Method for scanning an area using a multiphoton laser scanning microscope and the multiphoton laser scanning microscope

Abstract

The present invention relates to a method for scanning an area using a multiphoton laser scanning microscope with an acousto-optic deflector. The method comprises scanning an area of ​​a disk located in an XY plane perpendicular to the optical axis of the microscope objective by applying a frequency drive signal to the deflector, the drive signal having a first term targeting the center of the disk and a second term for scanning the periphery of the disk. The invention also relates to a multiphoton laser scanning microscope with a drive system configured to perform this method.

Description

[Technical Field]

[0001] The present invention relates to a method for scanning an area using a multiphoton laser scanning microscope, and further to such a laser scanning microscope. [Background technology]

[0002] Scanning methods, especially three-dimensional laser scanning methods, play a major role in examining biological samples, including, among other things, mapping the structure of the biological sample, the distribution of fluorescent markers, or the surface receptors of cells.

[0003] Known scanning procedures generally use three-dimensional laser scanning microscopes, which at present are mainly multi-photon (typically two-photon) microscopes.

[0004] Multiphoton laser scanning microscopes operate with lower photon energy but higher intensity laser light, from which the simultaneous absorption of several, typically two, photons is required for the excitation of a fluorophore. The emitted fluorescent photons can then be detected using a detector. The same scanning technique is also suitable for sample manipulation, e.g., photostimulation and activation.

[0005] Although the above-mentioned techniques can achieve three-dimensional scanning by moving the stage using a stepper motor, this method is rarely used in practice due to its slowness and mechanical uncertainty.When examining biological samples, the focal point of the laser beam is moved instead of the sample, so that the laser beam is deflected and focused at the desired position.

[0006] There are several known techniques for deflecting a laser beam: deflecting mirrors mounted on galvanometer scanners have previously been used as deflection elements, and the depth of the focal plane was controlled by moving the objective lens along the Z axis, but this too can be slow when multiple distant regions within a sample must be scanned nearly simultaneously, for example in nerve impulse transmission measurements.

[0007] One of the fastest known laser beam deflection techniques is the use of acousto-optic deflectors. WO 2006 / 042130 A2 describes a solution using two pairs of acousto-optic (AO) deflectors to focus a laser beam of a laser scanning microscope to any stationary point within an octahedral spatial region. The first pair of AO deflectors deflects the focal point in the XZ plane defined by the optical Z axis and the X axis perpendicular to it, while the second pair of AO deflectors deflects the focal point in the YZ plane defined by the Z axis and the Y axis perpendicular to the Z and X axes. The deflectors actually deflect the laser beam, converging or diverging it so that a focusing lens system (typically a microscope objective) following the deflectors forms a focal point. Therefore, the deflector's effect appears as a change in the position of the focal point. The AO deflector includes an AO crystal and a transducer attached to the end of the AO crystal. The transducer converts an electrical drive signal into a mechanical signal, which generates acoustic waves within the AO crystal. In this way, the transducer excites acoustic waves within the AO crystal at a frequency corresponding to the frequency of the electrical drive signal. Because the instantaneous frequency of the electrical drive signal determines the frequency of the acoustic waves the transducer excites within the AO crystal, the electrical drive signal is usually called a frequency drive signal or simply a frequency signal. The excited acoustic waves propagate along the longitudinal axis of the AO crystal toward the opposite end of the transducer. As the acoustic waves propagate through the AO crystal, the refractive index of the material changes spatially according to the frequency of the acoustic waves. If the frequency of the frequency drive signal changes over time, this results in the frequency of the acoustic waves also changing along the longitudinal axis of the AO deflector. In this way, the AO crystal behaves as a spatially and temporally varying diffraction grating, deflecting a passing laser beam to converge or diverge.

[0008] In the aforementioned patent document WO 2006 / 042130 A2, transducers are arranged at both ends of each member of a plurality of AO deflector pairs, thereby generating counter-propagating acoustic waves within the members of the AO deflector pairs. To generate a stationary focal spot, a frequency drive signal with a constant slope (commonly known as a frequency chirp signal, or simply chirp signal) is applied to the members of the AO deflector pairs, i.e., the transducers that form part of the AO deflectors, thereby generating counter-propagating acoustic waves with a constant frequency gradient. The possible spatial positions of the focal spot are limited by the bandwidth of each AO deflector (or more precisely, the transducers that form part of it), within which the frequency of the chirp signal can be varied. This results in the octahedral-shaped scannable area presented in the patent document, within which a stationary focal spot can be generated at any point by appropriately setting the chirp signal.

[0009] Other deflector arrangements are known in comparison with those described in the above-mentioned patent documents, for example according to WO 2008 / 032061 A2, deflectors deflecting in the XZ and YZ planes are arranged alternately, so that a deflector deflecting in one plane is always followed by a deflector deflecting in the other plane, and between adjacent deflectors a telecentric relay (afocal telescope) images the previous deflector onto the next deflector.

[0010] In the configuration disclosed in WO2010 / 076579A1, a deflector deflecting in the XZ plane is placed between deflectors deflecting in the YZ plane, so that a single common telecentric relay between two deflectors deflecting in the XZ plane and two deflectors deflecting in the YZ plane can ensure imaging. Due to the negative (typically -1) lateral magnification of the telecentric relay, the transducers are placed at the same end of each deflector deflecting in the same plane, and the excited acoustic waves travel in the same direction relative to each other.

[0011] A prior art mode of laser scanning microscopy in which a pair of AO deflectors is used to generate a stationary focus with the aid of a frequency chirp signal driving the deflectors is called random access point scanning mode. As can be seen from the above references, this can be achieved in known ways using different arrangements of the AO deflectors.

[0012] In random-access point scanning mode, a stationary focal spot is used to scan the sample so that the two-dimensional or three-dimensional area of ​​the sample being examined is scanned point-by-point using successively created discrete focal spots. Although acousto-optic deflectors are very fast devices and can be used to generate focal spots at desired stationary positions very quickly (in as little as 10–20 microseconds), scanning is still a time-consuming process if the area to be scanned needs to be covered by a series of stationary points. To enable the laser beam to focus to successive stationary points, a corresponding chirp signal must be applied to the AO deflector for each stationary point. However, when transitioning between two successive chirp signals used to target different locations, the focal spot at one stationary point collapses while a new focal spot gradually appears at the other. During a period of transition, none of the focal spots will be of sufficient intensity or size to produce detectable fluorescence excitation.

[0013] To solve this problem, several solutions have been developed.

[0014] WO2020 / 035710A1 describes a solution in which several acoustic wave portions are generated in a single AO crystal by concatenating several chirp signals that are simultaneously focused at different points, so that one AO ​​deflector can measure at several points simultaneously.

[0015] In the already mentioned patent document WO2008 / 032061 A2, instead of generating one stationary focal point, the focal point is moved continuously along a straight line in the focal plane by controlling the frequency difference between the multiple acoustic waves generated in the multiple AO deflectors.

[0016] EP2800995 B1, as well as patent application WO2018 / 042214 A2 and the paper "High-Speed ​​3D Imaging of Spines, Dendrites, and Neuronal Ensembles in Behavior Animals" (Szalay et al., 2016, Neuron 92, 37-72, paper 372) all disclose scanning systems in which the focal spot is not fixed but moves continuously (drifts) with the help of four successive AO deflectors. The focal spot is moved in the XY plane by controlling the frequency difference between the acoustic waves generated in each deflector, while the axial position of the focal spot along the Z axis is changed by controlling the tilt of the chirp signal generated in the deflectors. This will be referred to as the drift mode hereafter.

[0017] The continuous scanning of special lines is also known from the prior art.

[0018] The paper "An Acousto-Optic Scanning System with Very Fast Nonlinear Scanning" (Friedman et al. OPTICS LETTERS / Vol. 25, No. 24 / December 15, 2000) describes scanning a circular arc in the XY plane. The paper aims to show how aberrations can be reduced during rapid scanning by using a pair of AO deflectors filled with counter-propagating acoustic waves instead of a single AO deflector deflecting in each plane. Results are presented in relation to continuous scanning along a circular arc. Although the paper speaks of "very fast" scanning, this is only very fast compared to scanning with a single AO deflector instead of two pairs. The T of the AO deflector access The filling time, i.e. the time required for the acoustic wave to fill the aperture corresponding to the width of the light beam passing through the AO deflector, is compared to the T scan The period is Taccess The scan speed is not considered to be fast at all, since it is an order of magnitude larger than the filling time. access The fill time was 0.62 μs, T calculated from the highest scan frequency shown in Figure 4. scan The period is 5 μs, which is T access This is almost 10 times the fill time. The authors did not recommend or consider scanning faster than this because they were looking for a solution at a speed that would not cause the focal spot to widen or collapse, the latter phenomenon being considered undesirable.

[0019] In the paper "Acousto-Optic Lenses with Very Fast Nonlinear Scanning" (Kaplan et al. OPTICS LETTERS / Vol. 26, No. 14 / July 15, 2001), a system consisting of two acousto-optic deflectors is presented, which can be used to harmonically move the focal spot along the Z axis, which corresponds to the optical axis. Here, the authors point out, in particular, that proper focusing is achieved by T scan ≫T acces He points out that this can only be achieved if s.

[0020] In biological measurements, simultaneous scanning of a cell population is often required. This is usually solved by using a random-access point scanning mode to sequentially create a stationary focal spot at the center of each cell, thus measuring the cell center. However, this has several drawbacks. On the one hand, the signal to be measured is not necessarily strongest at the center of the cell. On the other hand, in living samples (in the case of in vivo measurements), cells may be moving, so the focal spot may not be at the center of the cell but at its edge or even outside the cell. The aforementioned patent application WO 2018 / 042214 A2 and the paper "High-Speed ​​3D Imaging of Spines, Dendrites, and Neuronal Ensembles in Behavioral Animals" (Szalay et al., 2016, Neuron 92, 723-738) provide a solution to these problems. In particular, they recommend scanning a square in the XY plane that covers the biological object to be examined (e.g., cell body, dendritic spine). The square consists of short parallel lines along which the focal spot is continuously moved in drift mode. However, this method is not fast enough when quasi-simultaneous measurements of many targets are required. For example, a 20 µm x 20 µm area with a focal spot size of 0.5-1 micron can be scanned with 20 mini-drifts, requiring 20 AO cycles. A typical AO cycle time is 30 µs (for a 15 mm incident beam diameter), so 20 cycles take 20 x 30 µs. [Prior art documents] [Non-patent literature]

[0021] [Non-Patent Document 1] Szalay et al., 2016, Neuron 92, 37-72 [Non-patent document 2] Friedman et al. OPTICS LETTERS / Vol. 25, No. 24 / December 15, 2000 [Non-patent document 3] Kaplan et al. OPTICS LETTERS / Vol. 26, No. 14 / July 15, 2001 [Patent documents]

[0022] [Patent Document 1] WO2008 / 032061 A2 [Patent Document 2] WO2010 / 076579 A1 [Patent Document 3] WO2020 / 035710A1 [Patent Document 4] EP2800995 B1 [Patent Document 5] WO2018 / 042214 A2 Summary of the Invention

[0023] It is an object of the present invention to provide a scanning method that uses an AO deflector to scan a small area (typically less than 20 μm x 20 μm) in a plane perpendicular to the optical axis of a laser scanning microscope much faster than has been possible with conventional methods. It is another object of the present invention to provide such a laser scanning microscope.

[0024] The present inventors have discovered that when scanning along a circle or ellipse, significantly increasing the scanning speed compared to that proposed in the aforementioned paper "An Acousto-Optic Scanning System with Very Fast Nonlinear Scanning" (Friedman et al. OPTICS LETTERS / Vol. 25, No. 24 / December 15, 2000) allows aberration phenomena previously considered undesirable to be exploited in an unexpected way to scan the entire area within the circle or ellipse. The present inventors have found that the scanning time T along the circle or ellipse (T in the aforementioned paper) can be significantly increased compared to that proposed in the aforementioned paper "An Acousto-Optic Scanning System with Very Fast Nonlinear Scanning" (Friedman et al. OPTICS LETTERS / Vol. 25, No. 24 / December 15, 2000) allows aberration phenomena previously considered undesirable to be exploited in an unexpected way to scan the entire area within the circle or ellipse. scan time) is the filling time T of the AO deflector. acc (T in the above paper accessThey found that when the time required for the laser beam to be scanned is shortened to an order of magnitude (corresponding to the scanning time), the resulting aberrations produce satellite spots toward the center of the circle or ellipse, and the intensity of these spots is high enough to enable information collection from the inside of the circle or ellipse, i.e., scanning by multiphoton or two-photon excitation.

[0025] Accordingly, the present invention relates to a method for scanning an area with a multiphoton laser scanning microscope comprising at least one laser source and a focusing lens system defining an optical Z-axis, an optical beam path between the at least one laser source and the focusing lens system, and first and second acousto-optic deflectors disposed in the optical beam path for deflecting the laser beam exiting the focusing lens system in an XZ plane defined by the optical Z-axis and an X-axis perpendicular to the optical Z-axis, and third and fourth acousto-optic deflectors disposed in the optical beam path for deflecting the laser beam exiting the focusing lens system in a YZ plane defined by the optical Z-axis and a Y-axis perpendicular to the optical Z-axis and the X-axis.

[0026] In the present invention, a disk (disk part) located in a plane perpendicular to the XY plane defined by the X-axis and the Y-axis is scanned, and the scanning is performed by applying a frequency drive signal function f defined by the following equation to the first, second, third, and fourth acousto-optic deflectors, respectively: X1 (t), f X2 (t), f Y1 (t), f Y2 (t) while guiding the laser beam along an optical beam path from the laser source to the focusing lens system. TIFF2026502149000002.tif6150 TIFF2026502149000003.tif6150 TIFF2026502149000004.tif6150 TIFF2026502149000005.tif6150 where f 0X1 (t), f 0X2 (t), f0Y1 (t), f 0Y2 (t) is the frequency drive signal term for focusing the laser beam on the center of the disc in random access point scanning mode, where the coordinates of the disc center are x, y, z, and Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) is a frequency drive signal term that determines the outer circumference of the disk, and is defined by the following equations: TIFF2026502149000006.tif9150 TIFF2026502149000007.tif9150 TIFF2026502149000008.tif9150 TIFF2026502149000009.tif9150 In the above formula, s X1 , s X2 , s Y1 , s Y2 is chosen between +1 and -1, and f ampX is a first multiplying factor that determines a first radius Rx of the disc along the X-axis, and f ampY is a second multiplying factor that determines the second radius Ry of the disk along the Y axis, φ is an arbitrary phase shift, and T is the time required to scan the disk. In the present invention, the value of time T is set to 0.25 T acc From 1.6 T acc where T acc is the fill time of the deflector.

[0027] The method according to the invention works in any plane with any z coordinate, and therefore also in the plane where z=0 (which is included in the above expression, since the XY plane defined by the X and Y axes is also parallel to itself). The above expression therefore also includes the possibility of scanning a disc lying in the XY plane defined by the X and Y axes, where the z coordinate of the centre of the disc is 0.

[0028] Those skilled in the art will recognize that ΔfX1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 Without the (t) term, f is used so that the four deflectors create a stationary focal spot at a position with coordinates x, y, z corresponding to the center of the disk. 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 The method of selecting the term (t) is well known (this corresponds to the random access point scanning mode). The description of the state of the art lists, among others, such documents, the contents of which are incorporated herein by reference.

[0029] The frequency drive signal component Δf that determines the periphery of the disk X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 Although (t) is given as a cosine modulation for the first and second deflectors and as a sine modulation for the third and fourth deflectors, it is clear that this is equivalent to using sine modulation for the first and second deflectors and cosine modulation for the third and fourth deflectors. As reflected in the above equation, it is even possible to include a common phase shift φ in the arguments of each sine / cosine term. This phase shift parameter φ can be used to control where on the disk the scan begins, for example, at the top or right edge of the disk. If the phase shift φ is chosen to be 90°, the sine becomes a cosine, and vice versa.

[0030] Deflector filling time T acc is the effective filling time, and its value is T acc =H / V, where H is the diameter of the laser beam entering the deflector if the diameter of the laser beam is smaller than the aperture of the acousto-optic deflector, or the diameter of the aperture of the deflector if the diameter of the aperture is smaller than the beam diameter, and V is the acoustic phase velocity of the sound waves in the acousto-optic deflector. When different deflectors are used, the fill time Tacc of the deflector is calculated as the fill time T of the first deflector located along the optical beam path.acc means.

[0031] An optical beam path refers to the beam path determined by optical elements along which a laser beam propagates from a laser source to a focusing lens system (usually through a rear aperture). Any optical element capable of deflecting a laser beam or changing its diameter, divergence, polarization, power, etc. can be used to create this optical beam. Examples of such optical elements include mirrors (motorized or manually adjustable and fixed), beam splitters, beam combiners, lenses, lens systems, prisms, irises, Faraday isolators, polarizers, polarization rotation or manipulation optical elements (e.g., corrugated plates), acousto-optic deflectors, modulators or filters, electro-optic modulators, dichroic mirrors, spectral or neutral density filters, etc. Each optical element defining an optical beam path is positioned within the beam path created by the optical elements located upstream of it and defines the immediate portion of the optical beam path downstream of it.

[0032] The AO deflector itself can be understood to be an optical element that shapes the light beam path by being positioned within the beam path defined by other optical elements located upstream of the AO deflector.

[0033] It should be noted that the laser beam entering the focusing lens system is, from a scanning perspective, only the "useful" part of the laser beam leaving the laser source. This is because the original laser beam is attenuated by the time it reaches the rear aperture of the focusing lens system for several reasons. For example, for technical reasons, it may be necessary to split off part of the laser beam, for example by using a beam splitter. By definition, the beam path of the split laser beam is no longer part of the optical beam path between at least one laser source and the focusing lens system.

[0034] Another reason why the useful portion of the laser beam does not coincide with the laser beam exiting the laser source is losses that occur during the operation of the AO deflector. Deflection in an AO deflector operates on the principle of diffraction, and AO deflectors are typically optimized to significantly increase the intensity of the beam belonging to the +1 or -1 diffraction order compared to other diffraction orders, making this diffraction order optimally usable for scanning. Other diffraction orders (including the undeflected zeroth diffraction order) can be separated by filters operating on polarization or spatial filtering principles or by the spatial arrangement of the AO deflector. By definition, separated orders do not belong to the "useful" beam from a scanning perspective. For this reason, AO deflectors are defined not through their effect on the laser beam entering the AO deflector, but through their effect on the laser beam exiting the focusing lens system. For example, no AO deflector will deflect the portion of the laser beam that exits as the zeroth diffraction order. These considerations are obvious to those skilled in the art, and are used and presented, for example, in the references and patent documents mentioned above. See, for example, WO 2008 / 032061 A2 and WO 2010 / 076579 A1.

[0035] Parameter s X1 , s X2 , s Y1 , s Y2 The value of s is +1 or -1, which is selected depending on the direction of deflection that the deflector causes in the focal plane of the lens (e.g., in the sense of how the focal spot is deflected in the XY plane as a result of a 1 MHz increase in frequency). For example, if the first AO deflector (X1) and the second AO deflector (X2), which deflect in the XZ plane, change the X coordinate of the focal spot under the objective lens in opposite senses in response to a 1 MHz increase in the frequency of the drive signal that drives them, then s X1 and s X2 The sign of s should also be chosen to be opposite. For example, s X1 =1 and s X2 = -1, or vice versa, s X1 =-1 and s X2However, if the deflectors X1 and X2 cause the X coordinate of the focal spot to change with the same sign as the frequency of the drive signal increases, then s X1 and s X2 The signs of the s must also be the same. For example, s X1 = s X2 =1, or s X1 = s X2 = -1. It will be clear to one skilled in the art how, for the deflectors used in a given arrangement, the signs must be chosen so that the individual deflectors in a deflector pair move the focal spot in opposite directions in the XY plane.

[0036] The time T is 0.5 T acc and 1.5 T acc Between 0.75 T and 1.0 T acc and 1.25 T acc is selected to be between

[0037] The disk may be circular or elliptical depending on whether the radius Rx and the radius Ry are the same or different. Preferably, both the radius Rx and the radius Ry are between 2 μm and 20 μm, for example between 5 μm and 10 μm.

[0038] Multiplier f ampX The value of is selected so that the radius Rx has a first value and the multiplying factor f ampY The value of is chosen such that the radius Ry has a second value. The first and second values ​​may be the same (this is the case when the disk is circular).

[0039] If the center of the disk is more than 50 μm away from the focal plane of the objective lens (assuming an objective lens with an effective focal length of 10 mm), the disk is scanned in multiple cycles by scanning a different part of the disk periphery during each cycle's intermediate time window of the cycle time. The intermediate time window of the cycle time can be understood as the time interval between the first third and the last third of the cycle time. To ensure that the intermediate time window of the cycle time is at a different part of the disk periphery, the starting point of the scan can be shifted along the disk periphery by appropriately selecting the phase shift φ as described above.

[0040] The laser scanning microscope is preferably a two-photon microscope.

[0041] The invention also relates to the use of the method according to the invention for scanning a cell body (soma), by selecting the center of a disk with coordinates x, y, z to coincide with the center of the cell body, and by adjusting the multiplying factor f so that the value of the radius Rx is equal to half the diameter of the cell body along the X axis. ampX and the multiplying factor f so that the value of the radius Ry is equal to half the diameter of the cell body along the Y axis. ampY The value of

[0042] The invention further relates to a laser scanning microscope as claimed in claim 11. Further preferred embodiments of the invention are defined in the dependent claims.

[0043] Further details of the invention will now be described, by way of example only, with reference to the following drawings, in which: [Brief explanation of the drawings]

[0044] [Figure 1] FIG. 1 is a schematic diagram of an example embodiment of a laser scanning microscope according to the present invention. [Figure 2]Figure 2 shows the measurement results of scanning an exemplary disk with submicron-diameter fluorescent beads, where the scanning time of the disk is three times the filling time of the deflector (T = 3 · Tacc). [Figure 3] FIG. 3 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is twice the filling time of the deflector (T=2·Tacc). [Figure 4] FIG. 4 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 1.8 times the deflector filling time (T=1.8·Tacc). [Figure 5] FIG. 5 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 1.6 times the deflector filling time (T=1.6·Tacc). [Figure 6] FIG. 6 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 1.4 times the deflector filling time (T=1.4·Tacc). [Figure 7] FIG. 7 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is the same as the filling time of the deflector (T=Tacc). [Figure 8] FIG. 8 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 0.75 times the deflector filling time (T=0.75·Tacc). [Figure 9] FIG. 9 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 0.5 times the deflector filling time (T=0.5·Tacc). [Figure 10] FIG. 10 shows the measurement results of scanning an exemplary disk, where the scanning time of the disk is 0.25 times the deflector fill time (T=0.25·Tacc). [Figure 11] FIG. 11 shows the measurement results of scanning an exemplary disk, where the scan time of the disk is 0.125 times the deflector fill time (T=0.125·Tacc). [Figure 12] 12a-12c show further exemplary scans of a circle where the center of the circle is not in the focal plane of the objective lens. DETAILED DESCRIPTION OF THE INVENTION

[0045] FIG. 1 shows a schematic diagram of one embodiment of a two-photon laser scanning microscope 10 according to the present invention. In this embodiment, the microscope 10 is a two-photon microscope with a single laser source 12; however, microscopes 10 with multiple laser sources 12, capable of generating laser beams of different wavelengths, are also known. The microscope 10 has a focusing lens system that defines an optical Z-axis, which in this example is a microscope objective 14. Between the laser source 12 and the microscope objective 14 is an optical beam path 16, shown by a dashed line in FIG. 1, that runs from the exit aperture of the laser source 12 to the rear aperture 14a of the microscope objective 14. The optical beam path is created by various optical elements, which in this embodiment are a mirror 20, a beam manipulator 22, a lens 24, a telecentric relay 26, and a beam splitter 28, all of which are shown in FIG. 1. The beam manipulator 22 can include, for example, a Faraday isolator, a dispersion compensation module, a beam stabilizer, and a beam expander, which are individually shown, for example, in Patent Document No. WO2016 / 079547A1 and are well known from the state of the art. The microscope 10 also includes one or more detectors 30, which in this example are a photomultiplier tube (PMT) detector 31 and a CCD camera 32. A beam splitter 28 is used to direct the fluorescence emitted from the sample and passing through the objective lens 14 toward the detector 30. Of course, in addition to these, other known optical elements can also be used to create the light beam path 16.

[0046] First and second acousto-optic deflectors X1 and X2 are disposed in the optical beam path 16 and are used to deflect the laser beam 18 exiting the objective lens 14 in the XZ plane defined by the optical Z axis and an X axis perpendicular to the optical Z axis. Additionally, third and fourth acousto-optic deflectors Y1 and Y2 are disposed in the optical beam path 16 to deflect the laser beam 18 exiting the objective lens 14 in the YZ plane defined by the optical Z axis and a Y axis perpendicular to the optical Z axis and the X axis. The laser beam 18 is focused by the objective lens 14 to a focal point 19, which is actually a single focal spot, but in the case of the method of the present invention, is simultaneously a single focal spot and multiple satellite spots.

[0047] The X and Y axes are preferably defined in the focal plane of the objective lens 14, such that the origin of the coordinate system defined by the X, Y and Z axes also lies in the focal plane.

[0048] Each of the deflectors X1, X2, Y1, and Y2 includes an AO crystal 40 and a transducer 42 disposed at one end thereof. In the arrangement shown in Figure 1, the transducers 42 are located at the same end of the AO crystal 40 for deflectors X1 and X2 and at the same end of the AO crystal 40 for deflectors Y1 and Y2, so that the generated acoustic waves propagate in essentially the same direction within deflectors X1 and X2 and also within deflectors Y1 and Y2.

[0049] The microscope 10 further includes a drive system 50 that drives a region of the disk that lies in a plane perpendicular to the XY plane defined by the X and Y axes, with a frequency drive signal function f for each of the first, second, third, and fourth acousto-optic deflectors, defined as X1 (t), f X2 (t), f Y1 (t), f Y2 (t) is configured to scan by generating: TIFF2026502149000010.tif6150 TIFF2026502149000011.tif6150 TIFF2026502149000012.tif6150 TIFF2026502149000013.tif6150 In the above formula, f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 (t) is the frequency drive signal term for focusing the laser beam in random access point scanning mode at the center of the disk defined by coordinates x, y, z, and Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) is the frequency drive signal term that determines the outer circumference of the disk, and is defined by the following equations: TIFF2026502149000014.tif9150 TIFF2026502149000015.tif9150 TIFF2026502149000016.tif9150 TIFF2026502149000017.tif9150 where s X1 , s X2 , s Y1 , s Y2 is chosen between +1 and -1, and f ampx is a first multiplying factor that determines the first radius Rx of the disk along the X axis, and f ampY is the second multiplying factor that determines the second radius of the disk along the Y axis, φ is the arbitrary phase shift, and T is the time required to scan the disk, which has a value of 0.25 T acc From 1.6 T acc Between now and T acc is the fill time of the deflector.

[0050] Deflector filling time T acc is T acc=H / V, where H is the diameter of the laser beam 17 entering the first acousto-optic deflector X1 downstream of the laser source 12 (which is different from the diameter of the laser beam 18 exiting the objective lens 14, as mentioned above), because the diameter of the deflector X1 is larger. V is the acoustic phase velocity in the AO crystal 40 of the acousto-optic deflector X1. It should be noted that in this embodiment, the four acousto-optic deflectors X1, X2, Y1, and Y2 have the same structure.

[0051] In this embodiment, all four acousto-optic deflectors X1, X2, Y1, and Y2 are optimized for the −1 diffraction order. Therefore, in the arrangement shown in FIG. X1 =-1 and s X2 =1, and s Y1 =-1 and s Y2 A value of =1 is used and the phase shift φ is chosen to be 0. Substituting this into the above equation gives the following Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) frequency drive signal term is obtained: TIFF2026502149000018.tif9150 TIFF2026502149000019.tif9150 TIFF2026502149000020.tif9150 TIFF2026502149000021.tif9150

[0052] f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 How to choose the term (t) to determine Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2It is well known to those skilled in the art how, in the absence of the (t) term, the former term will create a stationary focal spot at the centre of the disc with coordinates x, y, z (this is the random access point scanning mode, see for example WO2006 / 042130A2).

[0053] The frequency drive signal term Δf defines the outer periphery of the disk X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) theoretically moves the focal point 19 around a central point with coordinates x, y, z along the periphery of a disk defined by radii Rx and Ry. ampX = f ampY In the case of , the disk is circular (Rx = Ry) and f ampX ≠f ampY In this case, the disk is elliptical (Rx ≠ Ry).

[0054] The scanning time T of the disk is equal to the filling time T of the deflectors X1, X2, Y1, and Y2. acc , several satellite spots appear in the area of ​​the disk next to the focal point 19, the intensity of which is already high enough to cause the simultaneous absorption of several photons required for the excitation of fluorophores. In this way, it is possible to simultaneously excite fluorophores located not only at the periphery of the disk but also in the interior of the disk, and the resulting emitted fluorescence photons can also be detected simultaneously. An advantage of the method according to the invention is that the interior area of ​​the disk is automatically scanned at the same time as the periphery of the disk is scanned, so that a separate scan of the interior of the disk is not necessary. Since several satellite spots appear in the area of ​​the disk (towards the center of the disk), a suitable multiplying factor f ampX and f ampY The scanned area can be easily determined by selecting

[0055] In the following, we will present the appearance of satellite spots used for area scanning through measurements taken with the microscope 10 shown in FIG.

[0056] A series of measurements shown in Figures 2 to 11 show the dependence of the resulting scan pattern on the disk scan time. For better illustration, a circular disk was scanned in these measurements. The wavelength of the laser was 920 nm, the acoustic phase velocity was 705 m / s, the effective focal length of the objective lens 14 was 9 mm, and the diameter of the beam entering the first deflector X1 was 15 mm, which resulted in a fill time of T acc =21.3μs. The multiplying factor is f ampX = f ampY =0.2123 MHz. This value was obtained by scanning a circle with a radius of about 5 μm. To simplify the measurement, the center of the circle was at the point with coordinates x=y=z=0. This means that f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 This was achieved by choosing the value of the (t) term to correspond to the center frequency of the frequency band. Measurements were performed on submicron (170 nm in this example) fluorescent polystyrene beads, a standard test target for two-photon microscopes. The measurements shown in each figure were obtained for different scan times.

[0057] In the measurement in Figure 2, the circular scanning time is T = 3 T acc The pattern is a circular ring on which small horizontal stripes appear due to mechanical vibration of the sample. The thickness of the ring is approximately 0.5 μm, in accordance with the resolution of the microscope.

[0058] In the measurement in Figure 3, the circular scanning time is T = 2 T acc The pattern is similar to the previous one, essentially a single ring, without any substantial artifacts.

[0059] In the measurement in Figure 4, the circular scanning time is T = 1.8 T acc Here the ring is slightly blurred, but the intensity of the satellite spots is still too small to cause a significant two-photon effect.

[0060] In the measurement in Figure 5, the circular scanning time is T = 1.6 T acc At this scan rate, internal "order" begins to emerge (although it is so faint that it is not clearly visible in the black and white image). In this case, significant two-photon absorption occurs within the circular ring, and emitted fluorescence photons are also detected from within the circle by detector 30.

[0061] In the measurement in Figure 6, the circular scanning time is T = 1.4 T acc The previously distinct circular ring can be seen to begin to morph into a square resting on one of its vertices, revealing additional "order" within the circle. More and more fluorescent information is coming from the region within the circular ring.

[0062] In the measurement in Figure 7, the circular scanning time is T = T acc The scanning time of the circle, T, is the filling time, T acc When it reaches this point, the number of satellite spots increases, causing the center of the circle to be illuminated and clearly visible knots to appear along the outer edge of the circle.

[0063] In the measurement in Figure 8, the circular scanning time is T = 0.75 T acc In this case, the intensity of the focal spot scanning the circumference decreases while the intensity of the satellite spots appearing around the center of the circle increases, resulting in the area around the center of the sample being the brightest.

[0064] In the measurement in Figure 9, the circular scanning time is T = 0.5 T acc By further increasing the scanning speed, the knots in the circular ring become increasingly clearly defined, so that less light reaches the "fuzzy" intermediate areas between the knots, but the circle is still nearly entirely illuminated.

[0065] In the measurement in Figure 10, the circular scanning time is T = 0.25 T accAt this scanning speed, the center appears dark again and the knots turn into multiple distinct dots that form a dot grid, but still illuminate an area of ​​the circular disk.

[0066] In the measurement in Figure 11, the circular scanning time is T = 0.125 T acc In this case, the frequency modulation required to create the circle is too rapid to have any significant effect, resulting in a bright center, the complete disappearance of the satellite spots, and the focal spot appearing undeflected at the center of the circle (the point determined by the coordinates x=y=z=0 in this example).

[0067] The scanning pattern shown above is a conventional random access point scanning mode where a point with arbitrary x, y, z coordinates is scanned using f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 It can also be obtained by targeting the term (t). In this case, the term that draws the circle, Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) deflects the focal spot around a circle, and the point in question will be the center of this circle. By shortening the time T for scanning the circle, i.e., by increasing the scanning speed, a similar pattern can be observed around the center with coordinates x, y, and z. ampX ≠f ampY , the pattern becomes an ellipse instead of a circle, and its minor and major axes are multiplied by two factors, f ampX and f ampY has the same ratio as the ratio of

[0068] If the frequency of the signal driving the deflector is changed by Δf, the angle of the diffracted beam will change by: TIFF2026502149000022.tif9150 where λ is the wavelength of the laser beam and V is the acoustic phase velocity in the acousto-optic crystal.

[0069] As a result, the effective focal length F obj The displacement of the focal spot in the focal plane of the objective lens 14 having (assuming the absolute value of the effective lateral magnification of the intermediate optics, e.g., relay, is 1) varies as follows: TIFF2026502149000023.tif9150 In the case of disk scanning, for example, deflectors X1 and X2 deflect by the same absolute amount, and they are added (although the signs of the modulation terms are opposite, because there is also a relay with a -1 multiplication factor between deflectors X1 and X2). Therefore, instead of Δf, we have f amp and multiplying the displacement by two, the predicted radius of the circle is: TIFF2026502149000024.tif9150 For example, for the parameters applied in Figures 1-11, λ = 920 nm, V = 705 m / s, f amp =0.2123 MHz, F obj = 9 mm and the radius of the scanned circle is R = 4.98 μm.

[0070] For an ellipse, the above formula will result in different radii along the X and Y axes.

[0071] Note that the size of this radius becomes more and more "nominal" as the focal spot moves faster and faster around the circle (ellipse) and the satellite spots fill more and more of the interior of the disk.

[0072] When scanning a larger disk, for example with a radius of 10-30 μm, on the one hand, the RF power, which determines the diffraction efficiency of the deflectors X1, X2, Y1, Y2, must be increased in order to be able to compensate for the loss of intensity due to the scanning of a larger circle, and on the other hand, in order to scan a circle with a larger diameter, the deflection of the beam by the first pair of deflectors must be made to a greater extent, and this more deflected beam will be diffracted less by the second pair of deflectors (due to the fixed acceptance angle of the deflectors).

[0073] The inventors have found that the size of the scannable circle (or ellipse) is limited only by the RF power, which determines the diffraction efficiency of the deflectors X1, X2, Y1, Y2, other than the limitations imposed by the bandwidth and acceptance angle of the deflectors X1, X2, Y1, Y2 (which also limit the field of view).

[0074] 12a-12c show the displacement of the scanned disk along the Z axis, i.e., when the disk (and its center) is not in the focal plane. Note that the focal plane is always a plane parallel to the XY plane, and it is desirable to select a coordinate system such that the focal plane coincides with the XY plane.

[0075] For simplicity, these measurements were also for the scan of a circle, the centre of which was at a point with coordinates of the form x=y=0 and z≠0.

[0076] The following measurements were also performed on submicron beads and the following parameters: ampX = f ampY = f amp = 0.2 MHz is used, and the scan time is T = T acc The laser wavelength was 920 nm and the beam diameter was 15 mm.

[0077] The measurement shown in Figure 12a gave a z coordinate of z = 50 μm (the origin of the coordinate system was located in the focal plane), Figure 12b shows the measurement at z = 100 μm, and Figure 12c shows the measurement at z = 150 μm.

[0078] To shift the circle center more and more along the Z axis, as is well known in random access point scanning mode, it is necessary to create increasingly larger beam deflections with each deflector. This causes the deflected beam to strike deflectors X2 and Y2 at angles different from the optimal angle, thus reducing the diffraction efficiency. A larger shift of the circle center along the Z axis results in a reduction in the effective aperture and therefore a reduction in beam intensity, resulting in less brightness inside the circle while increasing the depth of focus. On the other hand, as the deflection along the Z axis increases, the effective time window during which there is still significant excitation becomes shorter. This is because deflectors X2 and Y2 receive the incident beam at angles closer to the optimal angle for less and less time, and therefore a larger portion of the circle becomes dark. While this effect is not noticeable at z=50 μm, it can be observed that at z=100 μm the upper quarter of the circle is dark, and at z=150 μm half of the circle is dark. This effect can be eliminated as follows. That is, if the center of the disk is more than 50 μm from the objective lens focal plane, multiple scans of the disk are performed, with each scan cycle scanning a different portion of the disk's circumference during an intermediate time window within the cycle time. This is achieved by shifting the phase of the cosine or sine modulation so that the circular (or elliptical) scan begins at a different rotational position in each cycle, allowing the entire circle to be scanned in multiple cycles, so that the dark areas are always in different locations. The cycle time is preferably the same as the AO deflector cycle time. For example, assuming a beam diameter of 15 mm, a typical AO deflector cycle time is approximately 20–30 μs, although longer cycle times are possible for some applications.

[0079] In fact, it is noteworthy that this method allows for much faster scanning of the disk than prior art methods. For a shift of ±150 μm along the Z axis, scanning a disk approximately 10-20 μm in diameter within a 300 μm thick area requires only two AO deflector cycle times, compared to 10-20 or more cycle times that would be required with conventional drift scanning techniques.

[0080] The same situation applies to scanning an ellipse shifted along the Z axis.

[0081] When the center of the disk was shifted parallel to the XY plane, no such phenomenon was experienced.

[0082] From the perspective of practical application, the method according to the present invention provides a good solution for simultaneous stimulation of, for example, cell bodies (i.e., scanning of cell bodies). Of course, instead of cell bodies, other small biological objects (ideally biological objects with a diameter of less than 20 μm) can also be scanned, possibly together with their surroundings (ideally, the biological object with its surroundings does not exceed an area of ​​20 μm in diameter). The latter may be necessary due to motion artifacts, as described, for example, in patent application WO 2018 / 042214 A2 and in "Fast 3D Imaging of Spine Columns, Dendrites, and Neuronal Aggregates in Behavioral Animals" (Szalay et al., 2016, Neuron 92, 723-738).

[0083] The method of the present invention allows for extremely rapid, nearly simultaneous scanning of multiple spatially dispersed measurement regions (regions of interest) with diameters of 20 μm or less. This is because each measurement region can be scanned during a single AO cycle. Therefore, n regions (n>1, n>5, or n>10, but preferably n<100) can be scanned within a distance of 50 μm from the objective focal plane within n times the AO cycle time. Measurement regions at distances of 50 to 150 μm from the objective focal plane can be scanned independently within a maximum of two AO cycle times. Even at larger distances, scanning each measurement region requires only a few AO cycles. Considering that the AO cycle time for a typical beam diameter of 15 mm is typically about 30 μs, a 20 μm x 20 μm region can be scanned within one to two cycle times, i.e., 30 to 60 μs, depending on the measured distance from the objective focal plane. Up to 10 measurement regions can be scanned in a few hundred μs. In contrast, a state-of-the-art minidrift takes approximately 20 x 30 μsec = 600 μsec to scan a single 20 μm x 20 μm area as described in the prior art.

[0084] Another possible application, e.g., for in vivo measurements, is to scan dendritic spikes by scanning a disk that covers the dendritic spikes. This ensures that the dendritic spikes remain within the scanned disk even if the living sample (e.g., animal) moves during the scan. For example, this can be achieved by scanning a disk (preferably a circular disk) with a diameter of about 5 μm.

[0085] It will be obvious to those skilled in the art that various modifications to the above disclosed embodiments can be conceived without departing from the scope of protection determined by the appended claims. [Explanation of symbols]

[0086] 10: Two-photon laser scanning microscope 12: Laser source 14: Microscope objective lens 16: Light beam path 17, 18: Laser beam 19:Focus 20: Mirror 22: Beam manipulator 24: Lens 26: Telecentric relay 28: Beam splitter 30: Detector 40:AO crystal 42: Transducer 50: Drive system X1,X2,Y1,Y2: Acousto-optic deflector

Claims

1. 1. A method of scanning an area with a multiphoton laser scanning microscope comprising: at least one laser light source and a focusing lens system defining an optical Z-axis, an optical beam path between the at least one laser light source and the focusing lens system; and further comprising: first and second acousto-optic deflectors disposed in the optical beam path for deflecting the laser beam exiting the focusing lens system in an X-Z plane defined by the optical Z-axis and an X-axis perpendicular to the optical Z-axis; and third and fourth acousto-optic deflectors disposed in the optical beam path for deflecting the laser beam exiting the focusing lens system in a Y-Z plane defined by the optical Z-axis and a Y-axis perpendicular to the optical Z-axis and the X-axis, the method comprising: The disk, which is located in a plane perpendicular to the X-Y plane defined by the X-axis and the Y-axis, is scanned by guiding the laser beam from the laser light source to the focusing lens system along the optical beam path, and during the scanning, the first, second, third and fourth acousto-optic deflectors are respectively supplied with frequency drive signal functions f defined by the following equations (1) to (4): X1 (t), f X2 (t), f Y1 (t), f Y2 (t) is assigned: where f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 (t) is a frequency drive signal term for focusing the laser beam in a random access point scanning mode on the center of the disk having coordinates x, y, z, and Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) is a frequency drive signal term that determines the outer circumference of the disk, and is defined by the following equations (5) to (8), respectively: where s X1 , s X2 , s Y1 , s Y2 is chosen between +1 and -1, and f ampX is a first multiplying factor that determines a first radius Rx of the disc along the X-axis, and f ampY is a second multiplying factor that determines a second radius Ry of the disc along the Y axis, φ is an arbitrary phase shift, T is the time required to scan the disc, and The value of the time T is set to 0.25.T acc From 1.6 T acc Choose a time between t and t, where t is the time acc is the fill time of the deflector.

2. 2. The method of claim 1, wherein the value of the time T is 0.5·T acc From 1.5T acc Between, preferably 0.75 T acc From 1.25 T acc The selection is characterized in that the value is selected to be between

3. 3. A method according to claim 1 or 2, characterized in that the disc is a circle or an oval.

4. 4. A method according to claim 3, characterized in that both the radius Rx and the radius Ry are between 2 μm and 20 μm, preferably between 5 μm and 10 μm.

5. 5. The method of claim 3, wherein the multiplying factor f is adjusted so that the radius Rx has a first value. ampX and select a value for the multiplying factor f so that the radius Ry has a second value. ampY The value of

6. 6. The method according to claim 5, wherein the value of the radius Rx is the same as the value of the radius Ry.

7. 7. A method according to any one of claims 1 to 6, characterized in that the centre of the disc is at a distance of 50 μm or more from the focal plane of the objective lens, and the disc is scanned in multiple cycles, each cycle scanning a different part of the outer periphery of the disc during an intermediate time window within the cycle time of that cycle.

8. 8. The method according to any one of claims 1 to 7, characterized in that the laser scanning microscope is a two-photon microscope.

9. 9. Use of the method according to any one of claims 1 to 8 for scanning a region of interest in a biological sample, wherein the center of the disk having coordinates x, y, z is selected to coincide with the center of the region of interest, and the multiplying factor f ampX is selected so that the value of the radius Rx is equal to half the diameter of the region of interest along the X-axis, and the multiplying factor f ampY The value of Ry is selected so that the value of the radius Ry is equal to half the diameter of the region of interest along the Y axis.

10. 10. A method according to claim 9, characterized in that a plurality of regions of interest are scanned successively, the centers of which lie in different planes parallel to the XY plane.

11. 1. A multiphoton laser scanning microscope comprising at least one laser light source and a focusing lens system defining an optical Z-axis, with an optical beam path between the at least one laser light source and the focusing lens system, the microscope further comprising first and second acousto-optic deflectors disposed in the optical beam path for deflecting a laser beam exiting the focusing lens system in an X-Z plane defined by the optical Z-axis and an X-axis perpendicular to the optical Z-axis, and third and fourth acousto-optic deflectors disposed in the optical beam path for deflecting a laser beam exiting the focusing lens system in a Y-Z plane defined by the optical Z-axis and a Y-axis perpendicular to the optical Z-axis and the X-axis, the multiphoton laser scanning microscope comprising a drive system comprising: The first, second, third and fourth acousto-optic deflectors are configured to scan a disk located in a plane perpendicular to an X-Y plane defined by the X-axis and the Y-axis, and are provided with a frequency drive signal function f defined by the following equations (1) to (4), respectively: X1 (t), f X2 (t), f Y1 (t), f Y2 A drive system configured to generate (t): where f 0X1 (t), f 0X2 (t), f 0Y1 (t), f 0Y2 (t) is a frequency drive signal term for focusing the laser beam in a random access point scanning mode on the center of the disk having coordinates x, y, z, and Δf X1 (t), Δf X2 (t), Δf Y1 (t), Δf Y2 (t) is a frequency drive signal term that determines the outer circumference of the disk, and is defined by the following equations (5) to (8), respectively: where s X1 , s X2 , s Y1 , s Y2 is chosen between +1 and -1, and f ampX is a first multiplying factor that determines a first radius Rx of the disc along the X-axis, and f ampY is a second multiplying factor that determines a second radius Ry of the disc along the Y axis, φ is an arbitrary phase shift, T is the time required to scan the disc, and 0.25 T acc From 1.6 T acc where T acc is the fill time of the deflector.

12. 12. The multiphoton laser scanning microscope according to claim 11, wherein the value of the time T is 0.5·T acc From 1.5 T acc Between, preferably 0.75 T acc From 1.25 T acc The selection is characterized in that the value is selected to be between

13. 13. A multiphoton laser scanning microscope according to claim 11 or 12, characterized in that the disk is circular or elliptical.

14. A multiphoton laser scanning microscope according to claim 13, characterized in that both the radius Rx and the radius Ry are between 2 μm and 20 μm, preferably between 5 μm and 10 μm.

15. 15. A multiphoton laser scanning microscope according to claim 13, wherein a multiplying factor f is set so that the radius Rx has a first value. ampX and select a value for the multiplying factor f so that the radius Ry has a second value. ampY The value of

16. 16. The multi-photon laser scanning microscope according to claim 15, wherein the radius Rx and the radius Ry have the same value.

17. 17. A multi-photon laser scanning microscope according to claim 11, wherein the laser scanning microscope is a two-photon microscope.

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