DE102016123258A1 - Luminescence detector arrangement, fluorescence microscope and method for detecting a luminescence signal - Google Patents

Abstract

The invention relates to a luminescence detector arrangement for detecting a luminescence signal having a sensor element (118) with at least one multitap pixel having at least a first memory cell and a second memory cell, wherein the sensor element (118) is adapted to operate the multitap pixel in a first time interval ( 208) by means of the first memory cell and to read in a second time interval (209) different from the first time interval (208) by means of the second memory cell. Furthermore, the luminescence detector arrangement has a light source (104) which is set up to emit a first emission light in at least one first spectral range and independently emit a second emission light in a second spectral range, wherein the first spectral range and the second spectral range are at least partially mutually distinct wherein the light source (104) is synchronized with the sensor element (118) to emit the first emission light in the first time interval (208) and to emit the second emission light in the second time interval (209); and wherein the luminescence detector arrangement is further configured to detect a luminescence signal caused by the first emission light and by the second emission light with the sensor element (118). In a further aspect, the invention relates to a fluorescence microscope and a method for detecting a luminescence signal.

Description

The present invention relates to a luminescence detector assembly, a fluorescence microscope, and a method for detecting a luminescence signal. The invention is particularly in the field of fluorescence microscopy, in particular in the field of wide-field microscopy.

State of the art

In the field of far-field microscopy, in particular in fluorescence microscopy with far field detection, the simultaneous imaging of several luminescent objects, in particular several fluorophores with different spectral properties, is often of central interest in order to investigate structural and functional correlations between different structures. In order to achieve sufficient sensitivity for fluorescence imaging, this type of microscopy often involves monochrome cameras, i. color sensitive cameras, since these are typically available with a higher sensitivity and / or a faster readability than other camera systems, which also offer a color resolution.

However, in order to be able to carry out color-sensitive measurements with monochrome cameras or to be able to carry out measurements in different spectral ranges, conventionally various methods can be used.

For example, for this purpose changing devices for assemblies with excitation spectral filters, a dichroic beam splitter and an emission spectral filter are used, which can be combined in so-called fluorescence filter cubes. These fluorescence filter cubes can then be exchanged for measurements in different spectral ranges, for example for optical excitations and / or detections at different wavelengths, according to the respective requirements. This can have the disadvantage that a replacement of such fluorescence filter cubes can be time-consuming, in particular if the fluorescence filter cubes to be exchanged have a large mass.

Furthermore, conventionally, dichroic multiband beam splitters with matching emission and excitation filter sets have been used. These often have relatively small filters, which can be moved, for example, by means of fast filter wheels, so that a filter change can possibly be carried out faster than when changing from fluorescence filter cubes. However, even such a mechanical change process still takes considerable time. In addition, in both conventional methods, for some measurements, a disadvantage may also result from the fact that the images of different fluorophores are recorded at different times at different spectral excitation regions and / or luminescence regions and thus a comparability of the measurement data from different spectral regions can be reduced. Especially with fast processes this can complicate the interpretation of spatial correlations of the examined objects.

Also, the use of multiband filter sets and color cameras is known. However, often the separation of the individual color channels can be severely compromised.

Furthermore, the use of Bayer masks for color sensors can reduce the sensitivity due to their filter effect, since the intensity values of the signal of individual pixels are determined in part mathematically from the neighboring pixels, which can often complicate a quantitative, scientific evaluation of the detector signals

Also multi-camera methods can be used. However, these are often cost-intensive and expensive, since several cameras must be purchased and the different cameras also synchronized and their images must be overlapped, which can often be associated with tolerance-related different lateral layers and geometric distortions with a great deal of effort.

Furthermore, camera splitters are known in which a microscopic field of view is spectrally divided and imaged on different areas of the same sensor. Such systems are often non-telecentric and also suffer from overlap problems, such as the multiple camera methods mentioned above.

In addition, the use of cameras or area sensors with multiple pixel taps is known, ie cameras with so-called multitap or two-tap pixels, such camera systems being used, for example, as FLIM cameras (pco.flim, PCO AG, MEMFLIM, TELEDYNE DALSA PROFESSIONAL IMAGING) or time-of-flight cameras (epc660, ESPROS PHOTONICS CORPORATION). In this case, an electrical charge resulting from the detection process in a detector pixel is collected in two or more memory cells, the integration time per memory cell being in a fixed phase range of a high-frequency modulation frequency (FIG. Pixel switching frequency). For example, such cameras are mostly used for range measurements and measurements by fluorescence lifetime microscopy.

It is therefore the object of the invention to provide a luminescence detector arrangement, a fluorescence microscope and a method for detecting a luminescence signal, which enable a simplified detection of luminescence signals at different excitation wavelengths at short time intervals.

Disclosure of the invention

According to the invention, a luminescence detector arrangement, a fluorescence microscope and a method for detecting a luminescence signal with the features of the independent patent claims are proposed. Advantageous embodiments are the subject of the respective subclaims and the following description.

In a first aspect, the invention relates to a luminescence detector arrangement for detecting a luminescence signal, which has a sensor element with at least one multitap pixel having at least a first memory cell and a second memory cell, wherein the sensor element is adapted to the multitap pixel in a first time interval by means of read the first memory cell and read in a second time interval different from the first time interval by means of the second memory cell. Furthermore, the luminescence detector arrangement has a light source which is set up to emit a first emission light in at least one first spectral range and independently emit a second emission light in a second spectral range, wherein the first spectral range and the second spectral range are at least partially different from one another ( Overlaps not excluded). In this case, the light source is synchronized with the sensor element in order to emit the first emission light in the first time interval and to emit the second emission light in the second time interval. The luminescence detector arrangement is also configured to detect a luminescence signal caused by the first emission light and by the second emission light with the sensor element.

In a further aspect, the invention relates to a fluorescence microscope with a luminescence detector arrangement according to the invention, wherein the fluorescence microscope is adapted to at least partially optically excite an object to be examined by means of the light source and at least partially detect a fluorescence signal generated by the optically excited object to be examined as a luminescence signal.

In a further aspect, the invention relates to a method for detecting a luminescence signal with the steps of a) providing a sensor element having at least one multitap pixel with at least one first memory cell and a second memory cell; b) providing a light source; c) emitting a first emission light in at least a first spectral range by means of the light source in a first time interval and reading out the at least one multitap pixel by means of the first memory cell in the first time interval; and d) emitting a second emission light in at least a second spectral range by means of the light source in a second time interval and reading out the at least one multitap pixel by means of the second memory cell in the second time interval. In this case, the first time interval and the second time interval are different from one another and the first spectral range and the second spectral range are at least partially different from one another.

An emission light is preferably in particular excitation light for excitation of an object to be examined or of fluorophores in the object to be examined and / or illumination light for illuminating the object to be examined. The first Sepktralbereich and the second spectral range are at least partially different from each other, i. the first and the second Sepktralbereich do not overlap each other completely, with a partial spectral overlap is possible but not mandatory.

A luminescence signal can, for example, have a fluorescence signal and / or a phosphorescence signal which occurs, for example, by an optical excitation of a fluorescent and / or phosphorescent object to be examined, or is emitted by the object.

In this case, multitap pixels can be pixels which are connected to a plurality of memory cells in order to be read out at different times from in each case one of the several memory cells. This can serve, for example, to accelerate the read-out process of the multitap pixel compared to the read-out process of conventional pixels with only one memory cell. For example, a multi-tap pixel may be in the form of a two-tap pixel having two memory cells. However, other embodiments of multitap pixels may also have any other number greater than 1 of memory cells, for example 3, 4, 8 or 16 memory cells.

As a light source is to be understood a source of electromagnetic radiation. Accordingly, it should be understood as light or emission light electromagnetic radiation. This may, but not necessarily, in the visible optical spectral range, but may for example include ultraviolet and / or infrared and / or X-rays. A spectral range is understood to be a wavelength range or frequency range of the frequencies of the electromagnetic radiation, which in the case of monochromatic light may be individual wavelengths or frequencies or, in the case of more or less broadband radiation, a continuous wavelength range or frequency range which extends the spectrum of electromagnetic radiation.

The invention offers the advantage that for the detection of the luminescence not necessarily spectral filters, such as excitation spectral filters for selecting the excitation wavelength (s) and / or emission spectral filters for selecting the wavelength (s) to be detected must be used. As a result, in particular, a time-consuming change of spectral filters can be dispensed with, as a result of which the measurement duration can be shortened and, in particular, several measurements in different spectral ranges which follow one another at very short time intervals can take place. For example, this can make it possible to carry out detections of luminescence in different spectral ranges within a few microseconds or milliseconds. In particular, the invention makes it possible to emit only emission light of the desired spectral range at certain time intervals and to detect the luminescence produced thereby and to emit emission light of another desired spectral range at other specific time intervals and to detect the luminescence produced thereby. In other words, the emission of certain emission radiation and the detection by means of the sensor element can be synchronized such that a preferably complete assignability of the detected signal to a specific emission radiation or excitation radiation is possible, although the sensor element is optionally color insensitive, in particular has no color filter matrix and also a spectral filtering of the excitation light and / or the luminescence radiation does not take place and in particular no excitation spectral filters or emission spectral filters are used.

This offers the advantage that a luminescence detector arrangement can be provided which can be operated without a cost-intensive spectral filter and which can be operated in particular without mechanically changing spectral filters and / or without manipulating filter wheels. Furthermore, the invention offers the advantage that no color-sensitive cameras must be used, but a use of a monochrome sensor element is sufficient. This makes it possible to use particularly sensitive and / or particularly quickly readable sensor elements.

Preferably, during a measurement, the first time interval and the second time interval each pass at least twice. That During a measurement, at least twice the first emission light and the second emission light are emitted and the at least one multitap pixel is read out at least twice by means of the first and the second memory cell. On the other hand, the measurement is preferably carried out over a measurement period or a measurement period which is such that a lifetime or decay time of the object to be measured luminescence, which depends, for example, on a decay of the fluorescence of the fluorophore in the object to be examined , the detection of the luminescence or the measurement signal is not substantially affected. For example, it is possible to select a measurement duration which is longer, in particular significantly longer, than an average decay duration of the luminescence of the object to be detected. In the case of fluorescence processes, the radiating life on the time scale of a few nanoseconds, so that a measurement period in the microsecond range is unaffected by the radiating life of the fluorophore, on the time scale of cellular processes in the millisecond range, however, still allows simultaneous detection of multiple fluorophores.

Preferably, the sensor element has a plurality of multitap pixels, which are preferably arranged in a matrix. This offers the advantage that a spatial resolution of the detected luminescence signal can be achieved. In this case, for example, the provision of optical elements may be advantageous in order, for example, to image the luminescence signal onto the sensor element. By way of example, the sensor element may have a CMOS matrix in which a plurality of multitap pixels is arranged, for example, in a rectangular matrix. For example, the sensor element may comprise a matrix comprising 1008 × 1008 multitap pixels, wherein other matrix sizes and / or sensor element sizes are also possible.

The luminescence detector arrangement is preferably set up to synchronize the sensor element and the light source by means of an externally provided synchronization signal. The synchronization signal can be provided, for example, by an electrical signal transmitter and / or an electric pulse generator. This has the advantage that the readout of the memory cells and the emission of the emission light by the light source can be synchronized in a particularly simple manner.

Alternatively or additionally, in accordance with a preferred embodiment, the luminescence detector arrangement can have a synchronization signal generator, which is preferably set up to provide a synchronization signal. The synchronization signal can in particular be set up to synchronize the sensor element and the light source by means of the synchronization signal. This has the advantage that it may be possible to dispense with the provision of a synchronization signal by an external synchronization signal source or an external synchronization signal generator. For example, the sensor element may comprise the synchronization signal generator. In other words, the synchronization signal generator is optionally integrated in the sensor element. This has the advantage that, for example, the sensor element provides an internal synchronization signal to control the readout of the memory cells, which can also be used externally for the synchronization of other components, such as the light source.

Preferably, the light source is modulated. Furthermore, the light source is preferably configured to emit the first emission light and / or the second emission light in response to the synchronization signal. In this case, the light source can have one or more emission elements. In particular, the light source can be designed such that it has a single emission element for the first and the second emission light, or that it has a separate emission element for each emission light. This offers the advantage that with a single light source, the first emission light and the second emission light can be provided or emitted and not necessarily multiple light sources must be provided. As a result, on the one hand a compact construction of the luminescence detector arrangement can be achieved. On the other hand, this can be advantageous with regard to the synchronization of the light source with the sensor element, since in this way only a single light source has to be synchronized with the sensor element.

Preferably, the synchronization signal comprises a periodic signal, which preferably has a repetition frequency which is in a range of 100 Hz to 100 MHz, preferably in a range of 1 kHz to 10 MHz, most preferably in a range of 10 kHz to 1 MHz , For example, the synchronization signal may be configured as an electrical voltage signal or comprise such. In particular, the synchronization signal may have a sinusoidal and / or rectangular and / or sawtooth-shaped voltage signal. The amplitudes of the voltage signal can be between 0.1 V and 20 V, for example. For example, the sensor element and / or the light source can be set up such that they are triggered by the synchronization signal at times when the synchronization signal has a rising edge and / or a falling edge. This has the advantage that the synchronization signal can be provided in a simple and reliable manner, without requiring high demands on the hardware of a synchronization signal generator.

The light source preferably has at least one semiconductor light source. In particular, at least one emission element of the light source can have a semiconductor light source. For example, the semiconductor light source may have at least one light emitting diode (LED) or light emitting diode and / or a laser diode and / or a diode laser. For example, a plurality of semiconductor light sources can be provided, which emit at mutually different colors or emission spectra. Particularly preferably, a plurality of different types of semiconductor light sources are integrated in the light source. The use of semiconductor light sources offers the advantage that they can be modulated very quickly compared to other light sources, i. can be switched on and off particularly quickly. This can be advantageous, in particular, in the case of particularly short and / or rapidly successive measurement intervals in order to achieve a rapid transition from emission light of the first spectral range to emission light of the second spectral range. The individual semiconductor light sources may further include bandpass filters for adjusting the emitted spectral range.

Preferably, a fluorescence microscope according to the invention is set up such that the optical excitation of the object to be examined takes place without the use of an excitation spectral filter. More preferably, the fluorescence microscope is set up such that the fluorescence signal generated by the optically excited object to be examined is carried out without the use of a luminescence spectral filter. Although the use of such spectral filters is not excluded, it is still advantageous that such filters can be dispensed with. This offers the advantage that it is possible to dispense with cost-intensive acquisition of such spectral filters. Also eliminates the need to perform time-consuming filter changes between measurements at different spectral ranges. Moreover, this offers the advantage that between measurements at different spectral ranges vibrations and / or mechanical shocks, which go hand in hand with a mechanical change of the spectral filters and can have negative effects on the microscopic measurement, can be avoided. But it is also possible To use luminescence spectral filters which block the overlap region of the luminescence spectra of the individual fluorophores, and thus improve the assignability of the individual Lummzenzzenzsignale to the individual fluorophores waiving the signal from the spectral overlap region. Such luminescence spectral filters need not be replaced during a measurement, so that no disadvantages caused thereby arise.

The readout of the at least one multitap pixel by means of the first memory cell is preferably synchronized with the emission of the first emission light and the reading of the at least one multitap pixel by means of the second memory cell is synchronized with the emission of the second emission light. According to further preferred embodiments, the at least one multitap pixel may also have more than two memory cells and the light source may have more than two different types of emission light, i. Emit emission light in more than two different spectral ranges. The readout of the multitap pixel by a specific memory cell particularly preferably takes place synchronously with the emission of determined emission light by the light source. This has the advantage that measurements can be carried out at different excitation spectral ranges in a very short time interval and a selective detection of the resulting luminescence is made possible.

Preferably, steps c) and d) are repeated alternately, preferably at a repetition frequency ranging from 100 Hz to 100 MHz, preferably in a range from 1 kHz to 10 MHz, most preferably in a range from 10 kHz to 1 MHz is.

For complete detection, the steps c) and d) are particularly preferably run through at least twice. This has the advantage that it can be measured over a longer period, i. that optionally light-faint luminescences can be detected and, in particular, short-lived luminescences can be detected in a time-dependent manner. Furthermore, an alternating or alternating excitation and detection at different spectral ranges a quasi simultaneous but separate measurements can be achieved, since the time differences between the measurements in the different emission lights in a very short time interval. This offers the advantage that the influence of possibly occurring temporal changes of the object to be examined on the measurement can be kept as low as possible

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 drawings and will be described below with reference to the drawing.

list of figures

• 1 shows a schematic representation of a fluorescence microscope according to a preferred embodiment.
• 2 shows an exemplary time sequence of operations of a method according to the invention according to a preferred embodiment.

1 shows a schematic representation of a fluorescence microscope 100 according to a preferred embodiment. The fluorescence microscope 100 is adapted to detect the luminescence or fluorescence of an object to be examined 102, in particular time-dependent or time-resolved. This is indicated by the fluorescence microscope 100 a light source 104 on, by means of which emission light, which can serve in particular as excitation light and / or as illumination light for optically exciting or illuminating the object to be examined, can be emitted in at least a first and a second spectral range. In particular, the emission light is emitted in such a way that the emission light is at least partially along an illumination beam path 106 spreads. Optionally, an excitation spectral filter can be used 108 in the illumination beam path 106 be arranged to optionally perform a spectral filtering of the emission light in the first and / or the second spectral range. The illumination beam path 106 Subsequently, the emission light at a beam splitter 110 at least partially in the direction of an objective 112 reflected. Preferably, the beam splitter 110 is designed as a multiband beam splitter, so that preferably the emission light is reflected both in the first spectral range and in the second spectral range with the highest possible reflectivity rate. If the emission light is emitted in more than two different spectral ranges, the beam splitter 110 be adapted to reflect the other spectral regions with the highest possible reflectivity. In particular, the beam splitter 110 to be configured not to reflect light in spectral ranges other than the emission light, but to transmit with the greatest possible transmissivity. For example, the beam splitter 110 be designed as a dichroic multiband beam splitter.

That of the beam splitter 110 reflected emission light is transmitted through the lens 112 on an area of the object to be examined 102 at least partially focused, so that in the area to be examined optionally present fluorescence and / or phosphorescence can be excited. The objective 112 is preferably configured as a microscope objective and is further for the collection of light or luminescence of the object 102 used for microscopic evaluation. That of the lens 112 collected luminescent light or fluorescent light, which optionally after excitation of the object 102 with the emission light from the object 102 is emitted through the lens 110 collected and spreads in the direction of the beam splitter 110 out, with the beam splitter 110 is adapted to transmit the light collected by the lens luminescence or fluorescent light. After the beam splitter 110 Optionally an emission filter 114 be arranged, if necessary, through the lens 112 to spectrally filter accumulated luminescent light. For example, the emission spectral filter 114 be designed as a multi-band spectral filter. Further, according to the illustrated embodiment, the luminescent light is detected by means of an optical element 116 on a sensor element 118 focused or along the beam path 116a imaged optically. The optical element 116 For example, it can be designed as a convex optical lens or a condenser lens, in particular as a tube lens or, more generally, a tube optic.

The sensor element 118 For example, it may be formed as a camera, which preferably has a plurality of multitap pixels arranged in a matrix. According to the embodiment shown, the sensor element 118 and the light source 104 by means of a control unit 120 controlled. In this case, the control unit in particular that of the sensor element 118 receive signals received or detected or read from the memory cells included therefrom and optionally further processed and / or store. In particular, by the control unit 120 a synchronization signal may be provided to synchronize the light source with the sensor element in time. Alternatively, the control unit 120 one from the sensor element 118 provided synchronization signal and directly or after an optional processing to the light source 104 hand off. Optionally, the sensor element 118 for providing the synchronization signal, a synchronization signal generator 121 exhibit.

2 shows an exemplary, schematic time sequence of operations of a method according to the invention according to a preferred embodiment, which are plotted against the time t. The uppermost graph shows a progression of the pixel switching frequency 201 with a period length 202 , an emission of the first emission light in the first spectral range 203 , an emission of the second emission light in the second spectral range 204 , a readout of the first memory cell 205, a readout of the second memory cell 206 , as well as a period of image acquisition 207 , The pixel switching frequency in frequency and phase preferably corresponds to the synchronization signal. The applied amplitudes claim no quantitative comparability, but the graphs are intended to illustrate only a relative, temporal sequence. For example, means in the graph 203 . 204 . 205 and 206 a large amplitude value that the corresponding emission light is emitted or the corresponding memory cell is read, while the small amplitude value means that the corresponding emission light is not emitted or the corresponding memory cell is not read out. Analog applies to the graph 207 in that, with a large amplitude value, image acquisition occurs and no image acquisition takes place with a small amplitude value. The dashed lines indicate the first time interval 208 and the second time interval 209 in one period 202 ,

As with conventional fluorescence lifetime microscopy techniques, in accordance with the illustrated embodiment of the invention, the pixel switching frequency 201 set such that a complete exposure and detection of the overall image, ie the entire period of image acquisition 207 or the recording of an image, a plurality of complete cycles or periods 202 the pixel switching frequency 201 includes or persists, preferably more than two cycles. However, preferably, the pixel switching frequency becomes 201 so low or the period 202 chosen so large that an influence of changes due to a decay of the fluorescence lifetime of fluorophores in the examined object has no significant influence on the imaging properties or the measurement and thus can be neglected. In other words, the pixel switching frequency 201 is chosen such that a suitable compromise results from a total measurement duration which is long enough to neglect temporal changes in the fluorescence or luminescence, but nevertheless to perform a plurality of measurement cycles in order to obtain a sufficient time To ensure integration to obtain signal intensity with quasi-simultaneity ("real time"). For example, the pixel switching frequency may be in the range between 10 kHz and 1 MHz. Furthermore, according to the embodiment shown, the emission of the first and second emission light 203 . 204 the modulatable light source 104 phase synchronous to the detection of the light through the different memory cells of the multi-tap pixels, or for reading the first memory cell 205 or for reading the second memory cell 206 set.

As accordingly quickly modulated light sources 104 For example, semiconductor light sources, such as diode lasers or light emitting diodes (LEDs), are provided so that the method may provide different geometric flux for, preferably, wide field fluorescence microscopy methods with coherent and / or incoherent illumination. This also offers the possibility of using the invention optionally in epifluorescence microscopy, in TIRF microscopy, in structured illumination microscopy, in light-sheet microscopy, in multifocal confocal microscopy (eg by spinning disk) and in multifocal multiphoton microscopy ,

The modulation of the light source 203 . 204 For example, it can also be separated from the pixel switching frequency 201 respectively. For example, a modulation of a light source which emits light continuously over time can be effected by electro-optical and / or acousto-optical and / or electromechanical (eg chopper) or other methods. The reading out of the memory cells 205 . 206 is preferably such that the signal over more than two complete cycles or periods 202 the pixel switching frequency 201 is integrated, so that there is a shortest possible time interval between the measurements at different optical excitations and thereby a possible simultaneous measurement at different emission light types or excitation wavelengths can be done. This can be done by means of a conventional, simple mutual synchronization of the illumination to the exposure time of the camera, ie with a complete image acquisition 207 with the first emission light and only then a complete image acquisition 207 with the second emission light, can not be achieved. The higher the number of cycles of pixel switching frequency 201 within a total exposure time of image acquisition 207 is chosen, the lower the influences of the time-dependent changes of the object to be examined and the better the comparability of the measurements with emission light or excitation light in different spectral ranges.

LIST OF REFERENCE NUMBERS

100

fluorescence microscope

102

object

104

light source

106

Illumination beam path

108

Anregungsspektralfilter

110

beamsplitter

112

lens

114

Emissionsspektralfilter

116

optical element, tube optics

116a

beam path

118

sensor element

120

control unit

121

Synchronization signal generator

201

Pixel switching frequency

202

Period of pixel switching frequency

203

Emission of the first emission light

204

Emission of the second emission light

205

Reading the first memory cell

206

Readout of the second memory cell

207

image capture

Claims (15)

A luminescence detector arrangement for detecting a luminescence signal, comprising: a sensor element (118) with at least one multitap pixel having at least a first memory cell and a second memory cell, wherein the sensor element (118) is adapted to operate the multitap pixel in a first time interval (208 ) by means of the first memory cell and to read in a second time interval (209) different from the first time interval (208) by means of the second memory cell; - A light source (104) which is adapted to emit a first emission light in at least a first spectral range and independently emit a second emission light in a second spectral range, wherein the first spectral range and the second spectral range are at least partially different from each other; wherein the light source (104) is synchronized with the sensor element (118) to emit the first emission light in the first time interval (208) and to emit the second emission light in the second time interval (209); and wherein the luminescence detector assembly is further configured to cause a light caused by the first emission light and the second emission light Detecting the luminescence signal with the sensor element (118). Luminescence detector arrangement according to Claim 1 in which the luminescence detector arrangement is set up to pass through the first time interval and the second time interval in each case at least twice, in particular alternately. Luminescence detector arrangement according to Claim 1 or 2 wherein the sensor element (118) comprises a plurality of multi-tap pixels, which are preferably arranged in a matrix. Luminescence detector arrangement according to one of the preceding claims, wherein the luminescence detector arrangement is adapted to synchronize the sensor element (118) and the light source (104) by means of an externally provided synchronization signal. Luminescence detector arrangement according to one of Claims 1 to 3 , further comprising a synchronization signal generator (121) which is adapted to provide a synchronization signal, wherein the synchronization signal is adapted to synchronize the sensor element (118) and the light source (104) by means of the synchronization signal. Luminescence detector arrangement according to Claim 5 wherein the sensor element (118) comprises the synchronization signal generator (121). Luminescence detector arrangement according to one of Claims 4 to 6 wherein the light source (104) is modulatable and configured to emit the first emission light and / or the second emission light in response to the synchronization signal. Luminescence detector arrangement according to one of Claims 4 to 7 wherein the synchronization signal comprises a periodic signal which preferably has a repetition frequency which is in a range of 100 Hz to 100 MHz, preferably in a range of 1 kHz to 10 MHz, most preferably in a range of 10 kHz to 1 MHz , Luminescence detector arrangement according to one of the preceding claims, wherein the light source (104) comprises at least one semiconductor light source. Fluorescence microscope (100) with a luminescence detector arrangement according to one of the preceding claims, wherein the fluorescence microscope (100) is adapted to at least partially optically excite an object (102) to be examined by means of the light source (104) and to produce an object excited by the optically excited object to be examined Fluorescence signal to detect at least partially as a luminescence signal. Fluorescence microscope (100) according to Claim 10 wherein the fluorescence microscope (100) is arranged such that the optical excitation of the object to be examined (102) without using an excitation spectral filter (108) takes place and / or wherein the fluorescence microscope (100) is arranged such that from the optically excited to fluorescence signal detected without the use of an emission spectral filter (114). A method of detecting a luminescent signal comprising the steps of: a) providing a sensor element (118) with at least one multitap pixel having at least a first memory cell and a second memory cell; b) providing a light source (104); c) emitting a first emission light in at least a first spectral range by means of the light source in a first time interval (208) and reading the at least one multitap pixel by means of the first memory cell in the first time interval (208); d) emitting a second emission light in at least a second spectral range by means of the light source in a second time interval (209) and reading the at least one multitap pixel by means of the second memory cell in the second time interval (209); wherein the first time interval (208) and the second time interval (209) are different from each other and wherein the first spectral range and the second spectral range are at least partially different from each other. Method according to Claim 12 wherein the reading of the at least one multitap pixel is synchronized by means of the first memory cell with emitting the first emission light and wherein the reading of the at least one multitap pixel is synchronized by means of the second memory cell with the emitting of the second emission light. Method according to Claim 12 or 13 wherein steps c) and d) are repeated alternately and preferably at a repetition frequency in a range of 100 Hz to 100 MHz, preferably in a range of 1 kHz to 10 MHz, most preferably in a range of 10 kHz to 1 MHz repeated. Method according to Claim 14 , wherein for a complete detection, the steps c) and d) are each passed at least twice.

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