample holder space part 10 in the same way, only from the opposite direction. In the embodiment shown in the figure, the device according to the invention contains the coupling element 20 which maps the object wave from the holographic light source 16 onto the common optical axis and directs it towards the sample holder space 10, reflects the light of the holographic light source 16 and transmits the fluorescent emitted light.

As illustrated in Figure 1, the fluorescence sensor 14 and the holographic sensor 18 face the sample holder space part 10, i.e. with the help of these sensors, the equipment records the fluorescence properties of the sample holder space part 10 from one direction (i.e. for example a flowing through the measuring cell) as the sample volume, while a - preferably digital - holographic measurement is performed from the other direction.

Since in the applications of the method and equipment according to the invention (e.g. water quality control) the aim is usually to scan a large volume, preferably a flow-through measuring cell with a relatively large thickness (it can be as thick as 0.8 mm, even if a resolution of 1 μm is achieved on the holographic sensor) we apply (which includes the part of the sample holder under examination). Accordingly, the sample holder area for testing can reach a thickness of up to 0.8 mm, which corresponds to the extension along the common optical axis.

In order to be able to properly measure the emitted fluorescent light with a digital camera at a great depth within such a cell, imaging is preferably used, for example a small optical magnification or reduction. Such an embodiment is illustrated in Figure 1. In this embodiment, the device includes a fluorescence reduction lens 22 arranged between the fluorescence sensor 14 and the coupling element 20 on the common optical axis.

- 11 The reduction/enlargement ratio of the fluorescence reduction lens 22 is preferably between 0.51.5, more preferably between 0.9 and 1.1, especially approximately 1. Typically, we use a fluorescence reduction lens 22 with a reduction/enlargement ratio of around 1, i.e. it neither zooms in nor zooms out. If the depth of field of a lens with a magnification/reduction ratio of around 1 would be (much) smaller than the common optical axis extension of the 10 sample holder spaces, then by using a reduction lens with a smaller magnification/reduction ratio, the depth of field can be increased, and it can even be achieved that the depth of field is close to should be the same as the z-direction extension of the sample holder space part. If the depth of field would be greater than the z-direction extension with a lens with a magnification/reduction ratio of around 1, then the magnification/reduction ratio can be raised above 1, i.e. a magnifying lens can be arranged as a fluorescence reduction lens. However, there is no need to increase the magnification/reduction ratio, since in this case the depth of field corresponding to the magnification/reduction ratio of around 1 is also suitable.

What has been explained above is understood to mean that, in one embodiment of the method and equipment, by adjusting the reduction/enlargement degree, the depth of field of the fluorescence reduction lens is selected for the extension of the sample holder space in the direction of the common optical axis (hereinafter: z-direction), thanks to which a depth of field can be advantageously selected with which in the depth of the sample holder space, it can essentially be completely mapped onto the fluorescence sensor. Therefore, if the depth of field of the mapping essentially covers the sample holder in its depth direction, the objects to be examined remain detectable (they do not disappear due to lack of focus), wherever they are within the measuring cell.

Thanks to the use of the fluorescence imaging lens 22 shown in Figure 1 and its appropriate parameters (reduction/enlargement, depth of field), it can be imaged at the depth of the sample holder onto the fluorescence sensor (light beams emitted from the entire depth of the sample holder are imaged onto the sensor). An additional advantage of the arrangement of the fluorescence reduction lens 22 is that it can be used to overcome the (quadratic) weakening of the intensity of the emitted fluorescent light with distance, since the still sufficiently strong intensity

- 12 fluorescent light is mapped onto the fluorescence sensor by the fluorescence reducing lens 22. In known fluorescent sample testing equipment, in which the fluorescence recording and the holographic recording are recorded on the same sensor, it is usually not possible to arrange a fluorescence reducing lens that affects the depth of field as desired, all the more so because in such systems the holographic recording is expediently used it is advisable to use a high magnification lens.

Although the fluorescence recording obtained with low magnification is not suitable for recognizing fluorescent objects (the objects in the recording will be blurred due to the low magnification, low resolution (and possibly out of focus), the applied magnification is not sufficient for shape determination due to the size of the fluorescent objects that occur), the however, yes to their detection and identification, and preferably in the large depth of the sample holder. Essentially, only images taken with high magnification would be suitable for shape recognition. Then the depth of field would be very small. At the same time, it should also be taken into account that the emitted light of fluorescent objects can be detected with high reliability if the fluorescence recording is taken with a sufficiently long exposure time (otherwise, the object image of fluorescent objects will typically be dim). The relatively long exposure time, on the other hand, causes fluorescent objects to move during the exposure time, which would also impair the chance of being able to recognize them from a fluorescence recording. This is also why fluorescent flow cytometers are used essentially exclusively for counting fluorescent objects.

We will see below that the features of fluorescent image recording described above do not appear as disadvantages in the method and equipment according to the invention, since according to the invention, fluorescence recording is only used for the identification of fluorescent objects (not for their recognition), for which it is essentially sufficient if the fluorescent objects are imaged , the image of the object appears on the fluorescence recording. We will see below that, according to the invention, the relatively long exposure time required for taking fluorescence images is not a problem either.

- 13 For object recognition, morphological classification, and evaluation of the fluorescent content of the sample, the device according to the invention also contains a suitably designed holographic arrangement. If we also arrange a magnifying lens, we can essentially talk about a holographic microscope. Based on the image reconstructed from the holographic recording (spatial model of the investigated objects), objects with fluorescence can already be classified based on morphological aspects, since such objects can be identified based on their location on the reconstructed holographic image with the help of position determination on the fluorescence recording (determining the position of the object).

In contrast to known flow cytometers, in the method and equipment according to the invention, it is not a problem if several fluorescent objects are simultaneously present in the examined sample volume (the amount of sample in the measuring cell/sample holder during the measurement). As mentioned above, it is possible to examine the fluorescence of the objects present in the sample with a camera with a suitable sensitivity (see below) (in the known solutions where fluorescence recording and holographic recording are recorded with the same sensor, it is not possible to adjust the sensitivity of the sensor for the application, i.e. choose to take a fluorescence or holographic recording).

Based on the recording of the camera for recording the fluorescence recording, it is therefore possible to determine the position of the fluorescent objects in a given examined volume of the sample (more precisely, the 2D projection of their 3D position). Since the fluorescence recording and the holographic recording are made of the same condition - i.e. a specific examined sample volume - the content of the two recordings can be assigned to each other, and the information belonging to the same object can be found (registered) in the two recordings. The exposure time of the fluorescence recording is typically much longer than that of the holographic recording, during this exposure time we make the holographic recording with a much shorter exposure time; the two recordings are therefore made overlapping in time. This is facilitated by the determination of the position of the fluorescent object on the fluorescence recording, because as a result - the two recordings correspond to each other

- If 14 is registered - the given object can be identified in the holographic recording with the help of the position.

The sensor for recording fluorescence is preferably of high sensitivity (so that we can detect the relatively weak fluorescence signal as best as possible), but relatively low resolution. The holographic recording is preferably made with a high-resolution sensor, the sensitivity of which is low compared to the fluorescence sensor (in the case of the holographic sensor, a high-sensitivity sensor is not required, since the light intensity of the holographic light source (e.g. laser or LED) used to create the recording is not low). In an exemplary embodiment, the sensitivity of the fluorescence measuring (color) camera was 5.3 Volts/Lux-sec, while that of the digital holographic camera used in the holographic arrangement was 0.724 Volts/Lux-sec.

In one embodiment of the device and method according to the invention, a fluorescence sensor with a sensitivity of 4-7 Volts/Lux-sec is used and/or a holographic sensor with a sensitivity of 0.5-2 Volts/Lux-sec is used. In this embodiment, the fluorescence or holographic sensor can preferably be chosen so that they have the appropriate sensitivity for the given application, especially preferably the sensitivity of both (fluorescence and holographic) sensors is chosen from the above ranges, so that in the case of both sensors of the equipment, the most suitable for the application sensor can be used.

In one embodiment of the invention, an exposure time of 40-200 ms (Tf) is used for the preparation of the fluorescence recording, and/or an exposure time of 0.55 ms (Th) is used for the preparation of the holographic recording.

If the exposure time Tf is chosen from the specified range, the fluorescence recording will most likely include the generation of the light beam from weakly emitting fluorescent objects. Within the range, of course, the choice is determined by the strength of the excitation light and the extent to which the emitted fluorescent light weakens upon reaching the fluorescence sensor. If the exposure time Th is chosen from the above range, then there is a good chance of holographic recording

- 15 can be properly sharpened, avoiding the displacement of the fluorescent objects. If both exposure times are chosen respectively from the above ranges, then both advantages appear, i.e. they can be adjusted according to the application of both sensors.

In addition to the longer exposure time used for fluorescence recording, it is advisable to use a sensor with a lower resolution to record the fluorescence information, because we do not need a high-resolution result, but due to the low resolution, such a recording can be read from the sensor very quickly. In addition, low-resolution sensors typically have high sensitivity due to relatively large pixel sizes, so a low-resolution sensor can meet all the requirements of a fluorescence sensor. A fluorescence sensor with a resolution of 0.5-3 megapixels/25 mm ² is preferably used for this. Even more preferably, a sensor with a resolution of 1-2 megapixels/25 mm ² , particularly preferably approximately 1.3 megapixels/25 mm ² , can be used.

The light emitted by fluorescent objects is imaged on this sensor. As explained above, for imaging, we preferably use a reducing lens, which can be a reducer/enlarger. Typically, the degree of reduction/enlargement is close to 1, i.e. essentially neither enlargement nor reduction takes place. In this case, the reducing lens projects an image onto the fluorescence sensor approximately as large as the extent of the examined space perpendicular to the common axis. This mapping typically does not fill a 5x5 mm sensor, so we only use a part of it during the test, for example 256x256 pixels or 320x320 pixels.

The holographic recording is preferably taken with a short exposure time, choosing the exposure time from the above range. However, since we need a high-resolution recording for the reconstruction with the appropriate detail in the case of the holographic recording, we use a sensor with a higher resolution than the sensor used for the fluorescence recording as a holographic sensor, which, however, has a longer readout time than that of the fluorescence sensor. The holographic sensor preferably has a resolution of 5-20 megapixels/25 mm ² ; yet

- 16 preferably with a resolution of 10-15 megapixels/25 mm ² , particularly preferably with a resolution of approximately 12 megapixels/25 mm ² . With a sensor with such a resolution, a recording with sufficient detail can be taken so that it can be reconstructed in good quality. Relatively high-resolution sensors typically have a lower sensitivity, but are fully adequate for recording the light of the holographic light source.

The longer exposure time of the fluorescence recording and the longer reading time of the holographic recording can be coordinated (they are of the same order of magnitude); until the next recording is taken - that is, when the volume to be examined is replaced - both recordings can be downloaded. In many cases, even more time passes until the next recording is made.

The method according to the invention significantly speeds up the processing compared to the traditional digital holographic microscope, since it is necessary to reconstruct and analyze the shape of all objects. On the other hand, according to the invention, the holographic recording belonging to the given fluorescence recording is reconstructed only if (at least one) fluorescent object could be identified in the fluorescence recording. In the event that a fluorescent object could not be identified on the fluorescence recording, the corresponding holographic recording is ignored and not reconstructed. The evaluation of the sample is also aided by the fact that by determining the object position(s) on the fluorescence recording, the circle of the fluorescent object(s) found in the given recording can be determined (in most cases, typically only one such object in one recording, for a sufficiently rare sample). With the help of the object positions, the reconstructed information belonging to such objects, i.e. essentially their three-dimensional model, can be easily separated in the reconstructed holographic recording.

A great advantage of the method and equipment according to the invention compared to flow cytometers is that, according to the invention, it is possible to create a high-resolution image (model) of the fluorescent (autofluorescent or dyed fluorescence) objects in the flowing sample and thus help with more accurate classification.

- 17 For registering the recordings of the cameras - i.e. the fluorescence sensor and the holographic sensor (i.e. assigning them to each other, determining the common area they image, and in case of possible different orientations, i.e. if they do not completely face each other in the device according to the invention) preferably test objects we use The test objects include, for example, some highly fluorescent objects that can also be easily identified with the holographic system. As a test object, we use, for example, test beads. When a sufficient amount (for example, three) of these test beads are detected together, they can be identified based on their reconstructed arrangement. Based on the object positions measured on both the fluorescence and holographic sensors, the rotation, shift and magnification (selection of the appropriate ROI [region of interest found to be interesting]) required to match the images of the two sensors can be obtained with the help of a suitable algorithm, i.e. the registration of the two sensors can be performed. The resulting settings can also be advantageously tested with new test objects in a similar way. This is a semi-automatic calibration procedure (with human intervention), but a fully automated calibration procedure can also be used or developed.

Calibration can also be carried out based on the movement pattern of individual objects, i.e. in the case of continuous shots, by following objects that do not move very regularly (also continuously determining the position in both images).

For a good volumetric detection (high depth of field) of a fluorescent signal, it is therefore necessary to use a reducing lens with a small optical magnification/reduction or without reduction/magnification. If reduction/enlargement is not used, the fluorescence sensor (detector) must be in the immediate vicinity of the measuring cell (sample holder). In the case of a sensor arranged relatively far from the objects to be measured, the measurement is made difficult by the fact that the strength of the fluorescent light decreases squarely with the distance and appears in an extensive spot (in this case, its detection and localization would become more and more difficult with the increasing distance). To avoid this, the reduction lens according to the invention can be used in such cases, with the use of which the tested sample holder space and the fluorescence sensor can be arranged further away from each other.

- 18 Some known holographic systems, especially those in which fluorescence and holographic imaging are performed with the same sensor, are lensless, since it is not possible to arrange a lens suitable for both the fluorescence sensor and the holographic sensor. A solution without a lens can increase the measurable surface area, but it is much more difficult to ensure adequate and uniform high-intensity fluorescent lighting on a large surface area. In addition, in order to achieve the required resolution of the holographic reconstruction in the known solutions without a lens, since the pixel size basically limits the resolution that can be achieved in the case of a system without a lens, it is necessary to use some kind of super-resolution method. This requires more recordings, which significantly slows down sampling and complicates the registration of fluorescence recording, recordings and recordings made for super-resolution.

As shown in the embodiment according to Figure 1, by using suitable optical elements, objectives, and lenses, sufficiently strong illumination can be easily ensured in the entire (smaller) examined volume.

Due to the separate fluorescence and holographic sensor, according to the invention, it is possible to use the appropriate magnification, with which the required resolution can be achieved much more easily in the holographic system and thus the measurement can be much faster. Accordingly, the device according to the invention in the embodiment illustrated in Figure 1 contains the holographic imaging lens 24 arranged between the sample holding space part 10 and the holographic sensor 18. The magnification of the holographic reduction lens 24 is preferably between 1.5 and 10, more preferably between 2 and 3, especially approximately 2.5. By using such magnification, a sufficiently high resolution can be ensured for high-quality reconstruction.

Although the measurable surface and volume may seem smaller when lenses are used, the larger sample volumes examined without lenses cannot be used at all due to the large number of exposures required (in the case of super-resolution, it increases quadratically with the increase in resolution) and the low depth of field of the fluorescence recordings.

- 19 In accordance with the above, the demands placed on fluorescence and holographic sensors are quite different. In the case according to the invention, the fluorescence camera (sensor), which requires a small magnification in order to achieve a high depth of field, preferably has a low resolution (has a large pixel size), but is very sensitive, and is also preferably capable of high amplification with low noise (high sensitivity). It is useful if the camera can also detect colors, so that different colored fluorescences can be distinguished.

In the case of the holographic camera, since adequate lighting can be provided more easily (since we typically use a laser or LED light source), the important thing is not the sensitivity, but the small pixel size and the highest possible resolution. A monochrome camera (sensor) can also be used here, since the color information can be replaced by fluorescent signals (it can be determined preferably if several different fluorescent emitted light beams are present) and colored illumination is hindered by fluorescent measurement anyway.

It can be seen that the various demands on the sensors could only be met at the cost of major compromises.

Therefore, it is advisable to use the optical arrangement according to the invention, which realizes a measuring device that is much more efficient than the known solutions. In the case of holographic measurement, in order to achieve a high-resolution reconstruction, appropriate magnification may be necessary (in the case without a lens, the pixel size of the sensor usually limits the available resolution), for which in the case of the invention it is possible to use a lens. (It should be noted that the lensless arrangement significantly limits the achievable resolution. Only the lensless system is suitable for processing non-moving or barely moving, low-resolution, not very thick samples.)

With the method and equipment according to the invention, it is advantageous to detect multiple fluorescent responses at the same time. If the wavelengths emitted by fluorescent objects are far from each other, they can be separated with a simple color camera (e.g. a Bayer pattern). But if the emission peaks are close to each other, so be it

- The use of 20 more fluorescence cameras is conceivable, and their light paths are preferably separated by dichroic mirrors (eg, if both emit red, one is only 30 nm higher).

In the method and equipment according to the invention, the sample flows through the measuring cell (sample container). The sample is relatively rare, with autofluorescence (for example, chlorophyll, phycocyanin, phycoerythrin, etc.), or marked with some fluorescent dye (FDA - fluorescein diacetate: a dye suitable for separating living and non-living things, FITC - fluorescein isothiocyanate: fluorescent dye, etc.) contains objects.

The appropriate wavelength fluorescent lighting is provided by one or more obliquely placed excitation light sources 12a, 12b, which illuminate the measured volume well, evenly and with adequate brightness (cf. Figure 1). In the embodiment according to Figure 1, the excitation light source 12a, 12b is suitable for illuminating the sample holder-space part 10 from a direction other than the common optical axis (i.e. obliquely and preferably at a small angle relative to the common optical axis, preferably approx. 30-60°) is arranged. In the present embodiment, the device therefore contains excitation light sources 12a, 12b suitable for emitting excitation light of different wavelengths.

The excitation light sources can of course be LEDs, but a laser or a properly filtered halogen light source can also be used as an excitation light source.

In the embodiment of the device according to the invention illustrated in Figure 1, it contains excitation filters 26a, 26b (excitation filters) arranged between the excitation light source 12a and 12b and the sample holder space part 10, allowing the excitation light to pass through (filtering out other wavelengths as best as possible). By using them, it can be ensured that the illumination is optimal for triggering the appropriate fluorescence and does not interfere with the holographic and fluorescent measurements, i.e. only the excitation light of the appropriate wavelength can reach the 10 sample holder space parts.

Since the fluorescence excitations occur obliquely, but focused on the measured area, they can be individually tuned, modulated, and filtered. Thus, they are able to provide high light intensity and do not interfere with each other.

- 21 The emitted (auto- or dyed) fluorescence is recorded by one (or more) high-sensitivity fluorescence detectors (cameras) 14 through a coupling element 20 with suitable optics. The emission filter 28, which is conveniently placed in front of it and allows the fluorescent emitted light to pass through (filters out other wavelengths as much as possible), ensures that only the fluorescent signal enters the camera, so that the light of the excitation light and the holographic light source are also filtered out. In the present embodiment, the device therefore includes an emission filter 28 arranged on the common optical axis between the fluorescence sensor 14 and the coupling element 20, which allows the fluorescent emitted light to pass through.

The illumination with the appropriate coherence length required for the holographic system is provided, for example, by a laser. The wavelength of this must be such that it does not interfere with the fluorescence measurement, and with a suitable filter, used in the embodiment according to Figure 1, arranged between the holographic sensor 18 and the sample holder space part 10, which transmits the light of the holographic light source 16 ( filter out other wavelengths as much as possible) should be well separated with 30 first filters. The filter 30 ensures that only the diffracted image of the objects illuminated by the laser is recorded by the holographic camera and the fluorescent excitation light is completely cut out.

The selection of objects showing fluorescence in the method according to the invention (i.e. finding them on a fluorescence image) significantly increases the processing speed of the method and equipment according to the invention, as it is not necessary to reconstruct all holographic images, but only those that are of interest to us, i.e. those that contain at least one three-dimensional model of fluorescent object according to the determination of the identification step performed on the fluorescence recording. This enables the use of a higher resolution sensor, higher magnification and the accompanying smaller field of view, since the speed of flow and the volume that can be scanned are no longer determined by the speed of evaluation of (digital) holograms. The fluorescent signal designates where a specific fluorescent object is in the examined field of view. Thus, the reconstruction is only for the given object

- 22 associated holographic recordings must be made. The parameters of this reconstruction can preferably be precisely determined by processing a reduced-resolution image before reconstructing the high-resolution recording. More precisely, the optimization of the processing times makes it possible in the case of known solutions. In this context, it is very important to define the reconstruction distance as precisely as possible. If there is time to determine it, the image of the reconstructed objects can be much better. The method according to the invention makes this possible. The holder of the object (the location of the volume that encloses the object) can also be determined more precisely when using the method according to the invention, since up to an order of magnitude more processing time remains for the reconstruction of individual truly interesting objects. The samples subjected to fluorescence examination are typically so rare in terms of fluorescent objects that, if the sample is fed into the sample holder in a flow-through system, a fluorescent object will be found only every 10th recording or even less often. This increases the processing time for the reconstruction of the selected holographic images. If an available sample is denser than this, it can even be subjected to dilution for more efficient processing and evaluation for fluorescent objects. However, since the method and equipment according to the invention are used, for example, to check the efficiency of water purification (to determine how many fluorescent objects remain in a water sample after water purification), typically due to the nature of the use, a small number of fluorescent objects must be counted for the entire sample. Fluorescent staining is used to check whether all organisms have been successfully exterminated during water purification and disinfection.

In one embodiment of the method according to the invention, after taking at least two fluorescence images, the average of the (previous and current) fluorescence images is subtracted from the last (currently) taken fluorescence image to create a difference fluorescence image, and the fluorescent object identification step is performed on the difference fluorescence image . In the present embodiment, the background fluorescence is preferably averaged by subtracting the average formed by previous fluorescence recordings, and thus only the deviation from this is examined.

- 23 So permanent fluorescent reflections, bubbles, cracks, and camera errors are automatically eliminated by subtracting the average, because their signal is constant over time.

In the fluorescent object identification step of the method, a threshold value (with respect to the light intensity that can be measured in the fluorescence recording) is advantageously set, below which the light intensity values found during the search are not considered as a signal belonging to a fluorescent object. When searching for fluorescent objects, we look for the maximum light intensity on the fluorescence image. If there is and is greater than the predetermined threshold, then we also examine the measured values in one of its surroundings, which may also belong to the given object. In this way, multiple detections can be ruled out, because the areas belonging to a given object are excluded from the search for additional fluorescent objects. The search is preferably performed pixel by pixel, examining the light intensity recorded in the given pixel. If we examine adjacent objects (they can also be behind each other in space) (which would be interpreted as a single fluorescent object in the fluorescent object identification step), this will be revealed during the holographic reconstruction, and then it can be analyzed separately how in the case of the low-resolution fluorescence image understand segmentation. In the case of rare samples, fluorescent objects rarely come together in this way.

The 2D position of an object is detected on the fluorescence camera in the examined sample holding space (measuring cell). When several objects are reconstructed within the examined area based on the holographic recording (e.g. even at different depths), it is necessary to determine which ones are fluorescent and which ones are not. This can be done by comparing and jointly analyzing the two measurements as described above: in essence, the fluorescence recording marks the fluorescent objects (and accordingly the non-fluorescent objects can be ignored). It also happens that the fluorescent object is extensive, and in this case, according to the invention, the information of the two recordings can be conveniently compared (for example, we can examine whether the contents of both recordings refer to an extensive object).

- 24 Fluorescent objects can be distinguished from background noise by applying the threshold value. Since non-moving objects are removed by averaging the recordings and subtracting the average from the current recording, only moving fluorescent objects (moved in a flow cell or floating on their own) are preferably visible on the differential fluorescence image obtained by difference formation. The threshold value used is a function of the fluorescence signal (if the signal is small, the threshold is also small). Accordingly, the threshold value must be set for the current illumination. It should be noted that in the case of FDA staining (when the objects are artificially made fluorescent by staining), where the fluorescence of the background increases during the measurement, this must be taken into account and followed when choosing the threshold, i.e. it may be necessary to use a time-varying threshold, i.e. in this embodiment the given we use a variable threshold value depending on the background fluorescence of the sample volume. In the case of auto-fluorescence measurement, the background fluorescence does not change, but even then it is necessary to check how big the background is (which may not result from fluorescence, but from background light infiltration). The threshold depends on the lighting, the sensitivity and dynamic range of the camera, the gain of the equipment, the signal-to-noise ratio, and the strength of the signal. Any of these can change, for example, by replacing one component.

The timing of recording is illustrated in the already mentioned figures 2A-2C. The fluorescence and holographic recordings are made in a time-overlapping manner. This is illustrated in figures 2A and 2B, which respectively show the exposure time Tf for fluorescence recording and the exposure time Th for holographic recording. Figures 2A and 2B are to be interpreted in relation to each other, they show the relationship between the typical length of the exposure times Tf and Th and also illustrate that the recording of the two recordings is started roughly simultaneously, i.e. their temporal overlap is advantageously ensured in this way. During the long T f exposure time, the holographic recording can also be taken with other timings. For fluorescence recording, we typically use a much longer exposure time, but since the required camera has a lower resolution, it typically takes about the same amount of time to prepare and read the two images (in the case of fluorescence recording, the exposure time is long, but the

- 25 readings are fast, and in the case of holographic recording, the exposure time is short, but the reading takes longer due to the higher resolution). Since the objects can move during the long exposure, we expect a little motion blur (motion blur). However, this is negligible precisely because of the low resolution, i.e. the identification of the given object in the holographic recording is essentially unaffected.

The appearance of motion blur can be advantageously used in one embodiment as illustrated in Figure 2C below. In this embodiment, in one sub-period Tf2 of the exposure time Tf of the fluorescence recording, the given sample volume is illuminated with two excitation lights of different wavelengths, and in another sub-period Tfi, the given sample volume is illuminated with only one of the two excitation lights, the fluorescent excitation lights the fluorescent emitted light emitted as a result is mapped, and in the case of identification of at least one fluorescent object in the fluorescent object identification step, based on the displacement of the object image of at least one fluorescent object in the fluorescence recording, the fluorescent object excited by the excitation light used in both subperiods and/or only the subperiods in one of them, we identify a fluorescent object excited by the applied excitation light.

Such an illumination scheme is illustrated in Figure 2C, where the total exposure time Tf is indicated; in the case shown in the figure, a single excitation illumination is used in the sub-period of length Tfi (for example, only the excitation light source 12a is operated), however, in the sub-period Tf2, we switch on the other excitation as well, so that the sample volume under examination is illuminated with two excitation lights characterized by different wavelengths. It is also possible to use two illuminations simultaneously in a first subperiod, and to switch off one of them in the subsequent second subperiod and use only one illumination. The steps of the method according to the present embodiment can also be performed on a recording made in this way.

- 26 The present embodiment can therefore be used to detect fluorescent signals that cannot otherwise be distinguished with the fluorescence sensor (camera) without a separate special filter (for example, the red fluorescence of chlorophyll (680nm), which is triggered by blue illumination (470nm), and the red fluorescence of phycocyanin (650nm ) fluorescence, which is triggered by amber (595 nm) illumination) can be distinguished. In the example, cells containing phycocyanin can be distinguished based on the amber lighting turned on at the appropriate time, for which the motion blur shows a special pattern (the motion blur for the two objects will be different, since the motion recorded in the recording is of different duration, so the motion blur showing displacement during the exposure time different degrees).

Figures 3A-3B illustrate exemplary related fluorescence and holographic images, and Figure 3C is a reconstruction of the holographic image shown in Figure 3B. In Figure 3A, images of three objects are displayed, i.e. in the test corresponding to the example, three fluorescent objects are present in the tested sample holding space at the moment of recording. In the given example, green and red fluorescence can be distinguished based on the fluorescence recording (in the example, the red fluorescence comes from autofluorescence, and the green from the fluorescence obtained by staining), the image of the leftmost object is red, the other two are green. The individual object images are marked with arrows.

Figure 3B is a holographic image of the fluorescence image shown in Figure 3A. In Figure 3B, it can be seen that, in addition to the image of the fluorescent objects, the images of several other objects are also displayed (ie, the corresponding interference pattern). A great advantage of the invention is that the images belonging to fluorescent objects can be selected from the large number of object images shown in the holographic recording of Figure 3B using the method according to the invention. As can be seen from the comparison of Figures 3A and 3B, based on the position of the object images of the fluorescence recording, those belonging to the fluorescent objects can be selected from among the object images of the holographic recording.

- 27 Figure 3C shows the reconstruction of the object image (interference pattern) surrounded by a white square in Figure 3B, on which the reconstructed shape of the fluorescent object can be observed. In the reconstructed image according to Figure 3C, rings are visible due to the twin image created due to the in-line arrangement used to create the holographic recording. The disturbing effect of the twin image can be reduced using known methods, however, morphological examination, i.e. the examination of the three-dimensional model obtained on the fluorescent object, is not affected by the presence of rings (the removal of the twin image is slow, so it is not used).

Figure 4 shows a fluorescence image taken with a relatively long exposure time. Due to the movement of the fluorescent object, the object image "draws a line behind it", i.e. motion blur occurs. According to the above, this can be suitable for separating objects that fluoresce with the same color by changing the fluorescence excitation at the appropriate time.

In order to avoid a greater degree of motion blur, in the case of a flowing sample, the sample preferably flows very slowly during the recordings (the above, advantageously exploitable small amount of motion blur typically occurs even in the case of a stationary sample). After taking the shots, however, by using a pump, the movement of the liquid can be sped up and then slowed down again for the next shot. The exposures are therefore advantageously made after the sample moved (with the pump) stops and slows down. The time for this is not long compared to the exposure and download times used, and does not significantly slow down the operation of the equipment, i.e. the recordings can preferably be made in relatively quick succession. In an exemplary implementation of the equipment, exposure occurs every second, but a speed of 3-4 Hz is probably also available (in this case, the exposure time for the fluorescence recording and the download time for the holographic recording can be coordinated and can be approximately the same).

A known device that uses the same sensor for detection cannot show a similar operation. Measuring the fluorescent signal requires a long exposure time, high gain and long exposure, while holographic recording requires high resolution and short exposure times (possibly color

- 28 recordings are possible) For this reason, in the known devices, these cannot be done at the same time, but only one after the other. In this case, however, objects can only be identified if they are not moving or are moving very slowly. If super-resolution has to be used in such known equipment, which is often a requirement in the case of lensless systems, it further reduces the processing speed and makes the registration of fluorescence and holographic images even more difficult. In addition, the requirement that the holographic system, if lensless, provides good resolution at a small object-sensor distance cannot be met in the known equipment, while the dichroic mirror used to filter out the fluorescent excitation light (this is, for example, an interference filter, which can provide high optical density and extinction), which is then absolutely necessary to filter out the fluorescent excitation light, it is usually 2-3 mm thick due to its manufacturing technology, so a smaller object-sensor distance is not possible in such a known solution.

The flow rate should preferably be set in such a way that an object appears on only one hologram, but does not wash out, so that it travels a distance smaller than a pixel size during the exposure time. However, between taking two shots, the entire examined volume should be exchanged, since then we are not measuring the same objects.

In one embodiment of the method, a fluidly moving sample is used, and after the replacement of the given sample volume, the steps of the procedure are performed for at least one more given sample volume.

If, for example, the exposure time of the fluorescence sensor is 80ms (in practice, enough light is collected for accurate detection during this time, but it depends on the camera and fluorescent lighting), we want the object to move only a few pixels during that time. In the case of a 5-pixel displacement, this causes a displacement of 8x as much (2048/256=8) on the holographic sensor - if the resolution of the fluorescence sensor is, for example, 256x256 and that of the holographic sensor is 2048x2048 (2048/256=8), which is 40 pixels/80ms. For example, this is how much the sample flow rate should be slowed down. Since the holographic recording is short (1 ms

- 29) exposure time, this speed only causes negligible motion blur. After recording, the sample can be moved further, even at high speed, as this does not affect the scanned volume.

The holographic sensor is, for example, 10-15 megapixels (we can use a 2048x2048 sub-range of this as described above); an average sensor is 5x5 mm. Downloading images of this size to the computer typically takes approx. It takes place at a speed of 4-5 frames/sec; downloading one recording approx. 200 ms, but it can be up to 500 ms. There are cameras that are much faster than this, but they are very expensive and the processing time of the received images and holograms is much slower, so it is not worth using them. For example, holographic exposure is done by flashing lasers, which is of the order of milliseconds. So, during the recording, the objects and their hologram seem to stand still and do not move even by a single pixel (there is no motion blur that would spoil the resolution).

Due to the low resolution of the fluorescence sensor (e.g. 1-2 megapixels) and differences in magnification, we only use a small part of it (e.g. 300x300 pixels); a sensor is typically 5x5 mm. As above, relatively long exposure times and high gain must be used to collect enough light from the weak fluorescent signal. Here, of course, the exposure time that can be used also depends on the intensity of the fluorescent excitation light (stronger illumination requires a shorter exposure time). In accordance with the above, an exposure time of 40-200ms was typically used for fluorescence recordings. In the case of longer exposure times, the object could move more during the recording, which can hinder the determination of the exact position, but in the case of short exposures, this is practically negligible. Such exposure times are suitable for fluorescent object separation based on motion blur seen in fluorescence imaging (see Figure 2C).

In an exemplary case, a fluorescence image with 256x256 pixels and a holographic image with 2048x2048 pixels are taken. How much surface of the sensor is used also depends on the scale of reduction/enlargement of the maps used. Resolutions can be even higher than this

- a difference between 30, but this may impair the estimation of the location of the objects on fluorescence imaging. In the case of a rare sample, where the objects are far from each other and therefore do not overlap regardless of their fluorescence, we can even apply a higher resolution difference.

It is possible to simultaneously record several different fluorescent signals by applying suitable filters. It must then be ensured that the applied illuminations (exciting lights) do not interfere with each other and that the emitted wavelengths can somehow be separated from each other. For example, we can use 470 nm and 600 nm illumination (using 10 nm bandpass filters as excitation filters). The applied emission filter allows light above 500nm to pass through (this prevents the 470nm excitation light from reaching the fluorescence sensor), and a (20nm bandwidth) band-exclusion (so-called 'notch') filter is used for the 600nm to filter out illumination so that it cannot reach the fluorescence sensor. We are then able to detect FDA stained sample (~520 nm wavelength emitted light, for live-non-living test), chlorophyll B autofluorescence (~680 nm emission), phycocyanin emission (~650 nm emission, blue-green algae) and phycoerythrin (570 nm) emission, blue-green algae, red algae), while the holograms are recorded with, for example, 405 nm wavelength light.

The fluorescent lighting is preferably provided in the equipment (thus also in the embodiment according to Figure 1) obliquely, at a small angle. This makes it possible to use several different fluorescent lights at the same time and to reduce the requirement for the dichroic filters used (in the embodiment according to Figure 1, the coupling element 20 can be a dichroic filter for fluorescent emitted lights, as it preferably allows only these to pass through to the fluorescence sensor). That is, if the fluorescent lighting were to shine directly on the sensor, the dichroic filter should provide much greater extinction than if the sample is illuminated obliquely, since then we only need to filter out the scattered light. Since the individual fluorescent lights are separate modules (excitation light sources 12a and 12b), their proper focusing and bandwidth filtering can be ensured, i.e. the emission filter, which in the embodiment according to Figure 1 is 28 emission filters, can be optimized more easily. In the case of other arrangements, their convergence and focusing is complicated, expensive and not really efficient

- 31 optically. With the solution according to the present embodiment, there is no need to use multiple transmissive mirrors at the bottom or at the top, which are expensive, their efficiency is only around 90%, and thus can lead to a serious loss of light, and would impair the quality of (both fluorescence and holographic) imaging, as they break on several surfaces and the light used.

Afocal optics can preferably be used to record holograms. The great advantage of this arrangement over other arrangements is that it ensures that the magnification of the various objects within the system is constant, i.e. it does not depend on the position of the object, even in the case of non-plane wave illumination. Thus, it is not necessary to calibrate the applied magnification.

The essence of the used arrangement is that we do not use a lens (objective) to achieve the appropriate magnification, but a lens system, which is essentially arranged like a telescope. This is advantageous because the one-lens system together with holographic reconstruction generates magnification independent of the distance of the object, but only if the illumination is parallel. If this is not the case, the magnification of the reconstructed objects will be different depending on the distance (this can be calibrated, but it is not easy). When using an afocal arrangement, on the other hand, the magnification of the afocal arrangement can be counted on practically regardless of the lighting. This typically does not affect the segmentation of the objects, as it is currently performed from the reconstructions of the optimal object. The optimal reconstruction is determined by comparing the reconstructions at different distances (counting with a lower resolution, of course) and by using special focal measures. When using the method according to the invention, there is preferably even time to determine the distance for the reconstruction more precisely.

In the device according to the invention, 31 dichroic mirrors can also advantageously be arranged, the task of which is to deflect the fluorescent signal reflected from the microscope objective, i.e. the holographic reduction lens (the dichroic filter 31 is not arranged perpendicular to the common optical axis, but slightly oblique to it). Otherwise, this signal would also enter the fluorescence camera and there, an additional, recorded from another side (defocused, more obscure)

- It would give 32 images of the measured volume. Preferably, the filter 30 and the dichroic mirror 31 can be replaced by a single optical element arranged in place of the dichroic mirror 31, in which case the filter 30 can be omitted.

In the holographic arrangement of the equipment, for example, we use an in-line arrangement, i.e. the illumination also provides the reference beam. It is advisable to use such an arrangement if the examined sample is rare, i.e. the scattering on the measured objects can be treated as a small perturbation of the reference. The advantage of the layout is its simplicity, insensitivity to vibrations, and robustness. Other arrangements can also be considered, such as on-axis in-line and off-axis arrangements, but there it is necessary to ensure that the reference is led to the holographic sensor by a separate path. With these, more extensive, less transparent samples can be examined and the phase of the objects can be reconstructed more easily (simpler, faster numerical methods can be used), but it requires more complicated optical equipment and is very sensitive to vibrations.

It is advisable to strive for the wavefront used in the holographic arrangement to be regular (preferably well approximated with a plane wave), this can be ensured, for example, with a single-mode laser, where the emitting surface is preferably small, because the large extent could limit the achievable resolution.

In the device according to the invention, the coupling element is implemented, for example, with a dichroic low-transmission mirror, which reflects short-wavelength light while allowing longer ones to pass through. In our equipment, this is to provide coherent illumination for the holographic arrangement, to direct it onto the sample and then onto the holographic sensor (the light of the holographic illumination is reflected by the dichroic low-transmission mirror), while the fluorescently emitted light passes through it and the fluorescence camera through it you see the measuring cell. The dichroic low-transmission mirror is preferably arranged at 45° to the common optical axis, as can also be seen in Figure 1.

In the method according to the invention, some characteristics of the equipment presented above, in particular the use of reduction lenses, filters, the resolutions and sensitivity values advantageously used in the sensors, can be used in the method according to the invention independently of other characteristics of the equipment.

- 33 The method and equipment according to the invention can combine the fluorescence measurement capability of flow cytometers with the benefits of holographic (preferably digital holographic) recordings. Preferably by using digital holographic microscopy, it can also reconstruct a high-resolution image of the found objects, suitable for classification, in such a way that it selects the fluorescent objects based on the fluorescence image from the few dozen objects in the image taken by the holographic microscope.

Of course, the invention is not limited to the preferred embodiments presented in detail, but further versions, modifications and further developments are also possible within the scope of protection defined by the claims.

Claims (18)

1. Procedure for fluorescence examination of a sample, during which - a specific examined sample volume of the sample is illuminated with fluorescent excitation light and a fluorescence recording is made by mapping the fluorescent emitted light of the given sample volume under the influence of the fluorescent excitation light, and - a holographic recording is made of the given sample volume in a time-overlapping manner with the fluorescence recording, characterized by the fact that the procedure is carried out with equipment for fluorescence testing of a sample, which contains - the sample holder compartment (10) suitable for receiving a given sample volume of the sample, - one or more excitation light sources (12a, 12b) suitable for illuminating the sample holder space (10) with fluorescent excitation light, - a fluorescence sensor (14) suitable for recording fluorescence by mapping the fluorescent emitted light emitted by the fluorescent excitation light of the given sample volume in the sample holder space (10) during the test, - a holographic arrangement containing a holographic light source (16) and a holographic sensor (18) suitable for taking a holographic image of the given sample volume under investigation, and the fluorescence sensor (14) and the holographic sensor (18) are opposite to each other in relation to the sample holder space part (10) , together with the sample holder space part (10) are arranged on a common optical axis, as well as - the coupling element (20) that maps the object wave from the holographic light source (16) onto the common optical axis and directs it towards the sample holder space part (10), reflects the light of the holographic light source (16) and transmits the fluorescent emitted light, and fluorescent in the fluorescence recording an object identification step is performed, and if at least one fluorescent object is identified in the given sample volume in the fluorescent object identification step, then - we assign at least one object position to at least one fluorescent object in the fluorescence recording, and - by reconstructing the holographic recording, a reconstructed holographic recording is produced and the reconstructed model of the at least one fluorescent object is identified based on the position of at least one object on the reconstructed holographic recording. 2. The method according to claim 1, characterized by the fact that a fluidly moving sample is used and the steps of the procedure are carried out for at least one more given sample volume after the change of the given sample volume. 3. The method according to claim 2, characterized by the fact that, after taking at least two fluorescence images, the average of the fluorescence images is subtracted from the last fluorescence image to create a differential fluorescence image, and the fluorescent object identification step is called the differential fluorescence image. done in the picture. 4. The method according to claim 3, characterized in that a threshold value is used to identify a fluorescent object in the fluorescent object identification step. 5. The method according to claim 4, characterized in that a variable threshold value depending on the background fluorescence of the given sample volume is used. 6. The 1-5. A method according to any one of claims, characterized in that - in one sub-period (Tf2) of the exposure time (Tf) of the fluorescence recording, the given sample volume is illuminated with two excitation lights of different wavelengths, and in another sub-period (Tfi) the given sample volume is illuminated with only one of the two excitation lights , - we make the fluorescence recording by imaging the fluorescent emitted light emitted as a result of the fluorescent excitation lights, and - if at least one fluorescent object is identified in the fluorescent object identification step, based on the displacement of the object image of at least one fluorescent object in the fluorescence recording, the fluorescent object excited by the excitation light applied in both subperiods (Tfi, Tf2) and/or only the subperiods (Tfi, A fluorescent object excited by the excitation light used in one of Tf2) is identified. 7. The 1-6. Method according to any one of the claims, characterized in that an exposure time (Tf) of 40-200 ms is used for the preparation of the fluorescence recording and/or an exposure time of 0.5-5 ms is used for the preparation of the holographic recording. 8. Equipment for the fluorescence examination of a sample, which contains - the sample holder compartment (10) suitable for receiving a given sample volume of the sample, - one or more excitation light sources (12a, 12b) suitable for illuminating the sample holder space (10) with fluorescent excitation light, - a fluorescence sensor (14) suitable for taking a fluorescence recording by mapping the fluorescent emitted light emitted by the fluorescent excitation light of the given sample volume in the sample holder space (10) during the test, and - a holographic arrangement containing a holographic light source (16) and a holographic sensor (18) suitable for making a holographic recording of the given sample volume, which means that - the fluorescence sensor (14) and the holographic sensor (18) are arranged opposite to each other in relation to the sample holder space part (10), on a common optical axis together with the sample holder space part (10), and - contains the coupling element (20) which maps the object wave from the holographic light source (16) onto the common optical axis and directs it towards the sample holder space part (10), reflects the light of the holographic light source (16) and transmits the fluorescent emitted light. 9. The device according to claim 8, characterized in that it contains a fluorescence reduction lens (22) arranged on the common optical axis between the fluorescence sensor (14) and the coupling element (20). 10. The device according to claim 9, characterized in that the depth of field of the fluorescence reducing lens (22) is selected for the extension of the sample holder space part (10) in the direction of the common optical axis by adjusting the reduction/enlargement degree. 11. The device according to claim 9 or 10, characterized in that the degree of reduction/enlargement of the fluorescence reduction lens (22) is between 0.5 and 1.5. 12. The 8-11. Device according to any one of claims, characterized in that it contains a holographic reduction lens (24) arranged between the sample holder space part (10) and the holographic sensor (18). 13. The device according to claim 12, characterized in that the magnification of the holographic imaging lens is between 1.5 and 10. 14. The 8-13. The device according to any one of the claims, characterized in that it contains the device arranged on the common optical axis - the emission filter (28) arranged between the fluorescence sensor (14) and the coupling element (20), which allows the fluorescent emitted light to pass through, and/or - a first filter (30) arranged between the holographic sensor (18) and the sample holder space part (10), which transmits the light of the holographic light source (16). 15. The 8-14. The device according to any one of the claims, characterized in that it contains an excitation filter (26a, 26b) arranged between the excitation light source (12a, 12b) and the sample holder space part (10), which allows the excitation light to pass through. 16. The 8-15. Device according to any one of the claims, characterized in that the excitation light source (12a, 12b) is arranged to illuminate the sample holder space part (10) from a direction other than the common optical axis. 17. The 8-16. Device according to any one of claims, characterized in that it contains several excitation light sources (12a, 12b) capable of emitting excitation light of different wavelengths. 18. The 8-17. Device according to any one of claims, characterized in that the fluorescence sensor (14) has a resolution of 0.5-3 megapixels/25 mm ² and/or the holographic sensor (18) has a resolution of 5-20 megapixels/25 mm ² .