EP3465317A1

EP3465317A1 - Optical arrangement for pulsed illumination, method for pulsed illumination and microscope

Info

Publication number: EP3465317A1
Application number: EP17728139.1A
Authority: EP
Inventors: Bernd Widzgowski; Vishnu Vardhan Krishnamachari; Lars Friedrich; Manuel KREMER; Arnold Giske
Original assignee: Leica Microsystems CMS GmbH
Current assignee: Leica Microsystems CMS GmbH
Priority date: 2016-05-31
Filing date: 2017-05-31
Publication date: 2019-04-10
Also published as: WO2017207664A1; US20190339500A1

Abstract

The invention relates to an optical arrangement (11), a microscope and a method for illuminating a sample space (71) with a sequence (36) of laser light pulses (3). Known optical arrangements (11) allow a pulse repetition frequency (13) of generated pulses to be reduced, but are cost-intensive, may reduce the stability of the laser light source (1) and cannot be adapted to every illumination situation. The optical arrangement (11) according to the invention eliminates these disadvantages by providing at least one laser light source (1) for generating the sequence (36) of laser light pulses (3) along the optical beam path (15) and a wavelength-selective pulse picker (19) situated in the optical beam path (15), wherein the pulse picker (19) with a predefined illumination clock timing (41) synchronizable with the laser light pulses (3) has an open state (42), in which it is light-transmissive for at least one laser light pulse (3) toward the sample space (71). In this case, the open state (42) has at least two different transmission states (33) which differ with regard to the respective transmission spectrum (31) thereof, wherein the two transmission states (33) can be switched on and/or off independently of one another.

Description

Optical arrangement for pulsed illumination, method for pulsed illumination and

microscope

The invention relates to an optical arrangement for illuminating a sample space with a sequence of laser light pulses generated in a laser cycle, a microscope and a method for illuminating a sample space with a sequence of laser light pulses.

Although known from the prior art optical arrangements allow, the

Pulse repetition frequency, which corresponds to the laser clock to reduce the pulses generated, however, are costly, can reduce the stability of the laser light source and can not be adapted to any lighting situation. An optical arrangement with which this can be achieved is described, for example, in EP 2 081 074 B1. There, a so-called Pulsepicker (German: pulse selector) between a mode-locked fiber oscillator and an optical fiber amplifier of a supercontinuum laser is arranged. The provision of the Pulse Picker at this point - in the Supercontinuumlaser - can be disadvantageous and cause additional costs. The object of the present invention is thus to provide an optical arrangement, a microscope and a method which are inexpensive, allow versatile lighting applications and also do not affect the stability of a laser system.

Optical arrangements for illumination can be used, for example, in fluorescence microscopy, in particular using confocal or light-sheet microscopes. In these applications, pulsed lasers are used to excite dyes (luminophores or fluorophores) which have an afterglow following the excitation. The afterglow (luminescence or fluorescence) usually required the excitation in a certain

Spectral range, i. at a certain wavelength. If several luminophores are used simultaneously, it is desirable to be able to distinguish the luminescence of different luminophores, in particular if they have a different temporal behavior of the luminescence. These difficulties are known and are solved in the prior art, for example, by individual lasers, which each stimulate a luminophore and are driven separately

The above-mentioned optical arrangement for illuminating a sample space with a sequence of laser light pulses generated in a laser cycle solves the above objects in that the optical arrangement comprises an optical beam path, at least one laser light source for generating the sequence of laser light pulses along the optical beam path and having a wavelength selective pulse spreader located in the optical path. The Pulspicker points in one predetermined, synchronizable with the laser light pulses lighting clock on an open state in which the pulse picker is transparent for at least one laser light pulse to the sample space, the open state has at least two different transmission states that differ with respect to their respective transmission spectrum, and wherein the two

Transmission states are independently switched on and / or off.

The microscope according to the invention, in particular a PIE microscope, achieves the above objects with an optical arrangement according to the invention.

The method according to the invention mentioned at the outset solves the above objects by sending the laser light pulses through a wavelength-selective pulse picker, which is switched between at least two different transmission spectrums in synchronism with the laser clock.

The present invention differs from the previously known solutions that do not allow wavelength-selective, with the laser light pulses synchronized reduction of the pulse repetition frequency.

In the following, further embodiments of the invention will be described, the additional or alternative technical features are advantageous in each case and can be combined with each other as desired.

The laser light source used may emit light of multiple colors, and may preferably be a supercontinuum laser or a Raman comb laser. The supercontinuum laser may include a pump light source and a non-linear fiber. By a non-linear fiber is meant a fiber in which nonlinear optical effects, such as self-phase modulation, of the coupled laser pulses of the pumping light source occur. The laser light source emits pulsed light with at least one laser light pulse or laser pulse.

The optical beam path is to be understood as an optical path along which the

propagate electromagnetic waves of light, or in pulses of the electromagnetic wave packet. The optical path is rectilinear and can be changed by optical elements such as mirrors, prisms, gratings or the like. The optical beam path is essentially determined by the laser light source.

A transmission state is to be understood as an adjustment or a mode of the optical arrangement. Each transmission state is due to a respective transmission spectrum

characterized in that the transmission spectra preferably at least one transmission maximum exhibit. Different transmission states differ by their

Transmission spectra, wherein the transmission spectra of two different

Transmission conditions may be partially identical and only in defined

Can distinguish wavelength ranges. Preferably, with the plurality of possible transmission states, the emission spectrum of

Laser light source can be covered. In the case of a supercontinuum laser, for example, all wavelengths of the visible spectrum or an entire octave can be covered. By this is to be understood that different transmission states over the emission spectrum of the

Distributed laser source across transmission maxima may have, wavelength ranges without a transmission maximum of a transmission state, however, may also be present.

Switching between the at least two different transmission spectra comprises in one embodiment of the method the generation of a frequency sequence comprising at least one frequency and the application of the frequency sequence to a sound transducer and um

Add the frequency of the frequency sequence.

Since the open state can be synchronized with the laser clock, it is thus ensured that a laser pulse in the pulse picker encounters a combination of transmission states which remain unchanged at least until the next lighting cycle, which can correspond to the next laser cycle. The lighting clock of the pulse picker can have a lower frequency than the laser clock, so that illumination of the sample space is prevented between two cycles of the lighting clock, in other words no laser light pulse is transmitted to the sample space. However, the lighting clock can also correspond to the laser clock, so that at each cycle of the laser clock, a laser light pulse can illuminate the sample space. Under the aforementioned PIE microscope, a microscope is to be understood, which excites a sample with interleaved (English: interleaved), in other words time-shifted and temporally interlocking, pulse sequences (English: excite). With the microscope according to the invention, this so-called "pulsed interleaved excitation (PIE)" functionality, ie the excitation with interleaved pulses, can be realized in a particularly advantageous manner in conjunction with a pulsed multicolor laser or a plurality of mutually synchronized pulsed multicolor lasers or a supercontinuum laser an optical examination of a sample with a microscope, in particular with a scanning microscope and very particularly preferably with a confocal scanning microscope is used.

In a further embodiment of the optical arrangement, the pulse picker has at least three different transmission states, which can be switched independently of one another. This has the advantage that three luminophores can be used in the sample, the

different transmission spectra of the three independently switchable

Transmission states with the excitation spectra of luminophores substantially

can match.

Thus, the three luminophores can be excited at arbitrary times in the lighting cycle, any combination of the transmission states being possible. At three

Transmission states, which can be switched on or off, thus result in eight possible combinations.

In further embodiments, four, five, six or more integer transmission states may be possible. In a further embodiment of the optical arrangement, the wavelength-selective pulse sputterer has an electro-optical element. This has the advantage that it allows very short switching times in the range of a few nanoseconds up to a few hundred picoseconds. An electro-optical element is based on the Kerr effect in a crystal and results in a wavelength-dependent rotation of the polarization of the light passing through the electro-optical element, the wavelength dependence being variable by means of the voltage applied to the electro-optical element.

In a further embodiment of the optical arrangement according to the invention, the

wavelength-selective Pulspicker an acousto-optical element. An acousto-optical element also has short switching times. In contrast to the use of an electro-optical, the switching time of an acousto-optical element is dependent on the experimental conditions.

Since an acousto-optic element is based on a sound wave which is generated in a medium and propagates, among other things, the speed of sound in the medium used, as well as the diameter of the light beam to be modulated in the medium determine the switching time.

For example, when switching from a first frequency to a second frequency, the sound wave precedes with the first frequency of the sound waves at the second frequency, wherein the Phase front, which is located in the transition region between the first and the second frequency, must propagate over the entire beam diameter, until there is talk of a successful switching.

An acousto-optical element is further based on the diffraction of electromagnetic waves on the acoustic wave, which forms a density grating. Only the wavelengths which satisfy the Bragg condition are diffracted at the density grating.

In one embodiment of the invention, the remaining wavelengths which are not diffracted at the density grid and thus do not reach the sample space can nevertheless emerge from the pulse spreader and be used for other applications. Conceivable applications are, for example, the illumination and generation of a stroboscopic effect, wherein the appearance of the originally broadband, preferably white appearing light changes depending on the composition of the transmission states by bending individual wavelengths or wavelength ranges on the density grating.

In the simplest case, the non-diffracted light enters a beam trap and is completely absorbed in it.

In a further embodiment of the optical arrangement according to the invention, the acousto-optical element has a crystal, wherein at least one connected to the crystal

Sound transducer is provided. With the sound transducer, which can be mounted on the crystal to transmit motion, a sound wave is coupled into the crystal in a simple manner and transmitted in the crystal. In this case, acoustic longitudinal waves are preferably generated, whose

Oscillation takes place in the propagation direction.

Acoustic transversal waves are also conceivable, but these generally have a lower velocity of sound than longitudinal waves.

The crystal can be located between an input path, into which the laser light pulses can be coupled, and an output path, through which the at least one laser light pulse can be coupled out to the sample space

The use of crystals also has the advantage that by suitable cuts of the crystals a direction of propagation through the crystal can be established, whose optical properties, such as the speed of sound, are known. Thus, according to the invention, at least one electro-optical element and / or at least one acousto-optical element, preferably an acousto-optic tunable filter (AOTF), can be arranged at a point in the optical beam path which, based on the propagation direction of the light - the laser light source, ie, for example, the

Supercontinuum laser (white light laser, WLL) or the amankammlaser or a non-linear (photocrystalline) optical fiber or a correspondingly pulsed laser light source is arranged downstream.

The entire disclosure content of WO 2011/154501 A1 is hereby incorporated by reference, and all embodiments described herein also extend in the context of optical components such as e.g. AOTF, AOM, AOD, EOM, EOD, AOBM and AOBS, which are described in WO 2011/154501 AI on page 2, second paragraph to page 3, first paragraph.

Problematic with pulsed laser light sources emitting multi-wavelength light (e.g., supercontinuum laser or Raman ridge laser) is that typically only one pulse picker for selecting the individual light pulses for all colors at a time can be realized by e.g. between the pumping light source and a nonlinear fiber of a

Supercontinuumlasers an electro- or acousto-optical element is placed. Although the functionality of the repetition rate reduction occurs, it simultaneously affects all colors. That In particular, the following configuration is provided with a supercontinuum laser with commercially available pulse picker, e.g. from EP 2 081 074 B1, not possible: illumination with a first excitation light having a wavelength of 629 nm with a

Repetition rate of 40 MHz, a second excitation light having a wavelength of 485 nm with a repetition rate of 20 MHz and a third excitation light with a wavelength of 557 nm with a repetition rate of 40 MHz, wherein e.g. the sequence of light pulses of the second and third excitation light is time-shifted by a predeterminable period of time (e.g., 12.5 ns or 25 ns) relative to the pulse position of the first excitation light of wavelength 629 nm.

Such a time sequence of the first to third excitation light would be e.g. in a picture of a sample provided with fluorescent dyes using a microscope and in particular with a scanning microscope and very particularly preferably with a confocal scanning microscope, in particular for PIE, whereby the fluorescent dyes of the sample can be excited with the first to third excitation light and the sample with the microscope or microscope the

Scanning microscope or the confocal scanning microscope is mapped or examined. The problem can be solved in that an AOTF, in which the color selection is done by setting an oscillation frequency, is controlled synchronously to the laser repetition rate with the aid of a digitally mixed control signal, ie a digitally mixed frequency sequence.

Preferably, the frequency sequence can be generated with a digital frequency synthesizer (frequency sequence generator).

The electro-optical element and / or the acousto-optical element is / are in this case driven by a frequency sequence suitable for this purpose, which stands for the epetitionsrate of the laser light pulses of the laser light source in a predeterminable temporal relationship and in particular is synchronous thereto.

Such a suitable frequency sequence can be generated by means of digital synthesizing, as e.g. in WO 2011/154501 AI is described.

In this case, in a certain sequence, the frequency is set alternately between at least two values corresponding to the desired colors or wavelengths, and, due to the synchronization per light pulse of the laser, those respectively applied to the current one on the AOTF

Vibration frequency corresponding color selected. In one refinement of the optical arrangement, a drive unit is provided for generating a frequency sequence for the pulse picker, the transmission state of the pulse picker being dependent on the frequency of the frequency sequence.

The frequency sequence can be applied to the transducer of the Pulspicker, wherein the

Sound transducer with the frequency of the frequency sequence oscillates. Consequently, a sound wave oscillating at this frequency propagates in the crystal, which corresponds to the speed of sound in the crystal

Material, in particular in the crystal, a density grating with a spatial frequency forms, which is directly proportional to the frequency of the frequency sequence.

Furthermore, in a further embodiment of the optical arrangement, the frequency sequence can have a superposition of at least two partial signals of different frequencies. This has the advantage that each frequency of the sub-signals leads to a density grating of different spatial frequency. Two different wavelengths or wavelength ranges of a pulse which impinges on the pulse picker along the optical beam path can thus be switched on independently of one another. The switch-on corresponds physically to the fulfillment of the Bragg condition, in which case light of the respective wavelength is diffracted in a predetermined direction and preferably in this direction is an output of the pulse spicker, through which the diffracted light can pass.

Since the light entering the pulse spout is pulsed, one is also one according to

Transmittance state selected spectral component pulsed. The increase in the pulse duration due to a reduced bandwidth is not discussed here, since the luminescence generally has a rise time in the range of picoseconds to microseconds and thus the excitation of a luminophore with a pulse duration of 10 fs does not depend on the excitation with a pulse duration different from, for example, 300 fs. The drive unit may comprise an overlay unit with which at least two frequencies in the frequency sequence can be superimposed. In particular, the drive unit comprises a digital data processing device for generating a digital frequency sequence composed of a plurality of frequencies and at least one digital-to-analog converter for converting the digital frequency sequence into an analog frequency sequence. The digital

Data processing device may include a digital frequency calculation device for calculating and generating at least two digital frequency sequence components of different frequency. Furthermore, a digital superposition device for superimposing the at least two digital frequency sequence components and calculating the resulting digital frequency sequence can be provided in the drive unit. The analog frequency sequence generated by the digital-to-analog converter can be fed into an amplifier and be transmitted from this amplified to the sound transducer.

The drive unit can be designed as a digital frequency synthesizer and can have a plurality of channels, with each of which simultaneously different vibration frequencies can be generated and with which simultaneously an electro-optical element and / or an acousto-optical element can be acted upon.

Frequency sequence on a synchronized with the laser clock control clock. This has the advantage that the frequency sequence at the time of a laser pulse arriving in the pulse picker is not changed, and thus each laser pulse has a unique composition of transmission states in the laser pulse

Open state of the pulse picker learns.

The frequency sequence can be pulsed. In a further embodiment, changeable stored in the optical arrangement

Lighting parameters exist on which depend the frequency of the frequency sequence and / or the control clock. The illumination parameters can thus contain a sequence for each transmission state, which controls the switching on or off of the respective transmission state. The switching on or off can be done periodically or aperiodically to predetermined, done with at least one clock of the laser clock matching times. Everyone

Transmission state can have a different sequence of switching on or off.

In a further embodiment, at least one at least two-colored transmission state is present, in which the transmission spectrum is at least two separate from each other

Has wavelength maxima. This has the advantage that luminophores whose luminescence is not mutually influenced (no cross-talk) can be excited at the same time. One of the

Excitation wavelengths may be larger and a second of the excitation wavelengths may be smaller than an excitation wavelength of a third luminophore. Two separate

Wavelength maxima are advantageous when it is desired that the third luminophore not be excited together with the first or second luminophore.

The number of transmission states is dependent in a further embodiment of the number of different frequencies in the frequency sequence. Consequently, everyone can

Transmission state to be assigned a different wavelength maximum, in particular, each transmission state is associated with exactly one different wavelength maximum.

In other words, there is a superposition of several sound waves in the pulse picker. Each of the sound waves has a different frequency, which correlates with the spatial frequency of the formed density grating. Further, at each density grating of a spatial frequency becomes exactly one

Wavelength or a wavelength range diffracted, namely the one that meets the Bragg condition. The number of possible wavelength maxima can correspond at most to the number of different frequencies in the frequency sequence.

The optical arrangement for illuminating a sample with laser pulses can be a pulsed one

Laser system which emits pulses in at least two different wavelength ranges in a pulse sequence and which has a Pulstriggermodul for generating a synchronized with the pulses of the laser system trigger signal. Furthermore, a drive unit with stored, changeable illumination parameters and with a trigger input, to which the Pulstriggermodul the pulsed laser system is connected to be provided, the

Drive unit in response to the stored illumination parameters and the trigger signal generates a periodically changing frequency sequence, which is composed of at least two frequencies. Furthermore, the optical arrangement can be an acousto-optical component having an input path into which the light generated by the pulsed laser system can be coupled, an output path through which the light transmitted through the acousto-optic component can be coupled out in accordance with the illumination parameters, and a crystal which with at least one

Sound transducer is connected include, wherein the frequency converter can be applied to the sound transducer, and wherein the sound transducer, the frequency sequence representing density grating can be generated in the crystal.

In this case, at least one sounder can be attached to a crystal, wherein light of the

Laser system can be coupled into the crystal and at least partially transmitted through the crystal and emerges from the crystal. The drive unit can transmit the frequency sequence to the at least one sound generator and the sound generator generates acoustic waves in the crystal as a function of the frequency sequence.

The acousto-optic component of the optical arrangement may have a direction of propagation of an acoustic wave generated by the acousto-optic component and forming the density grating that does not run collinear with the input path. In other words, the acoustic wave and the pulses are at an angle to each other. The optical arrangement can have at least two sound transducers attached to the crystal, wherein the density grating produced thereby can preferably be a stationary density grating. The standing wave is created by the superposition of two acoustic waves that pass through the crystal. Typically, each transducer faces an absorber to minimize reflections of the acoustic waves, thereby minimizing switching cycle time. The density grating of the optical arrangement can have at least two spatial frequencies, whereby pulses with at least two different wavelengths can be decoupled from the acousto-optical component via the output path by diffraction at the respective spatial frequencies. The spatial frequencies can correspond to the two frequencies of the drive unit.

The density grating formed in the crystal may be stationary with respect to an envelope of the density grating upon passage of a pulse through the crystal. Since the density grating is moved in the crystal, the frequency sequence composed by interference is not stationary with respect to the crystal, however stationary to the envelope. Thus, the synchronization is between the incoming

Laser pulses and the density grid sections possible. Furthermore, it is ensured that at the time of the arrival of a pulse, the change of the frequency composition of the acoustic wave has already been completed and indefinite frequency states (harmonics or the like) do not occur.

The frequency sequence generated by the drive unit can be divided into isochronous signal sections of a constant frequency composition in each case, wherein a time duration of the signal sections can correspond to a period duration of the pulse train emitted by the laser system. The

Signal sections can be the same length (in time), wherein the time window of a signal section can be as long as the time between two consecutive pulses.

By the sound transducer can be made more constant from the signal sections

Frequency composition Density grating sections each more constant

Space frequency composition be generated.

Further, the input path and the density grating may intersect at an interaction area, and each laser pulse passing through the interaction area may pass through only one density grating section at a time.

The laser pulse may substantially center the density grating section with respect to the

Propagation direction of the acoustic wave happen.

The method of operating an optical device may include the steps of generating a sequence of equidistant pulses of at least two different ones

Wavelength ranges and outputting a laser clock synchronous with the pulse train; the reading out of illumination parameters and generating a frequency sequence by the

Superposition of at least two frequencies as a function of the illumination parameters and the laser clock; transmitting the frequency sequence to a transducer and generating a density grid representing the frequency sequence in a crystal; the coupling of the equidistant pulses into the crystal; and the wavelength-selective coupling out of pulses from the crystal. Furthermore, the method may include generating a further density grating and superposing the density grating with the further density grating to produce a standing density grating.

The temporal equalization of signal sections each having the same frequency composition to a period duration of the sequence of pulses, as well as the generation of a density grating section from the signal section can be provided as a respective further method step.

In the method, the passage of a pulse can be synchronized by exactly one respective density grating section.

Most advantageously, the concept is scalable. So it is e.g. it is possible for light pulses of four different colors (which originate from the same laser light source) to either couple a pulse sequence alternating between four corresponding oscillation frequencies into the excitation beam path of a microscope, or a frequency sequence with two frequencies at the same pulse alternating with two other frequencies for the next following pulse depending on how the specific application requires it.

Due to the freedom of a digital synthesizing the principle of any frequency mixtures and frequency sequences and special configurations of the pulse picker are possible, such. to cancel out two colors with each clock of the laser clock, in the third and fourth colors e.g. only every second pulse with a corresponding time shift for the "PIE" method, and only every fourth pulse in the case of the fifth color. "Color" means a wavelength or wavelength range which is due to a transmission maximum from the pulse sputterer to the sample space This is done by bending or bending out of the acousto-optical element.

The lattice changes in the acousto-optic crystal should be able to be changed correspondingly fast (in particular as a function of the pulse repetition frequency of the light whose pulse repetition frequency is to be reduced) in order to allow short switching times. Helpful measures include a suitable selection of materials with high acoustic speed. Alternatively or additionally, a reduction of the optical beam diameter can take place until it is focused into the AOTF crystal. This reduces the interaction area or the

Interaction volume of the light to be changed with the density grating in the acousto-optical crystal or makes it possible to keep this as small as possible. The pulse repetition frequency of the light (of at least one wavelength), whose pulse repetition frequency is to be reduced, is preferably reduced in such a way that only a part of the original pulses in an application can be used per time interval.

For example, in one embodiment of original 100 pulses in a time interval only 95 to 5 pulses per time interval can be used for an application.

As far as can be represented with the synthesized frequency sequence, this can be used to generate an almost arbitrary sequence of pulses of different wavelengths and their temporally predeterminable sequence.

Without limiting the generality, the present invention can be used for a variety of

Applications are used. In particular, in addition to the already mentioned application for microscopy, scanning microscopy and / or confocal scanning microscopy, the optical

Arrangement and the method can also be used for applications in spectroscopy.

In general, the optical arrangement or method according to the invention can be used for applications involving the light of a supercontinuum laser and / or Raman ridge laser and / or a light source with a non-linear

Use (photocrystalline) optical fiber and / or a correspondingly pulsed laser light source and require a predetermined sequence and / or combination of laser pulses of predetermined wavelengths.

In the following, an embodiment of the invention will be described in more detail with reference to the accompanying drawings. The same technical features and technical features with the same technical effect are the sake of clarity provided with the same reference numerals.

It shows:

Fig. 1 shows a schematic structure of the optical arrangement according to the invention in one

possible design. 1 shows an exemplary embodiment of the optical arrangement 11 according to the invention, which comprises a laser light source 1 which generates pulsed laser light 2. The laser light 2 is shown schematically on the basis of a sequence 36 of four light pulses 3 which propagate along an optical beam path 15. The light pulses 3, also called laser pulses 3 or laser light pulses 3, have in the embodiment shown a pulse repetition frequency 13 of 80 MHz, wherein the Pulse repetition frequency 13 corresponds to the reciprocal value of a period 17 and a laser clock 35 marks. In this laser clock 35, the individual laser light pulses 3 are generated and emitted by the laser light source 1. In the illustrated embodiment, the period 17 has a value of 12.5 ns. The optical beam path 15 is drawn with a dashed line and in this is a wavelength-selective Pulspicker 19. The Pulspicker shown in the embodiment shown 19 includes an acousto-optical element 21, which is configured as acousto-optic adjustable filter 5, short AOTF 5 , The acousto-optic element 21 has a crystal 21a to which a sound transducer 21b is attached. In other embodiments, not shown, the Pulspicker 19 may be configured as an electro-optical element 22.

The laser light 2 may preferably be broadband and the visible spectral range 23, i.

Wavelengths 25 from about 400 nm to 700 nm. An exemplary spectrum 27 of the laser light 2 is shown in FIG. This contributes an intensity 29 of the laser light 2 over the wavelength 25. In the spectrum 27 of the laser light 2 are four different transmission spectra 31, which are each associated with a transmission state 33. Each of the transmission spectra 31 has a different wavelength maximum 32.

FIG. 1 further shows a symbolic first partial representation 101 of four transmission states 33, which repeat along a time axis 37 and are symbolized by the associated transmission spectra 31. The transmission spectra 31 are plotted along a wavelength axis 39 which extends obliquely upwards into the plane of the drawing (see the spectrum 27).

With reference numerals, only four of the transmission states 33 or transmission spectra 31 that are repeated in the lighting cycle 41 and total of sixteen are provided.

The transmission states 33 occur in a lighting cycle 41, which in the example shown corresponds to the laser clock 35 and can also be referred to as a control clock 41a. In the lighting cycle 41, the AOTF 5 is in an open position 42.

In the first partial view 101 of the embodiment shown in FIG. 1 of the optical arrangement 11 with four possible transmission states 33, a totality of the possible on or

switched off transmission states 33 shown in time dependence. In other words, these sixteen transmission states 33 are available for lighting within a period of four lighting cycles 41, ie four open positions 42 and can be switched on and off independently in each of the open positions 42.

The transmission states 33 are thus temporarily discrete and bound to the lighting clock 41.

The laser light source 1 has a trigger output 43, via which a generated trigger signal 4 is transmitted by means of a trigger line 45. The trigger signal 4 is in a second

Partial representation 103 shown and depends on the pulse repetition frequency 13 of the laser light source 1 from. In particular, the frequency of the trigger signal 4 corresponds to the pulse repetition frequency 13. The AOTF 5 is arranged downstream of the laser light source 1 in the optical beam path 15, with reference to the propagation direction 16 of the light 2. The AOTF 5 is actuated by a component 6 designed as a drive unit 51, which acts on the AOTF 5 with a frequency sequence 47 output by the drive unit 51. The signal 7 shown in a third partial representation 105 is a frequency sequence 47 and is transmitted via a control line 49 from the drive unit 51 to the AOTF 5.

As a result, a corresponding oscillation wave 53 propagates or passes through the AOTF 5 in the AOTF 5, whereby a Bragg grating 55 forms, with which the laser light 2 interacts and on which the laser light 2 is diffracted when the Bragg grating 55 forms a suitable effective lattice spacing 57 and a suitable propagation direction. The Bragg grating 55 is to be understood as a density grating 59.

It should be noted that the orientations of the optical beam path shown in FIG. 1 before and after the AOTF 5 with respect to the oscillation shaft 53 as well as an angle 63 between a zeroth order 65 and a first order 67 are shown purely schematically.

In general, the optical beam path 15 is coupled into the AOTF 5 at an angle to the oscillation wave 53, the zeroth order 65 is not deflected by the AOTF 5, and only the first order 67 is diffracted.

The reciprocal value of the grating pitch 57 corresponds to a spatial frequency 61. After passing through the laser light 2 by the AOTF 5, therefore, at least the zeroth 65 and the first diffraction order 67 of the changed light 69 result, which is indicated schematically in FIG. 1 to the right of the AOTF 5.

Now, the zeroth 65 and / or the first diffraction order 67 of the modified light 69 could be used for an application (general type). In the concrete exemplary embodiment, however, only the first diffraction order 67 of the modified light 69 is used, namely this changed light 69 is fed to a sample space 71 of a microscope 73, with which a sample provided with fluorescent dyes (both not shown) can be imaged.

The drive unit 51 has a trigger input 75 into which the trigger signal 4 of the trigger output 43 of the laser light source 1 is input.

In the drive unit 51 is a device in the form of at least one digital

Frequency synthesizer 77 (frequency sequence generator) provided with which a digital synthesis is such that a suitable time sequence of at least one oscillation frequency 7a or frequency sequence is generated, in response to the trigger signal 4. In Fig. 1, two oscillation frequencies 7a in the frequency sequence 47 consecutively combined.

In a frequency sequence 7, different oscillation frequencies 7a can be sequenced in time (as in FIG. 1). In this case, several oscillation frequencies 7a can occur simultaneously, i. be superimposed.

The frequency sequence 7 generated in this way is amplified by an amplifier 79 provided in the drive unit 51 (in the form of an analogue amplifier in this example) and fed to the AOTF 5 via a suitable line, the control line 49 (see dashed line). To generate the frequency sequence 47, the drive unit 51 can read out illumination parameter 81 from a lighting parameter memory and make it available to the digital frequency synthesizer 77. The frequency sequence 47 is composed of two partial signals 47a and 47b, but may also be composed of more than two partial signals 47a, 47b.

Here causes the - only schematically indicated - higher-frequency portion 85 of the

Frequency sequence 7, that in each case a first light pulse 87 of a pulse train with a blue color, ie a first wavelength 89, is diffracted into the first diffraction order 67 and thus the microscope 73 can be fed. The other light pulse portions 97 of the modified light 69 at other wavelengths of this pulse train undergo the AOTF 5 unbent and may be fed to a beam trap (not shown) should they not be needed for an application.

With the low-frequency component 91 of the frequency sequence 7, a second light pulse 93 with a red color, i. a second wavelength 95, diffracted into the first diffraction order 67 and supplied to the microscope 73.

In this embodiment, every second light pulse 3 of the pulse repetition frequency 13 of the laser light 2 is diffracted with a blue or red wavelength 25 into the first diffraction order 67, with the blue out (the first light pulse 87) and the red light pulse deflected out (the second light pulse 93). each of the pulse repetition frequency 13 originate from a different laser pulse 3 of the laser light source 1 and are therefore offset in time with respect to one another at a time interval 99. In that regard, the diffracted into the first diffraction order 67 light pulses 87 and 93 of the same wavelength 25 each have a pulse interval 99 of 25 ns, wherein a blue light pulse (the first light pulse 87) after 12.5 ns a red light pulse (the second light pulse 93 ) follows.

reference numeral

1 laser light source

2 laser light

3 light pulse

4 trigger signal

5 acousto-optic adjustable filter

6 component

7 signal

7a oscillation frequency

11 optical arrangement

13 pulse repetition frequency

15 optical beam path

17 period

19 wavelength-selective pulse picker

21 acousto-optic element

21a crystal

21b sound transducer

22 electro-optical element

23 visible spectral range

25 wavelength

27 Spectrum

29 intensity

31 transmission spectrum

33 transmission state

35 laser clock

36 episode

37 Timeline

39 wavelength axis

41 lighting cycle

41a control clock

42 open position

43 trigger output

45 trigger line frequency sequence

control line

control unit

oscillatory wave

Bragg grating

grid spacing

density grid

spatial frequency

angle

zeroth order

first order

changed light

sample space

microscope

trigger input

digital frequency synthesizer

amplifier

Lighting parameter higher-frequency content of the first light pulse

first wavelength low-frequency component second light pulse second wavelength other light pulse component time interval first partial representation second partial representation third partial representation

Claims

claims

Optical arrangement (11) with an optical beam path (15) for illuminating a sample space (71) with a sequence (36) of laser light pulses (13) generated in a laser clock (35) with at least one laser light source (1) for generating the sequence (36) of laser light pulses (3) along the optical beam path (15) and with a wavelength-selective pulse spreader (19) located in the optical beam path (15), which can be synchronized with the laser light pulses (3)

Lighting clock (41) has an open state (42), in which the Pulspicker (19) for at least one laser light pulse (3) to the sample chamber (71) is transparent, the open state (42) at least two different transmission states (33), the differ with respect to their respective transmission spectrum (31), and wherein the two transmission states (33) are independently switched on and / or off.

An optical arrangement (11) according to claim 1, wherein the pulse spreader (19) has at least three different transmission states (33) which can be switched independently of one another.

An optical arrangement (11) according to claim 1 or 2, wherein the wavelength-selective Pulspicker (19) comprises an electro-optical element (22).

An optical arrangement (11) according to any one of claims 1 to 3, wherein the wavelength-selective Pulspicker (19) comprises an acousto-optic element (21).

An optical arrangement (11) according to claim 4, wherein the acousto-optic element (21) comprises a crystal (21a), at least one sound transducer (21b) connected to the crystal (21a) being provided.

Optical arrangement (11) according to one of claims 1 to 5, wherein a drive unit (51) for generating a frequency sequence (47) for the Pulspicker (19) provided and the

Transmission state (33) of the Pulspickers (19) depending on the frequency (7a) of the

Frequency sequence (47) is.

7. An optical arrangement (11) according to claim 5 or 6, wherein the frequency sequence (47) a

Superposition of at least two sub-signals (47a, 47b) of different frequency (7a).

8. An optical arrangement (11) according to any one of claims 5 to 7, wherein the frequency sequence (47) has a synchronized with the laser clock (35) control clock (41a).

9. An optical arrangement (11) according to any one of claims 5 to 8, wherein stored, changeable illumination parameters (81) are present, of which the frequency (7a) of the frequency sequence (47) and / or the control clock (41a) depend.

10. An optical arrangement (11) according to any one of claims 1 to 9, wherein at least one at least two-tone transmission state (33) is present, in which the transmission spectrum (31) at least two mutually separate wavelength maxima (32).

11. An optical arrangement (11) according to any one of claims 5 to 10, wherein the number of

Transmission states (33) of the number of different frequencies (7a) in the

Frequency sequence (47) is dependent.

12. Microscope (73), in particular PIE microscope with an optical arrangement (11) according to one of claims 1 to 11.

13. A method for illuminating a sample space (71) with a sequence (36) of laser light pulses (3) which are generated in a laser clock (35), wherein the laser light pulses (3) are passed through a wavelength-selective Pulspicker (19), the synchronous to the laser clock (35) between at least two different transmission spectra (31) is switched.

14. The method of claim 13, wherein the switching between the at least two

different transmission spectra (31) further comprises: generating a frequency sequence (47) comprising at least one frequency (7a) and applying the frequency sequence (47) to a sound transducer (21b) and switching the frequency (7a) of the frequency sequence (47 ).