DE102012020241A1 - Image pickup device and method for taking a picture sequence - Google Patents

Abstract

The invention relates to image recording devices such as microscopes with control unit, modulatable light source and optoelectronic transducer with global electronic shutter ("global shutter" or "frame shutter"). These are limiting because of their low readout speed for the recording of pairs of images with a short time interval. The new image pickup device is to be able to record immediately successive images with a shorter time interval between the images. Also, the sample load should be reduced. For this purpose, a transducer is used with an input for a signal (17) for triggering a simultaneous electrical integration in all detection elements and the control unit is configured to initiate and terminate in the transducer a first integration and thereafter, before entering the transducer Reading out a content integrated in all of the detection elements during the first integration, triggering a second integration and ending it only after the reading of the content integrated during the first integration from all the detection elements has ended in the converter, during each integration the same exposure time controls the light source for pulsed or continuous emission and intercepts the emission between the integrations.

Description

The invention relates to an image recording apparatus, in particular a microscope, having at least one illumination beam path (for illuminating a sample) comprising a modulatable light source, a detection beam path (for recording an image of the sample) comprising an optoelectronic transducer having at least a two-dimensional matrix of detection elements ( "Pixel"), and a control unit connected to the light source and the transducer, and a method for capturing an image sequence from a sample by means of at least one illumination beam path (for illuminating the sample) comprising a modulatable light source and a detection beam path (for capturing an image of the sample) comprising an opto-electronic transducer having at least a two-dimensional matrix of detection elements.

For the purposes of the invention, the term light comprises any electromagnetic radiation which can be manipulated by optical means, that is to say in particular ultraviolet, visible and infrared radiation. For the purposes of the invention, sample light is light which is emitted by fluorescence from the sample, excited by the illuminating light pulses. It may also contain components that result from reflection and / or scattering of illumination light at the sample. A modulatable light source may be directly modulatable - such as a light emitting diode (LED) or a laser diode - or it may be indirectly modulatable by including an additional modulator, such as an adjustable acousto-optic filter (AOTF). , An optoelectronic transducer integrated after triggering an image recording in each of its detection elements an electrical variable such as charge or voltage.

In the state of the art, for example, a light-disk microscope for single-plane illumination (SPI) is provided as an image recording device US 2006/033987 A1 known. Therein, a two-dimensionally spatially resolving camera with CCD sensor (CCD for "charge-coupled device") is used as a converter. The illumination with a lens (also referred to as a light sheet) has the advantage that the sample is illuminated only to a small extent and thus only slightly burdened. SPI microscopy (SPIM) is therefore preferably used for living samples.

Two-dimensional optoelectronic transducers are known in the art, for example with an electronic global shutter ("global shutter" or "frame shutter") US 2009/095986 A1 , again in the form of a CCD sensor, or off US 2001/035542 A1 in the form of a CMOS sensor (CMOS for complementary metal-oxide semiconductor). In addition, converters with a so-called electronic rolling shutter (English "rolling shutter") are known, for example US 2006/157758 A1 , Global-shutter CCD sensors are problematic for capturing short-spaced pairs of images because they are slow to read the electrical charges built into photoabsorption. Even with CMOS sensors with global shutter, reading out all the detection elements ("pixels") takes a relatively long time, depending on their number ("resolution").

The invention has for its object to improve an image pickup device of the type mentioned and a method for recording a sequence of images, so that immediately consecutive images can be recorded with a shorter time interval between the images. In particular, the sample load should be reduced compared to a continuous illumination.

The object is achieved by an image recording apparatus having the features specified in claim 1, and by a method having the features specified in claim 14.

Advantageous embodiments of the invention are specified in the subclaims.

• – Zurücksetzen der Detektionselemente vor Beginn der Integration,
• – Aufrechterhaltung der Integration über eine Dauer des Auslösesignals,
• – Verschieben des integrierten Inhalts der der betreffenden Detektionselemente in einen jeweiligen (lichtgeschützten) Zwischenspeicher der betreffenden Detektionselemente,
• – Auslesen eines jeweiligen elektrischen Signals aus den Zwischenspeichern der betreffenden Detektionselemente,
• – Digitalisieren der ausgelesenen elektrischen Signale und
• – Bereitstellen der digitalen Bilddaten an einer Schnittstelle.

According to the invention, it is provided on the one hand that a known (time window based) converter, such as in US 2009/095986 A1 or US 2001/035542 A1 is described, which includes (for providing a global electronic shutter) an input for a signal for triggering (and a signal to terminate) a simultaneous electrical integration in all detection elements and the triggering of a second integration after the end of a first integration allowed during readout. Such converters are designed in a known manner to carry out the following steps autonomously and independently (in particular from a converter-external clock) in response to the triggering signal, for example in the form of a rising edge of a TTL pulse:

• - reset the detection elements before starting the integration,
• Maintaining integration over a duration of the triggering signal,
• Shifting the integrated content of the respective detection elements into a respective (light-protected) buffer of the respective detection elements,
• Reading out a respective electrical signal from the latches of the respective detection elements,
• - Digitizing the read electrical signals and
• - Providing the digital image data at an interface.

The triggering of the second integration still during readout is referred to as overlapping mode ("overlapping mode"), since the read-out operation in the converter and the second integration overlap in time (in particular completely). If the second integration is completed before the end of the read-out, the contents integrated into the detection elements by photoabsorption until then are discarded, so that only the image data from the first integration is provided. To avoid this, the described converters output the status of the read-out process (lasts / ends) via an interface.

On the other hand, it is provided that the control unit is connected to the (integrating triggering) input of the converter and is adapted to trigger and terminate in the converter a first integration and thereafter, before reading out in the converter a content integrated during the first integration is terminated from all the detection elements to trigger a second integration and only to terminate after in the converter, the readout of the integrated content during the first integration of all detection elements is completed, during each of the integrations for the same (total or effective) exposure time Controlling light source for (periodically) pulsed or continuous emission of at least one wavelength and between the integrations (ie after the termination of the first and before the triggering of the second) the emission is interrupted (in particular for the entire duration between the completion of the first integration and the off solve the second integration).

The basic idea of the invention is to synchronize the overlapping mode with successive, similar illumination pulses in order to allow a shorter time interval between a pair of immediately successively recorded images and thereby to minimize the burden on the sample.

The emission during the exposure time can be pulsed or continuous. Accordingly, the term of the exposure duration in the sense of the invention encompasses various special meanings: The total exposure duration is the duration of that period in which the envelope of the light intensity resulting from the modulation is different from zero. The effective exposure time is the summed duration of those periods within the total exposure time in which the light intensity resulting from the modulation is different from zero. In the case of continuous emission over the entire exposure time, the effective exposure time is equal to the total exposure time.

Particularly preferred embodiments of the invention in which the second integration period is longer than the first integration period, in particular with formation of the control unit for triggering the second integration such that a time-overlapping with the reading portion of the second integration is longer than the first integration, wherein preferably, the second integration is completely overlapped with the read-out (in other words, terminated with the end or even due to the end of the read-out operation of the converter). This represents an advantageous extension of the overlapping mode. The extension of the second integration beyond the exposure time by a corresponding earlier start while the previous integration results are being read out by the converter makes it possible to determine the time interval between the image recorded in the first integration and the one in FIG further reduce the picture taken during the second integration. Due to the identical exposure during both integrations, the same amount of light is achieved in both recorded images despite different integration periods.

It can be advantageous if the image recording device provides an operating element for setting the second integration duration, a quotient between the second and the first integration duration or a difference between the first and the second integration duration. In particular, in embodiments in which the second integration period completely overlaps with the read-out (ending with or by the end of the read-out operation of the converter), the variable adjustment of a time interval between two successive images becomes smaller than the readout duration of the converter allows all detection element. The permissible range is limited by the user-specified exposure time and by the readout duration of the transducer for all detection elements.

In a specific embodiment, the first integration duration and the second integration duration are the same length. Here, the image acquisition corresponds to the simple overlap mode.

Preferably, the first integration is the same length as the (total) exposure time. By avoiding a deviation, a minimum sample load is achieved.

Embodiments in which the image acquisition device provides an operating element for selecting one of a plurality of operating modes provided by the control unit are advantageous, wherein in the first operating mode the second integration duration is longer than the first integration duration (and preferably completely overlaps with the readout), in particular at a constant first and constant second integration duration, and in the second mode of operation, the first integration duration and the second integration duration are the same length. This allows the user to easily switch between simple and extended overlap mode.

Particularly preferred are embodiments in which the control unit is adapted to terminate the second integration (exactly) at the end or due to the end of the readout of the converter. In this way, it can be ensured that the second integration overlaps in time completely with the read-out operation of the signals of the first integration from the buffer, so that a minimum time interval of the images is made possible. In addition, discarding the data of the second integration by the converter due to a premature termination of the second integration (namely, before the end of the read-out operation) can be avoided. For example, the completion of the second integration due to the end of the read operation is accomplished by the transducer outputting a signal indicating the status (in particular the end) of the read to the previous (first) integration and the controller terminating the second integration if and when she identifies the final signal. For example, it terminates the second integration only when and as soon as it identifies the end signal.

The coupling of the termination of the second integration with the end of the read-out process succeeds, for example, by a time measurement and the comparison with the (predetermined) readout duration of the transducer for all detection elements. For this purpose, the control unit can expediently be set up to initiate the second integration for a pause duration after the termination of the first integration. The pause duration defines the time interval between the picture taken in the first integration and the picture taken in the second integration. The control unit may preferably determine the pause duration on the basis of a period duration of a periodic process (specified by the user or a higher-level control unit) such that the pause duration plus the first integration duration corresponds to an integral multiple of the period duration. If the periodic process corresponds to the modulation of the light source and / or a positioning of the light focus in the sample, then a same brightness can be achieved in both images. In addition to the period of an external process, the control unit will usually determine the pause duration based on the user defined exposure time.

Preferably, the control unit is configured to determine the pause duration such that it is longer than or equal to the readout duration minus the second integration duration or the second integration duration such that it is longer than or equal to the readout duration minus the pause duration , This avoids that the second integration ends before the read operation of the converter and the associated image data are discarded.

• – Ermitteln einer (vorgegebenen, insbesondere vom Benutzer) Belichtungsdauer, einer Auslesedauer für alle Detektionselemente, einer ersten Integrationsdauer, einer Pausendauer (zwischen Ende der ersten Integration und Auslösen der zweiten), wobei die Pausendauer kleiner ist als die Auslesedauer, und einer zweiten Integrationsdauer (wobei die Pausendauer in Abhängigkeit der zweiten Integrationsdauer ermittelt wird oder umgekehrt)
• – Steuern der Lichtquelle zur (periodisch) gepulsten oder kontinuierlichen ersten Emission von Licht mindestens einer vorgegebenen Wellenlänge für mindestens die Belichtungsdauer,
• – Setzen des (integrationsauslösenden) Eingangs des Wandlers synchron zu einem Beginn der ersten Emission (insbesondere durch konstanten Zeitversatz zum Steuern der Lichtquelle),
• – nach Ablauf (insbesondere am Ende) der ersten Integrationsdauer: Beenden der Emission und Zurücksetzen des Eingangs,
• – Abwarten der Pausendauer,
• – am Ende der Pause: Steuern der Lichtquelle zu einer mit der ersten Emission übereinstimmenden zweiten Emission für dieselbe Belichtungsdauer und
• – Setzen des (integrationsauslösenden) Eingangs des Wandlers synchron zu einem Beginn der zweiten Emission (insbesondere durch konstanten Zeitversatz zum Steuern der Lichtquelle)
• – nach Ablauf (insbesondere am Ende) der zweiten Integrationsdauer: Beenden der Emission und Zurücksetzen des Eingangs.

In particular, the control unit may be configured to perform the following steps:

• Determining a (predetermined, in particular by the user) exposure duration, a readout duration for all detection elements, a first integration duration, a pause duration (between the end of the first integration and the triggering of the second), wherein the pause duration is less than the readout duration, and a second integration duration ( wherein the pause duration is determined as a function of the second integration period or vice versa)
• Controlling the light source for (periodically) pulsed or continuous first emission of light of at least one predetermined wavelength for at least the exposure time,
• Setting the (integration-triggering) input of the converter in synchronism with a start of the first emission (in particular by a constant time offset for controlling the light source),
• - after expiration (especially at the end) of the first integration period: stop the emission and reset the input,
• - waiting for the pause,
• At the end of the pause: controlling the light source to a second emission corresponding to the first emission for the same exposure time and
• Setting the (integration-triggering) input of the converter synchronously with a start of the second emission (in particular by a constant time offset for controlling the light source)
• - after expiration (especially at the end) of the second integration period: stop the emission and reset the input.

In a particularly preferred embodiment, the image recording device is an SPI microscope, wherein the illumination beam path and the detection beam path intersect (at least approximately) in a focal plane of the detection beam path and the illumination beam path has a beam former for generating at least one segment of a light sheet (or a complete light sheet), in particular by focusing on a focal line, wherein the focal line lies (at least approximately) in the focal plane of the detection beam path.

The invention is particularly advantageous in a microscope in which the sample can be illuminated sequentially from two sides ("left" and "right") and a respective partial image can be recorded by it. The partial images are calculated into a total image that has a higher image quality than the partial images. With time-varying samples and / or a movement of the sample, for example, to capture a stack of images, the lower the time interval between the sub-images, the higher the image quality of the overall image. The reduction in the time interval made possible according to the invention makes it possible to increase the image quality of the overall image.

The invention is also advantageous in microscopes in which the illumination beam path has a variably adjustable deflection unit for displacing the light sheet, in particular within the focal plane of the detection beam path. Expediently, the control unit here can be set up to control the deflection unit for periodically shifting the light sheet with a predetermined period duration (and determine the pause duration on the basis of the period duration), the (total) exposure duration, in particular also the duration of the first integration, being an integer Multiples of the period corresponds. This achieves equal brightness in both successive images. In particular, the control unit may be configured to modulate an intensity of the emission of the light source during the respective exposure period. Then the respective effective exposure time is shorter than the respective total exposure time.

Also advantageous are embodiments of the microscope with a sample holder, wherein the control unit is adapted to move the sample holder during the emission of the light source along an optical axis of the detection beam path.

• – eine Pausendauer (unter anderem) anhand einer (vom Benutzer) vorgegebenen Belichtungszeit und einer (vom Wandler/vom Benutzer für den Wandler) vorgegebenen Auslesedauer für alle Detektionselemente ermittelt,
• – die Lichtquelle für eine Dauer einer (vom Benutzer) vorgegebenen Belichtungszeit zur periodisch gepulsten oder kontinuierlichen ersten Emission von Licht mindestens einer vorgegebenen Wellenlänge (und danach zum Beenden der Emission) gesteuert und synchron zu einem Beginn der ersten Emission für eine erste Integrationsdauer der (integrationsauslösenden) Eingang des Wandlers gesetzt (und nach Ablauf der ersten Integrationsdauer zurückgesetzt),
• – die Pausendauer abgewartet und
• – die Lichtquelle für die Dauer der Belichtungszeit zu einer mit der ersten Emission (spektral und in ihrem zeitlichen Intensitätsverlauf) übereinstimmenden zweiten Emission (und danach zum Beenden der Emission) gesteuert und synchron zu einem Beginn der zweiten Emission für eine zweite Integrationsdauer der (integrationsauslösenden) Eingang des Wandlers gesetzt (und nach Ablauf der zweiten Integrationsdauer zurückgesetzt).

In the method according to the invention is preferably:

• A pause duration (inter alia) determined on the basis of a (user-defined) exposure time and a (by the converter / user for the converter) predetermined readout duration for all detection elements,
• The light source is controlled for a duration of a (user-specified) exposure time for periodically pulsed or continuous first emission of light of at least one predetermined wavelength (and thereafter to terminate the emission) and synchronous to a beginning of the first emission for a first integration period of the (integration-triggering ) Input of the converter set (and reset after expiration of the first integration period),
• - Waited the pause time and
• The light source is controlled for the duration of the exposure time to a second emission coinciding with the first emission (spectral and in its temporal intensity course) and then ending the emission synchronous to a beginning of the second emission for a second integration period of the (integration triggering) Input of the converter set (and reset after expiration of the second integration period).

Again, those embodiments are particularly preferred in which the second integration duration is longer than the first integration duration, wherein the pause duration is shorter than a readout duration for all detection elements minus the second integration duration, so that the second integration is terminated after or at least as soon as the converter completes the Reading the detection elements has finished.

In a specific embodiment, the first integration duration and the second integration duration may be the same length and the pause duration may be shorter than a duration of a readout duration for all Detection elements and at least, in particular exactly, equal to the readout duration for all detection elements minus the exposure time.

The invention also includes a computer program and a control unit, which are each set up to carry out a method according to one of the method claims.

The control unit can be set up, for example, to carry out the described steps by being configured according to the program. In particular, a corresponding software module can be set up for each step.

The invention will be explained in more detail by means of exemplary embodiments.

In the drawings show:

1 an SPI microscope,

2 a first signal diagram and

3 a second signal diagram.

In all drawings, like parts bear like reference numerals.

1 shows a lens microscope as an image pickup device 1 with a light source 2 , which is a broadband laser 2.1 and an acousto-optic tunable filter 2.2 for external wavelength selection and intensity modulation. For example, the laser 2.1 generate light pulses with a length of 20 ps in a wavelength range from 450 nm to 700 nm. The repetition rate of the pulses is for example 40 MHz. At the output radiation of the laser 2.1 it may be a white light continuum, from which then the wavelengths used in the illumination radiation are externally filtered out, for example by the acousto-optic adjustable filter 2.2 , or the laser 2.1 may itself include a suitable means for wavelength selection and intensity modulation, such as an internal acousto-optic tunable filter or a frequency conversion unit. At the laser 2.1 it can be a fiber-based supercontinuum laser. In the case that the laser 2.1 itself includes means for wavelength selection and intensity modulation can be applied to the acousto-optic tunable filter 2.2 be waived. A suitable laser is for example the company Fianium Ltd. commercially available.

The image pickup device 1 further comprises an illumination beam path B, in which the of the light source 2 generated illumination radiation via an optical switch 3 , For example, a micromirror field, switchably passes either in a left branch B _L or in a right branch B _R. In the illumination beam paths B _{R / L} is in each case a beam shaper 4 arranged, which has a cylindrical appearance 5 as well as imaging optics 6 which comprises shaping the illumination radiation into a light sheet 7 cause. In alternative embodiments (not shown), instead, the cylinder optics 5 and / or the imaging optics 6 in front of the optical switch 3 be arranged in the constant illumination beam path section B or completely omitted, wherein the light sheet 7 then by means of the deflection unit 11 is produced. In particular, the cylinder optics can be pivoted into and out of the beam path. The light sheet 7 is to the test 8th , For example, a biological sample, which is embedded in a transparent gel, directed and intersects them in a focal plane of the illumination beam path B. Depending on the position of the optical switch 3 the sample is illuminated either from one side (left) or from the other side (right).

The image pickup device 1 further comprises a detection beam path A, in which a detection objective 9 is arranged, over which of the sample 8th emitted or scattered sample light is recorded. The axis of the detection beam path A is substantially perpendicular to the axis of the illumination beam path B and in particular substantially perpendicular to the sectional plane of the light sheet 7 with the sample 8th , The sample 8th is thus illuminated approximately only in one plane. The detection lens 9 serves the focusing in a focal plane of the detection beam A. The illumination beam path B and the detection beam A are completely separated from each other and intersect only in the respective focal plane. Arrangements are also possible with a different angle between detection beam path and illumination beam path, which is for example between 0 ° and 90 °. It is crucial for SPIM that the illumination beam path B and the detection beam path A are not aligned in parallel.

The image pickup device 1 further comprises a device 10 for positioning the sample 8th relative to the light sheet 7 by displacement of the sample holder, so that by a relative movement of the sample 8th along the axis of the detection beam A and / or relative to the illumination beam B different optical sections of the sample 8th can be included, which can serve as a basis for three-dimensional image data sets. The relative movement can also be a rotation of the sample 8th include. In addition, the illumination beam path B has a deflection unit 11 to move the sheet of light within the sample 8th on, also from the control unit 15 is controlled.

The detection beam path A further comprises a tube lens 12 over which the from the detection lens 9 detected emission radiation to an optoelectronic transducer 14 For example, a CCD sensor with a global electronic shutter is supplied. Furthermore, in the detection beam A, an emission filter 13 be provided, which is about the sample 8th blocked in the detection beam A reflected or scattered illumination light components in the sample light. The emission filter 13 However, it is not required for all modes of operation of the microscope and is therefore preferably removable. In some operations, detection of reflected or scattered illumination radiation may be via the transducer 14 even be desirable.

The image pickup device 1 further comprises a control unit 15 that have signal lines 16 / 17 / 18 / 19 with the light source 2 , with the converter 14 and with the deflection unit 11 connected is. The control unit 15 generates modulation signals which are transmitted via the signal line 16 to the light source 2 , there in particular the AOTF 2.2 , and time window control signals for triggering and terminating the light integration, which are transmitted via the signal line 17 to a signal input of the converter 14 be transmitted, and deflection signals, which via the signal line 19 to the deflection unit 11 be transmitted. The converter 14 in turn transmitted over the signal lines 18 a status signal on the read-out of the data from the respective previous integration and - after the end of the read-out process - the digitized image data to the control unit 15 ,

In 2 and 3 are signal diagrams of an embodiment of image acquisition in the simple overlap mode ( 2 ) or in the extended overlap mode ( 3 ), in which the timing of the over the signal lines 16 / 17 / 18 / 19 / 20 transmitted signals is recognizable. The individual tracks contain the following information from top to bottom: Lane 1 Main trigger, which indicates the starting point for all time calculations Lane 2 Distortion Synchronization: Each stroke indicates the beginning of a period of oscillation of the deflection unit 11 Track 3 Signal for triggering / terminating the integration in the converter 14 Lane 4 Control signal for the driver of the AOTF 2.2 or a directly controlled light source Lane 5 Readout status of the converter 14 Lane 6 Integration status of the detection elements of the converter 14 Lane 7 Switching time between lighting side or between laser lines in a sequential multi-channel recording

The exposure (obliquely hatched) and the integration (horizontally hatched), which preferably occur synchronously as shown, are also due to the pause of the duration D2 _converter between the two integration processes with the periodic movement of the deflection unit 11 synchronized. Since the AOTF 2.2 has a certain inertia, may be its control signal 18 opposite to that in the converter 14 integration-triggering and integration-terminating signal 17 to be preferred by a constant time offset, so that the pause durations D1 _AOTF-start and D2 _AOTF-start by the startup duration of the AOTF is shorter than the pause durations D1 converter or D2 converter. Clearly recognizable are the identical exposure durations for all integrations. In other embodiments (not shown), the signals 17 and 18 can also be output simultaneously.

With the first reset of the trigger signal 17 The converter automatically copies the contents of all detection elements into the hidden buffer and starts reading and digitizing from the buffer, which it does by setting the read-out status 18 displays.

In simple overlap mode ( 2 ) is at a time, the shutter signal by one exposure time before the end of the first read 17 set for the second integration, so that the second integration of the converter 14 overlapping in time with the first read-out process is performed. The end of the first read can be the control unit 15 from the time of the first reset of the trigger signal 17 and for the converter 14 determine the fixed readout duration. Once the converter 14 completed the first read and the readout status 18 reset, the trigger signal 17 reset the second time and so the second read-out in the converter 14 initiated. The second integration takes exactly as long as the first integration.

The simple overlap mode allows the time interval between any number of consecutive images to be reduced so that the average frame rate increases over regular integration readout sequences by (nearly) seamless readout readout sequences with maximum sample conservation can be.

In extended overlap mode ( 3 ) becomes the trigger signal 17 set even earlier for the second integration in order to further reduce the time interval to the first integration. Again the trigger signal remains 17 set to the end of the first readout. Once the converter 14 completed the first read and the readout status 18 reset, the trigger signal 17 reset the second time and so the second read-out in the converter 14 initiated. The second integration period is thus significantly longer than the first integration period. However, due to the identical exposure times, the intensity and brightness ratios are comparable to those during the first integration.

The extended overlap mode, as opposed to the simple overlap mode, further reduces only the temporal distance within each pair of consecutive frames (corresponding to a local increase in frame rate), while the average frame rate can not be further increased. The local increase of the frame rate is particularly advantageous for variable samples, in particular for alternating bilateral field recordings. In systems with more than one transducer and appropriate division of the sample light, for example by beam splitters, even more images (2 × number of transducers) can be recorded in quick succession, if the signals of the extended overlapping mode for the individual cameras are delayed accordingly.

The user is informed by the control unit 15 a control 21 to provide the desired exposure time, the desired center frame rate, and the desired local frame rate. If the frame rate is set higher than a frame rate, which corresponds to the readout duration (signal 18 set), so the control unit uses 15 the extended overlap mode when the local frame rate is set lower than the center frame rate, and otherwise the simple overlap mode. The pause duration D2 _converter (and the derived pause duration D2 _AOTF-start ) determines the control unit, for example, based on the user defined exposure time, the user preset frame rate and the (constant, transducer own) readout duration.

The invention can be used not only in SPI microscopes, but in any image recording devices, even in those in which the illumination beam path and the detection beam path are parallel or even partially coaxial. It is also without distraction unit 11 and / or can be used without double-sided illumination, the signals 19 and 20 omitted.

LIST OF REFERENCE NUMBERS

1

Imaging device

2

light source

2.1

laser

2.2

AOTF

3

Optical switch

4

beamformer

5

cylindrical optics

6

imaging optics

7

light sheet

8th

sample

9

detection objective

10

positioning

11

Deflector

12

tube lens

13

emission filter

14

converter

15

control unit

16

Signal (line)

17

Signal (line)

18

Signal (line)

19

Signal (line)

20

Signal (line)

21

operating element

A

Detection beam path

B

Illumination beam path

D _scanner

Delay from the start of the deflection unit

a

dead

D1 _converter

Delay until triggering the first integration

D1 _{AOTF start}

Delay until turning on the AOTF for first emission

D _AOTF-end

Delay until turning off the AOTF

D2 _converter

Delay until triggering the second integration

D2 _{AOTF start}

Delay until turning on the AOTF for second emission

L2 _converter

dark integration

T _converter

Identical first and second integration duration

T1 _converter

First integration period

T2 _converter

Second integration period

T _AOTF

exposure time

D3 _converter

Delay until third integration

D3 _{AOTF start}

Delay until turning on the AOTF for third emission

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 2006/033987 A1 [0003]
• US 2009/095986 A1 [0004, 0008]
• US 2001/035542 A1 [0004, 0008]
• US 2006/157758 A1 [0004]

Claims (17)

Image pickup device ( 1 ), in particular a microscope, with at least one illumination beam path (B), which is a modulatable light source ( 2 ), a detection beam path (A) having an optoelectronic transducer ( 14 ) comprising an at least two-dimensional matrix of detection elements, and a control unit ( 15 ) with the light source ( 2 ) and the converter ( 14 ), characterized in that the transducer ( 14 ) an input for a signal ( 17 ) for triggering a simultaneous electrical integration in all detection elements and that the control unit ( 15 ) is arranged in the converter ( 14 ) initiate and terminate a first integration and thereafter, before in the converter ( 14 ) a readout of a content integrated during the first integration from all the detection elements is terminated, triggering a second integration and only ending it again after the converter ( 14 ) the readout of the content integrated in the first integration from all the detection elements is completed, whereby during both integrations, in each case for the same exposure time, the light source ( 2 ) for pulsed or continuous emission of at least one wavelength and interrupts the emission between the integrations. Image pickup device ( 1 ) according to claim 1, wherein the second integration period is longer than the first integration period, in particular with the formation of the control unit ( 15 ) for triggering the second integration such that a temporally overlapping portion of the second integration with the read-out is longer than the first integration. Image pickup device ( 1 ) according to the preceding claim, which is a control element ( 21 ) for setting the second integration period, a quotient between the second and the first integration period or a difference between the first and the second integration period. Image pickup device ( 1 ) according to claim 1, wherein the first integration period and the second integration period are the same length. Image pickup device ( 1 ) according to one of the preceding claims, wherein the first integration is the same length as the exposure time. Image pickup device ( 1 ) according to one of the preceding claims, which is a control element ( 21 ) for selecting one of several of the control unit ( 15 ), wherein in the first operating mode the second integration duration is longer than the first integration duration, in particular at constant first and constant second integration duration, and in the second operating mode the first integration duration and the second integration duration are equal. Image pickup device ( 1 ) according to the preceding claim, wherein the control unit ( 15 ) is arranged to terminate the second integration at the end or at the end of the readout of the converter. Image pickup device ( 1 ) according to one of the preceding claims, wherein the control unit ( 15 ) is set up to trigger the second integration for a pause duration after the completion of the first integration. Image pickup device ( 1 ) according to one of the preceding claims, wherein the control unit ( 15 ) determines the pause duration based on a predetermined period of a periodic process so that the pause duration plus the first integration period corresponds to an integer multiple of the period. Image pickup device ( 1 ) according to claim 8 or 9, wherein the control unit ( 15 ) is arranged to determine the pause duration such that it is longer than or equal to the readout duration less the second integration duration or the second integration duration such that it is longer than or equal to the readout duration less the pause duration. Microscope ( 1 ) according to any one of the preceding claims, wherein the illumination beam path (B) and the detection beam path (A) intersect each other in a focal plane of the detection beam path (A) and the illumination beam path (B) a beam shaper ( 4 ) for generating at least one segment of a light sheet, in particular by focusing on a focal line, wherein the focal line lies in the focal plane of the detection beam path (A) has. Microscope ( 1 ) according to the preceding claim, wherein the illumination beam path (B) comprises a variably adjustable deflection unit ( 11 ) for the displacement of the light sheet, in particular within the focal plane of the detection beam path (A). Microscope ( 1 ) according to the preceding claim, wherein the control unit ( 15 ) is adapted to the deflection unit ( 11 ) to control the periodic displacement of the sheet of light with a predetermined period, wherein the exposure time, in particular the duration of the first integration corresponds to an integer multiple of the period, in particular with formation of the control unit ( 15 ) for modulating an intensity of the emission of the light source during the respective exposure period. Method for capturing an image sequence from a sample ( 8th ) by means of at least one illumination beam path (B) having a modulatable light source ( 2 ), and a detection beam path (A) comprising an opto-electronic converter ( 14 ) comprising an at least two-dimensional matrix of detection elements, wherein: a pause duration is determined on the basis of a predefined exposure duration and a predefined readout duration for all detection elements, the light source is 2 ) is controlled for the exposure period for the periodically pulsed or continuous first emission of light of at least one predetermined wavelength and synchronous to a beginning of the first emission for a first integration period of the input ( 17 ) of the converter ( 14 ) - the pause duration is waited for and - at the end of the pause duration the light source ( 2 ) is controlled for the exposure time to a second emission corresponding to the first emission and synchronously to a start of the second emission for a second integration period of the input ( 17 ) of the converter ( 14 ) is set. Method according to the preceding claim, wherein the second integration duration is longer than the first integration duration, wherein the pause duration is shorter than a readout duration for all detection elements minus the second integration duration. Method according to one of the preceding method claims, wherein the first integration duration and the second integration duration are the same length and the pause duration is shorter than a readout duration for all detection elements and at least, in particular exactly, equal to the readout duration for all detection elements minus the duration of the exposure duration. Computer program or control unit ( 15 ) arranged for carrying out a method according to one of the method claims.

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