DE102017121483B3 - Method for testing a multispectral specimen, control unit therefor and microscope arrangement - Google Patents

Abstract

A method of inspecting a multispectral specimen (150) using a fluorescence microscope (100) with imaging optics (120),
wherein the sample to be examined (150) is illuminated by means of a multispectral light source (210) which can be actuated by means of a trigger signal,
wherein a beam path (160) generated by the imaging optics (120) from light emitted by the sample (150) is detected by means of an image recording device (230),
wherein between the imaging optics (120) and the image capture device (230) a filter device (220) is introduced, which has a plurality of different movable filters (222), each associated with a different wavelength range and optionally between the imaging optics (120) and the image pickup device (230) can be introduced,
characterized in that each of the plurality of different movable filters (222) is associated with a coding with respect to a predetermined position with respect to the beam path (160), and
in that a trigger signal (S1) is generated using the coding by the filter device (20) in each case when the predetermined position of the filter (222) is reached and the multispectral light source (210) is triggered via it,
wherein then an intensity (I) of each wavelength range used (λ) of the multispectral light source (210) is set to a predetermined value, and
wherein by means of the image recording device (230) for the respective filter (222) in each case upon reaching the predetermined position, an image acquisition is started.

Description

The present invention relates to a method for examining a multispectral sample using a fluorescence microscope, a control unit for carrying out the method and a microscope assembly with a fluorescence microscope.

State of the art

In microscopy, imaging techniques are used to obtain images of objects to be examined. For this purpose, light emitted by an object, in particular fluorescent light, is often also detected by means of a camera. In order to obtain more accurate information or better images, the light recorded by the camera can be spectrally split. Such methods are also known by the term "spectral imaging".

For this purpose, it is possible, for example, to use filters which filter or pass through specific spectral regions and are introduced into a beam path. In addition to simple glass filters and filter cubes, acousto-optical filters, prisms, grids or diffractive optics are also suitable here, for example. Also, light sources with, for example, up to four different colors can be used.

As a camera, for example, the so-called. "ORCA flash 4.0" of Hamamatsu in question. According to the data sheet (see https://www.hamamatsu.com/resources/pdf/sys/SCAS0080E_C11440-22CU_tec.pdf), individual filters can be successively introduced into the beam path, and with a filter inserted, the object can then be illuminated and to take a picture. Then another filter can be set.

The EP 1291627 A1 relates to a method for simultaneously generating time resolved fluorescence images and time resolved emission spectra based on time and location correlated single photon counting. For this purpose, essential components such as light source and detector must be synchronized with each other in time.

The US 2013/0310268 A1 describes a method for standardizing microscope measurements using a calibration slide. In the fluorescence microscope described there, among other things, the filter is controlled by a control unit.

In a fluorescence microscopy method according to the WO 2015/185661 A1 the filters of the fluorescence microscope are synchronized with the camera for multispectral imaging.

In the fluorescence microscopy method according to the publications DE 10 2010 013 223 A1 such as US 2017/0031149 A1 the irradiation of the excitation light of the light source is synchronized with the readout of an active region of the detector surface of an image sensor, wherein the active region of the detector surface can be limited to the region to which the fluorescence light emitted by the specimen is focused by the detection optics.

According to the DE 698 34 963 T2 Among other things, color conversion of the UV microscope is proposed in which light is separated into components by two or more filters, then passed through a sample, and then converted to visible multicolor light by a transducer such as an image intensifier. Subsequently, the light is recombined by a set of two or more filters into a multi-color image with the sets of filters synchronized with each other.

The publication DE 10 2015 216 570 A1 relates to a fluorescence microscopy method in which the control of the filter system depends on the selected operating mode. However, these systems have some disadvantages. So when using individual filters switching between different filters takes a relatively long time. Depending on how the microscope, on which the "Spectral Imaging" is to be performed, is designed, retrofitting is often complex, which is also reflected in high costs, and is sometimes not possible.

In addition, relatively few spectral ranges can be distinguished or discriminated. For example, with two or three filter cubes the spectral resolution is limited. When using color cameras, the incident light is divided into several chips, so that only part of the full intensity of each chip is eliminated. This leads to a deterioration of the signal-to-noise ratio or signal-to-noise ratio.

Moreover, when using conventional methods, there is usually some cross-talk, so that one wavelength range can contribute to the signal that does not originate from the desired fluorescence dye, which leads to measurement errors. The time resolution is usually too low, especially in long switching times for fast-running processes, for example, in living cells.

Different, wavelength-dependent intensities of the excitation light also require a different exposure time on the camera side. This leads to further delays and also to Signal distortions, since the exposure times can be different lengths.

Against this background, therefore, the task of specifying a way to capture spectrally resolved images of an object better and / or faster.

Disclosure of the invention

According to the invention, a method for the examination of a multispectral sample, a control unit and a microscope arrangement with the features of the independent patent claims are proposed. Advantageous embodiments are the subject of the dependent claims and the following description.

The invention is based on a method for examining a multispectral sample using a fluorescence microscope with imaging optics. The imaging optics may in particular comprise a lens and a tube for imaging the sample and in particular also an eyepiece (or a suitable adapter for an image recording device). By means of a multispectral light source, which can be actuated by means of a triggering signal, the sample to be examined is then illuminated, and by means of an image recording device, a beam path (or imaging beam path) generated by the imaging optics of light emitted from the sample is detected. In this case, a filter device is introduced between the imaging optics and the image recording device, which has a plurality of different movable filters, each associated with a different wavelength range and optionally between the imaging optics and the image pickup device can be introduced, This means that the filters are individually introduced into the beam path to capture only light passing through the filter in question.

According to the invention, it is now provided that each of the several different movable filters associated with a coding with respect to a predetermined position with respect to the beam path (or imaging beam path), and that using the coding by the filter device in each case upon reaching the predetermined position of the filter, a trigger signal generated and above the multispectral light source is triggered. This means that the light source is triggered by the filter device. Then, an intensity of each wavelength range of the multispectral light source used is then set to a predetermined value. By means of the image recording device, in addition, an image recording is started for each filter upon reaching the predetermined position. The movement of the filter device can in particular be carried out continuously or in sections continuously. The latter means that the filter device is moved, for example, by one or more filters, then stopped and then moved on again. Under a continuous, in particular a continuously continuous, movement is to be understood in particular a movement without stop or intermediate stop. This can preferably take place in that the filter device has a filter disk which rotates through the beam path with an uninterrupted movement.

By triggering the multispectral light source by a trigger signal generated by the filter device and the subsequent start of the image acquisition, an associated image of the sample can be recorded very quickly for each filter. In particular, a central master unit is no longer necessary in this way, by means of which a movement of the filter device and an image acquisition are controlled and coordinated. Such master units used hitherto also require reconfirmation of the individual components prior to triggering, which would bring about a further loss of time, which is avoided by the proposed method. In addition, manual switching of filters is no longer necessary. The filter device can also be moved in this way at a relatively high speed, whereby the individual filters are introduced in particular successively in the beam path. This allows you to take pictures of objects with high spatial and temporal resolution. In addition, such a method can be used very easily in fluorescence microscopes - in particular light microscopes, also inverse microscopes, in question - by retrofitting a suitable filter device, possibly with a suitable control unit.

Advantageously, the image acquisition by means of the image acquisition device is started by a further triggering signal generated by the multispectral light source, after the multispectral light source has been triggered (triggered by the filter device or the trigger signal generated therefrom). In this way, a cascade for taking an image of the sample is triggered. The image recording device is thus triggered only indirectly by the filter device. In this way, however, it is possible to optimally match the exposure and the image recording.

Alternatively, it is also preferred if the image acquisition by means of the image recording device is started by the triggering signal or a further triggering signal which is generated by the filter device. Then, the filter device or an associated trigger signal triggers both the light source and the image recording device directly out. Depending on the situation, a common triggering signal given to the light source and the image pickup device can be used here. It is also conceivable to use two separate trigger signals. In this way, for example, a simpler synchronization of light source and image pickup device is possible, for example, after each rotation or only after a certain number of cycles of the filter device.

It is advantageous if light is emitted by the multispectral light source when triggered in accordance with at least one predetermined parameter. Particularly suitable as such parameters are the following: a predetermined wavelength range, a predetermined intensity, a predetermined time duration and a predetermined time. For this purpose, the light source can in particular have different lamps with different colors, for example LEDs with different wavelengths, in particular in the form of a multi-color LED lamp house. It is also conceivable that the multispectral light source also has a filter device with a plurality of filters, so that for example different colors or wavelengths can be generated from white light. Such a filter device can then also have a filter disk, which can then be synchronized in particular with the other filter disk. The at least one predetermined parameter is then expediently predetermined individually for at least one, but in particular all, of the multiple filters. This allows a particularly individual vote of each image acquisition. For example, different, necessary illumination durations for different wavelength ranges or colors can be set in this way in order to obtain as uniform as possible image recordings.

The predetermined intensity, ie the illumination intensity, is generally not the same for different wavelengths. The wavelength-dependent illumination intensity may be dependent on the sample, for example, one fluorochrome, for example, may glow less than another. Then, a higher illumination intensity would allow a better signal-to-noise ratio, or a shorter exposure time (i.e., a different predetermined amount of time). On the other hand, the wavelength-dependent illumination intensity can be dependent on the experiment, so that a particular fluorochrome can fade if the illumination intensity is too high. The wavelength-dependent illumination intensity may also be dependent on the system itself, because the smaller the illumination intensity, the longer the exposure time required for good image acquisition, and the slower the filter device should move to achieve the required exposure time.

The predetermined time periods or image acquisition times may depend on multiple systemic constraints and are generally not the same length for the different wavelengths. They hang, for example Depending on the shape of the filter (round or not round), the number of filters (the more filters are present in the filter device, the smaller the filters must be, the shorter the filters, the shorter the time window in which the Therefore, the faster the filter device rotates, the shorter the possible exposure time, whichever is shorter can be set for a particular filter).

Furthermore, it is preferred if a control unit is used in which the at least one parameter is stored and by means of which the light is emitted in accordance with the at least one parameter. Such a control unit may in particular also be part of the light source or be integrated in it. In this way, no intermediate PC or the like (the above-mentioned master unit) is necessary, but the program for exposing the sample is integrated directly into the light source, whereby a much faster operation is possible. In addition, such a control unit can preferably also be used for further functions of the microscope arrangement used, for example for the image recording device and / or the filter device. However, it is also conceivable to use separate control units for the individual components.

As already mentioned, it is preferred if a filter disk is used as filter device. In particular, its rotational speed can then be set as desired or to a desired value. This allows an individual adaptation of the image recording, but in particular a change in the recording speeds both for different samples to be observed and during the observation of only one sample.

The coding of the filter device for the individual filters with regard to the predetermined positions can preferably be effected by means of encoders for triggering and / or at least one encoder for a synchronization. Such encoders can be designed, for example, as magnets, which then emits the trigger signal, for example when a suitable sensor is reached. this makes possible a particularly simple and, above all, also timed to the movement of the filter device triggering, for example, the multispectral light source. By means of a sender for a synchronization can, for example, using the sensor for triggering the trigger, each time or after a certain number of cycles, for example when a filter disk is used in a filter device, a synchronization with the other donors. It is also conceivable to provide a second encoder for synchronization on the other side of the filter disc, for example, to be able to balance the disc can. It is also conceivable to use only the encoder for synchronization without individually assigned to the filters donors. Here then, for example, after a calibration with the individual encoder and the time and taking into account the rotational speed can be detected when the respective filter enters the beam path. Here then also fractions of cycles can be considered.

Advantageously, a filter device is used which has at least three, preferably at least five, more preferably at least seven, different filters. It is also conceivable, however, a different number of filters such as eight or nine. By a larger number of filters, a better discrimination of the wavelengths can be achieved, resulting in an overall better spatial and temporal resolution in the image acquisition.

It is particularly preferred if the image recording device used is at least one camera, which is designed in particular as a single-chip camera. This is then in particular a so-called gray scale camera. In this way, better results are achieved because the full intensity of the light impinges on the chip and is not distributed among different chips.

Preferably, a triggering of the illumination of the sample by means of the multispectral light source and / or starting the image acquisition by means of an interruption means is temporarily interrupted if necessary. In the simplest case, such an interrupting means may for example be a switch for interrupting a signal line which is used to trigger the multispectral light source or the image recording device. The filter device can simply be moved further and does not need to restart, but unnecessary image recordings are avoided, so that in the meantime, for example, the sample and / or lens can be changed or adjusted.

With the proposed method, it is thus possible, in particular, to specify a specific program for taking images of a sample, in which the filter device is moved at a certain speed and, when a respective filter is introduced into the beam path, a respective desired light pattern with desired parameters from the multispectral Light source is generated so that an associated image is captured by means of the image pickup device. The light patterns can differ from filter to filter. It is even conceivable that in addition a distinction is made after each revolution, for example, a filter disk. Likewise, in particular, the speed of the filter device can be varied. Such a program can then be specified by a user, so that everything can run automatically when taking pictures.

The invention furthermore relates to a control unit for use with a microscope arrangement comprising a fluorescence microscope with imaging optics for generating a beam path (or imaging beam path), a multispectral light source which can be actuated by means of a trigger signal, an image recording device and a filter device which comprises a plurality of different movable filters each associated with a different wavelength range, each selectively interposable between the imaging optics and the image sensing device, and each associated with a coding with respect to a given position with respect to the optical path. The control unit is configured to be in use perform a method according to the invention with the microscope arrangement,

In particular, all components required for the method can be controlled with such a control unit, particularly preferably within the framework of a fully automatic, programmable sequential control, in which image captures are finally triggered by the filter device in a specific trigger or triggering sequence. Thus, for example, by the filter device sending a trigger to the control unit and then bringing the intensity of the light source and its color to a pre-set level, the sample can be exposed and then at a certain pre-programmed interval and in a certain manner the image pickup device controlled ,

It should be emphasized that the process, which is triggered by the trigger of the filter device, previously in the firmware of the components involved (eg camera and LED lamp) can be loaded so that the process of the method further autonomously (ie without PC) and can also run at maximum speed.

The control unit or the programs loaded into the control unit and the components involved should preferably also be dynamic be replaceable, ie the programs can be replaced or modified, for example, depending on an event.

The control unit or the programs loaded into the control unit and the components involved should preferably also link events back so that external programs or components can be informed about the status of the sequence.

The invention furthermore relates to a microscope arrangement comprising a fluorescence microscope with imaging optics for generating a beam path (or imaging beam path), a multispectral light source which can be actuated by means of a trigger signal, an image recording device and a filter device which has a plurality of different movable filters , which are each associated with a different wavelength range, each optionally between the imaging optics and the image pickup device can be introduced, and each associated with a coding with respect to a predetermined position with respect to the beam path, and a control unit according to the invention. In this case, the filter device between imaging optics and the image pickup device is introduced or introduced.

It is also particularly preferred if the filter device is exchangeable. In this way, the microscope assembly can be used for various applications with different requirements, with only the filter device to be replaced, the light source and the image pickup device can still be used.

With regard to further refinements and advantages of the control unit according to the invention and the microscope arrangement according to the invention, reference is made to the above explanations of the method according to the invention, which apply correspondingly here.

Further advantages and embodiments of the invention will become apparent from the description and the accompanying drawings.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

The invention is illustrated schematically with reference to an embodiment in the drawing and will be described below with reference to the drawing.

list of figures

• 1 schematically shows a microscope arrangement according to the invention in a preferred embodiment, with which a method according to the invention can be carried out.
• 2 schematically shows an image recording system as part of a microscope assembly according to the invention in a preferred embodiment.
• 3 schematically shows an image recording system as part of a microscope assembly according to the invention in a further preferred embodiment.
• 4 schematically shows a filter device as part of a microscope assembly according to the invention in a further preferred embodiment.
• 5 schematically shows a light source as part of a microscope arrangement according to the invention in a further preferred embodiment.
• 6 shows by way of example a program with various parameters for light for a multispectral light source.
• 7 schematically shows a filter device as part of a microscope assembly according to the invention in a further preferred embodiment in different positions.

In 1 schematically a microscope arrangement according to the invention is shown in a preferred embodiment. The microscope arrangement 300 has an image acquisition system 200 and a fluorescence microscope 100 on which are used together.

The fluorescence microscope 100 exemplifies a microscope body 110 , which in particular further, not in detail here, components or assemblies, such as in particular a tube may have, an imaging optics 120 of the microscope with at least one lens and an adapter 130 on. There is also a sample carrier 140 with a sample to be observed on it 150 shown. The sample 150 is placed under the lens.

The image acquisition system 200 has a multispectral light source 210 For example, in the form of a multi-color LED lamp house, a filter device 220 , for example, with a filter disk, and an image pickup device 230 , for example in the form of a single-chip camera. The filter means 220 also has an example of a drive or a motor 227 on to move the filter device and thus contained therein, individual filters.

By way of example, each of these three components has its own integrated control unit 218 . 228 respectively. 238 provided that can be used to control certain functions, as they will be explained in more detail later. It is understood that only one common control unit can be used.

The image acquisition system 200 is now so in relation to the fluorescence microscope 100 arranged that the multispectral light source 210 the sample 150 can illuminate and that of the sample 150 emitted light along a beam path (or imaging beam path) 160 of the fluorescence microscope from the image capture device 230 can be detected. Here is the filter device 220 arranged such that in each case a separate filter in the beam path 160 can be located, as will be explained in more detail below.

Furthermore, two signals or trigger signals S1 and S2 plotted the triggering of the multispectral light source 210 and the image pickup device 230 , starting from the filter device 220 demonstrate. By suitable triggering or suitable triggering at the right time, a continuous image acquisition series can be created. For further details, reference is made to the following statements.

There is also an interruption device 260 , here in the form of a switch provided to the signal S1 to interrupt. This can be prevented that the light source triggered, ie to illuminate the sample 150 is stimulated. In the case of the triggering cascade shown here, the triggering or starting of the image recording by means of the image recording device is also prevented in this way. The filter device can now continue to be in motion, however, this interruption can be used, for example, the sample carrier 140 and with it the sample 150 to move or the lens 120 switch.

In 2 is schematically an image acquisition system 200 as part of a microscope assembly according to the invention in a preferred embodiment, as also with respect to 1 has already been explained, shown in a different view. In particular, here is part of the filter device 220 a filter disc 221 (or a filter wheel) shown, which in the embodiment shown eight separate filters 222 which is on the edge of the filter disc 221 are arranged. For the sake of clarity, however, only one of these filters 222 provided with a reference numeral.

The filter disc 221 For example, by means of in 1 shown drive 227 moved, in particular rotated. For this purpose, a direction of rotation R of the filter disk 221 , here by way of example in a clockwise direction, indicated. Both the direction of rotation and the rotational speed can be adjusted in particular to a desired value and, in particular, the rotational speed can also be adjusted or adjusted during operation.

At the filters 222 For example, it may be recesses in the filter disk 221 act, in which appropriate filter elements, such as glass, are introduced. It is understood that the filter and the associated recesses or openings not only circular, as shown here by way of example, but also in other forms, such as elliptical or rectangular, may be formed.

The individual filters 222 are different, ie they can have different colors or be transparent to different wavelengths or wavelength ranges of light.

The beam path 160 is shown here by three arrows. From the multispectral light source 210 emitted light first illuminates the sample 150 and from the sample 150 emitted light, in particular fluorescent light, then passes through one of the filters 222 in the image pickup device 230 , in which then a picture of the sample 150 recorded or generated.

As based on the 2 can be seen, anyone can filter 222 optional and separate in the beam path 160 be introduced, by the rotation of the filter disc 221 , As the filter disc 221 can be continuously moved or rotated, a respective filter is always only for a certain period of time in the beam path, before - after a certain period of time in which no light is passed through the filter device - the next filter enters the beam path. In this context, also on the 7 and the related description.

In 3 is now schematically illustrated an image recording system as part of a microscope arrangement according to the invention in a further preferred embodiment. On the filter disc 221 are - compared to 2 - now additional donors 223 attached, of which for clarity, only one is provided with a reference numeral.

Every giver 223 is one of the filters 222 assigned. In this way, a coding is generated with respect to a predetermined position in the beam path. Next to the filter disc 221 is a sensor 225 arranged, which can also be part of the filter device, by means of which it can be detected when one of the donors during rotation of the filter disc 221 this sensor 225 is reached or moved past this. For this purpose, the encoders can be coded. This can be done for example by asymmetric arrangement. In 3 This coding is due to the different orientation of the donors 223 shown showing icons. In this way it is possible to detect when which of the filters 222 will enter the beam path.

From the filter device 220 , for example, using the mentioned control unit 228 , can now, based on the recognition of encoded donors 223 through the sensor 225 a signal S1 generated and sent to the multispectral light source 210 be transmitted. In the signal S1 can be transmitted when which filter will enter the beam path. This can also be the rotational speed of the filter disk 222 be taken into account.

It is also conceivable that the encoders themselves are not encoded, but a coding only in the formation of the signal S1 is generated, for example, based on the order of the encoder 223 in which they are on the sensor 225 be moved past.

The multispectral light source may then be in accordance with the signal S1 Emit light according to each of the filters predetermined parameters. The multispectral light source 210 and thus the illumination of the sample are thus triggered by the filter device in this way. The multispectral light source can in turn - as already mentioned - for example, represent a broadband light source with downstream filters. These filters can then be adjusted accordingly, for example by means of a suitable filter wheel, when triggered.

The multispectral light source 210 in turn, then, for example, using the mentioned control unit 218 , also a signal (eg another trigger signal), here the signal S2 , generate that to the image capture device 230 is transmitted. Here can in the signal S2 when the multispectral light source will emit light, so that the image pickup device can detect light emitted from the sample accordingly, ie the detection of the light emitted by the sample is triggered in this way.

It is conceivable that the signal S1 - if it contains relevant information - from the multispectral light source 210 simply to the image capture device 230 is passed on. Likewise, however, a separate, suitable signal can be generated.

In 4 schematically a filter device is shown as part of a microscope assembly according to the invention in a further preferred embodiment. In particular, here again the filter disc 221 and the sensor 225 shown. The givers 223 are here not symbolically represented in coded form, but with an exemplary placement on the filter disc 221 , The givers 223 can be formed, for example, in the form of magnets, which when passing by the sensor 225 generate an individual signal pattern.

There is also another donor 224 on the filter disc 221 attached, which can serve for synchronization. Exemplary is this additional donor 224 formed in the form of two juxtaposed magnets, a specific signal pattern in the sensor 225 can cause. In the case of a Hall sensor as a sensor 225 For example, a double pulse train is caused. In this way, it is particularly easy, the individual donors 223 due to their order to the individual filters.

For purposes of balancing the filter disc 221 In addition, for example, another donor in the type of donor 224 on the other or opposite side of the filter disc 221 be provided.

Furthermore, with the individual filters 222 now different letters a . b . c . d . e . f . G and H drawn, each with an individual filter 222 assigned. In the diagram at the bottom of the 4 For this purpose, it is shown which encoders in which order (over the time t) at a full rotation of the filter disc, starting at the position shown on the sensor 225 be moved past.

Besides the eight donors 223 that the individual filters a to H (starting here at G ), is also the encoder 224 (shown as a double magnet). That way, the signal can S1 are formed in which is specified when which filter will be introduced into the beam path.

In 5 is now shown schematically a light source as part of a microscope arrangement according to the invention in a further preferred embodiment. This is the multispectral light source 210 the signal S1 fed and it becomes the signal S2 as already stated in relation to 3 was explained in more detail.

Furthermore, now is a PC 240 shown with screen. This is to be explained by way of example, how a program P can be generated, which then in the light source 210 (or the aforementioned control unit) can be deposited.

Such a program P indicates with which parameters light on which filter from the multispectral light source 210 should be emitted when triggered. For each of the filters a to H (corresponding to fewer or more with a different number of filters) can be given by a user in each case a value for various parameters to generate the program P, which then in the multispectral light source 210 or whose control unit can be deposited. If necessary, d .H. when triggered by the filter device, then light is emitted according to the program.

It can also be provided that light is not emitted by the light source for each filter which is next introduced into the beam path. This can also be varied from revolution to rotation of the filter disk. In an extreme case, for example, during the first three turns only for the filter a Light with the predetermined parameters are emitted, for the next three revolutions only for the filter b and so on.

Furthermore, it is possible that during a period of time in which a filter is introduced into the beam path, a plurality of light pulses are emitted, each with different parameters, so as to allow a greater variety of possible image recordings. The desired program can be designed individually according to the wishes or needs of a user or an experiment.

In 6 is now exemplified such a program for a light source with different parameters for each filter shown. For this purpose, a time duration or pulse duration Δt, a wavelength or a wavelength range λ and an intensity I are predeterminable parameters for light to be emitted by the multispectral light source via the individual filters a to H , as already mentioned, applied. For example, the duration or pulse duration and the associated intensity (of a light pulse) can be matched to one another, wherein a shorter pulse duration can be compensated, for example, by a higher intensity.

Each of these parameters can be specified individually for each of the filters and stored as a program in the light source. It is expedient here to match the individual parameters to one another in such a way that the image recordings finally generated are as similar as possible.

In 7 schematically a filter device is shown as part of a microscope assembly according to the invention in a further preferred embodiment in different positions. From left to right, different positions are shown when the filter disc 221 as indicated, moves in a clockwise direction of rotation R.

There is a chip 231 , the part of the image pickup device 230 , in particular in the form of a single-chip camera, can be shown as it passes through one of the filters 222 is visible or how it can detect light that passes through this filter.

At time t1 is the chip 231 for the first time completely inside the opening of the filter, at time t2 it is in the middle of the opening and at time t3 it is for the last time completely in the opening. During the time difference t3-t1, therefore, the chip can be completely illuminated. Thus, the parameters for the light to be emitted by the multispectral light source, in particular the time duration or pulse length, can be aligned to this time difference in order to obtain an optimum quality for the image recording.

If a longer time difference is required for a desired pulse duration, the rotational speed of the filter disk would have to be reduced. However, it is also conceivable to increase the intensity of the corresponding light pulse. The time t1 can, for example, also be used as a parameter for the light to be emitted, both directly and as a reference for a start time of a light pulse.

With the proposed method or the corresponding microscope arrangement, for example, about 900 image spectra (eg Flash4 2048 x 8 pp) with a spectral spread of nine wavelength ranges (ie new filters) can be recorded. at 200 For example, images of 2048 x 1024 pp each and a filter disk with ten different filters can record 20 full spectra per second with a spectral spread of ten wavelength ranges. In this respect, spectrally resolved time-lapse recording (or "timelapse imaging") becomes possible.

In addition, the different, wavelength-dependent quantum yields of a chip used can be compensated by carrying out a test measurement without a sample but with a filter disk. If the intensities of the different wavelengths of the light source are known and the respective filter characteristic is known, the chip can be calibrated with regard to its sensitivity with respect to the wavelengths.

In addition, the intensity of the light source can be adjusted so that weakly light-emitting samples are automatically illuminated more intensely, preferably in such a way that sets a good signal-to-noise ratio on the camera.

Furthermore, it is possible, in particular under favorable experimental conditions, for the intensities to be adjusted automatically so that all channels of the spectral recording require the same exposure time.

The fact that both the sensitivity of the chip and the filter characteristics and also the intensities are known, also a quantitative, spectrally resolved time-lapse recording (or "timelapse imaging") is possible.

Another advantage is that once the microscope arrangement has been properly adjusted, the overall process can be done without external software (e.g., Windows). The triggering cascade of filter device via multi-spectral light source for image acquisition can run directly and can thus achieve a maximum and constant speed.

The experiment, once set, is essentially determined only by the rotational speed of the filter disk. If it rotates more slowly, the recording frequency of the image recording device automatically decreases without the multispectral light source or its parameters changing.

This allows new types of experiments, e.g. an experiment is initially relatively slow (i.e., few spectra per second, but also less sample loading), and then, for example, when a particular event occurs, the rotational speed can be increased to obtain a higher spectral time resolution.

It is also possible that a particular filter position will launch a pattern of programs, not just one but several programs. This presupposes that the corresponding filter is located "over" the chip over a sufficiently long period of time, which means that this chip can also be illuminated accordingly.

By storing a plurality of programs or program sequences in the light source or their control unit, which can be switched over at runtime, it can be achieved that the spectral image recording conditions can be changed over the time of the experiment sequence, such that at different times Experiments various program sequences in the multispectral light source can be activated.

Claims (14)

A method of examining a multispectral sample (150) using a fluorescence microscope (100) with imaging optics (120), wherein the sample to be examined (150) is illuminated by means of a multispectral light source (210) which can be actuated by means of a trigger signal in which an optical path (160) generated by the imaging optics (120) from light emitted by the specimen (150) is detected by means of an image recording device (230), wherein a filter device (220) is arranged between the imaging optics (120) and the image recording device (230). is introduced, which has a plurality of different movable filters (222) which are each assigned to a different wavelength range and in each case optionally between the imaging optics (120) and the image pickup device (230) can be introduced, characterized in that the several different movable filters (222) each one coding with respect to a predetermined position with respect to au f is associated with the beam path (160), and that using the coding by the filter device (20) each upon reaching the predetermined position of the filter (222) generates a trigger signal (S1) and above the multispectral light source (210) is triggered, Then, an intensity (I) of a particular wavelength range (λ) of the multispectral light source (210) is set to a predetermined value, and wherein by means of the image pickup device (230) for the respective filter (222) respectively upon reaching the predetermined position, an image recording started becomes. Method according to Claim 1 wherein the image acquisition is started by the image capture device (230) by a further trigger signal (S2) generated by the multispectral light source (210) after the multispectral light source (210) is triggered, or wherein the image acquisition by the image capture device (21) 230) by the trigger signal (S1) or another trigger signal generated by the filter device (220) is started. Method according to Claim 1 or 2 in which light is emitted by the multispectral light source (210) in response to at least one predetermined parameter (Δt, λ, I, t1). Method according to Claim 3 wherein the at least one parameter is selected from: a predetermined wavelength range (λ), a predetermined intensity (I), a predetermined time duration (Δt) and a predetermined time (t1). Method according to Claim 3 or 4 wherein the at least one predetermined parameter (Δt, λ, I, t1) is individually specified for at least one of the plurality of filters (222). Method according to one of the preceding claims, wherein a filter disk (221) is used as a filter device (220) whose rotational speed is set in particular to a desired value. Method according to one of the preceding claims, wherein a filter device with at least three, preferably at least five, more preferably at least seven, different filters (222) is used as filter device (220). Method according to one of the preceding claims, wherein at least one camera, which is designed in particular as a single-chip camera, is used as image recording device (230). Method according to one of the preceding claims, wherein a triggering of the illumination of the sample (150) by means of the multispectral light source (210) and / or starting the image acquisition by means of the image recording device (230) by means of an interruption means (260) temporarily interrupted if necessary. Method according to one of the preceding claims, wherein the filter device (220) is moved continuously continuously or in sections continuously. Method according to one of the preceding claims, wherein a plurality of LEDs with different wavelengths are used as multispectral light source (210). A control unit (218) for use with a microscope assembly (300) comprising a fluorescence microscope (100) having imaging optics (120) for generating a beam path (160), a multispectral light source (210) operable by a trigger signal, an image pickup device (230) and a filter device (220) having a plurality of different movable filters (222), each associated with a different wavelength range, each selectively interposable between the imaging optic (120) and the image capture device (230), and respectively associated with a predetermined position with respect to the beam path (160), the control unit (218) being adapted to perform a method according to any one of the preceding claims when used with the microscope assembly (300). Microscope arrangement (300) comprising a fluorescence microscope (100) with imaging optics (120) for generating a beam path (160), a multispectral light source (210) which can be actuated by means of a trigger signal, an image acquisition device (230) and a filter device (220) having a plurality of different movable filters (222) each associated with a different wavelength range, each selectively interposable between the imaging optics (120) and the image pickup device (230), and each having a coding with respect to a predetermined position is assigned to the beam path, and a control unit (218) according to Claim 12 wherein the filter device (220) is introduced or can be introduced between imaging optics (120) and image recording device (230). Microscope arrangement (300) according to Claim 13 wherein the filter device (220) is exchangeable.

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