Scanning microscope

Abstract

To reduce luminance unevenness that may occur in a generated image even when a scanning microscope has high resolution and high speed.SOLUTION: A scanning microscope samples light from a sample irradiated with pulsed light, and converts it into data for each pixel, where a sampling time per pixel is approximately an integer multiple of a pulse period of the pulsed light, and sampling start timing for each pixel is synchronized with a pixel synchronization signal.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a scanning microscope. [Background technology]

[0002] Conventionally, there is known a microscope that samples fluorescence in synchronization with the emission timing of laser light periodically emitted from a light source such as a pulsed laser (see, for example, Patent Document 1). This microscope can reliably sample a fluorescence signal including a peak. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2001-159734 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, even when sampling of fluorescence is performed in synchronization with the timing of laser light emission, the sampling time per pixel forming an image varies depending on the observation conditions (e.g., observation range, scanning speed, image resolution, etc.), and the emission cycle of the laser light depends on the light source. Therefore, it becomes difficult to sample pixels in synchronization with the emission cycle of the laser light, and the number of times the laser light is emitted varies for each pixel. As a result, even if the phosphor is homogeneous and uniform, the brightness varies for each pixel, causing a problem that brightness unevenness occurs in the generated image. In particular, the shorter the sampling time per pixel, the more prominent this problem becomes.

[0005] The direction of improving the performance of laser scanning microscopes is toward higher resolution and higher speed (higher frame rate). Both of these trends are aimed at shortening the sampling time per pixel. On the other hand, the emission period of laser light from the ultrashort pulse laser used in multiphoton excitation observation is determined by the ultrashort pulse laser, and cannot be changed arbitrarily. Therefore, when the sampling time per pixel is shortened and the number of times that laser light is emitted per pixel decreases, the brightness will fluctuate significantly even if the number of times that laser light is emitted per pixel differs by just one time. Since both the emission of laser light and the sampling of pixels are repeated at a certain frequency, a beat of the difference frequency occurs in the generated image, which can cause brightness unevenness (e.g., stripe noise) that interferes with observation.

[0006] An object according to one aspect of the present invention is to provide a technique capable of reducing uneven brightness that may occur in a generated image even when the resolution and speed of a scanning microscope are increased. [Means for solving the problem]

[0007] A scanning microscope according to one embodiment of the present invention is a scanning microscope that samples light from a sample irradiated with pulsed light and converts it into data for each pixel, wherein the sampling time per pixel is approximately an integer multiple of the pulse period of the pulsed light, and the sampling start timing of each pixel is synchronized with a pixel synchronization signal.

[0008] A scanning microscope according to another aspect of the present invention includes a laser light source that emits pulsed light, a scanner that scans a sample two-dimensionally with the pulsed light, a photodetector that detects light from the sample, and a control device that generates pixel data for each pixel that constitutes an image of the sample based on an output signal from the photodetector and a pixel synchronization signal synchronized with the scanning position of the scanner, sets the sampling time per pixel to approximately an integer multiple of the pulse period of the pulsed light, and synchronizes the sampling start timing of each pixel with the pixel synchronization signal. Effect of the Invention

[0009] According to the above-described aspect, even if the resolution and speed of the scanning microscope are increased, it is possible to reduce uneven brightness that may occur in the generated image. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 illustrates an example of a configuration of a scanning microscope according to an embodiment. [Diagram 2] 4 is a timing chart illustrating a pulsed laser beam, excited fluorescent light, and each signal. [Diagram 3] 11 is a timing chart illustrating each signal when the scanner is a resonant scanner. [Figure 4] 4 is a timing chart illustrating a pulsed laser beam and excited fluorescent light. [Diagram 5] FIG. 2 is a diagram illustrating an example of a hardware configuration of a computer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0012] FIG. 1 is a diagram illustrating a configuration of a scanning microscope according to an embodiment.

[0013] The scanning microscope 1 illustrated in FIG. 1 includes a laser light source device 10, a microscope body 20, and a control device 30.

[0014] The laser source device 10 includes a laser source 11, a light adjustment unit 12, and a scanner 13.

[0015] The laser light source 11 emits a pulsed laser beam (pulsed light). More specifically, the laser light source 11 emits a laser beam whose intensity changes so as to peak at a predetermined period. The laser light source 11 also outputs a laser pulse signal synchronized with the emission of the laser beam to the control device 30. The laser light source 11 is a pulsed laser such as a pulsed TiSa (titanium sapphire) laser, a high frequency superimposed LD (Laser Diode), or the like.

[0016] The light adjustment unit 12 adjusts the intensity of the laser light emitted from the laser light source 11 according to an instruction from the control device 30. The light adjustment unit 12 is an acousto-optical element, a shutter, a neutral density filter, or the like. The adjustment of the intensity of the laser light may be performed by controlling the drive current of the laser light source 11 by the control device 30. In this case, the light adjustment unit 12 can be omitted.

[0017] The scanner 13 scans the laser light modulated by the dimming unit 12. The scanner 13 has a pair of galvanometer mirrors 13a and 13b that are, for example, aluminum-coated, and is driven in a raster scan manner by changing the angles of the galvanometer mirrors 13a and 13b in response to instructions from the control device 30. This allows the laser light from the laser light source 11 to be two-dimensionally scanned on the sample (specimen, specimen) S. The scanner 13 may be such a galvanometer scanner, a resonant scanner such as a resonant scanner, an acousto-optical deflection element, or the like.

[0018] The microscope body 20 includes a lens 21 , a dichroic mirror 22 , a lens 23 , an objective lens 24 , and a photodetector 25 .

[0019] The lenses 21 and 23 relay the laser light from the laser light source device 10 to the objective lens 24. The objective lens 24 irradiates the sample S with the laser light, while collecting the light from the sample S. The light from the sample S is, for example, fluorescence generated in the sample S when excited by irradiation of the sample S with the laser light.

[0020] The dichroic mirror 22 reflects the laser light from the laser light source device 10 toward the objective lens 24, while transmitting the light from the sample S focused by the objective lens 24, thereby branching the optical path of the laser light from the optical path of the light from the sample S.

[0021] The photodetector 25 detects light from the sample S that has passed through the dichroic mirror 22. More specifically, the photodetector 25 includes a photoelectric conversion element 251 and a photodetection circuit 252. The photoelectric conversion element 251 photoelectrically converts the light from the sample S that has passed through the dichroic mirror 22, and outputs an electrical signal according to the luminance of the light. The photoelectric conversion element 251 is a PMT (Photo Multiplier Tube), an MPPC (Multi-Pixel Photon Counter), or the like. The photodetection circuit 252 A / D converts the output signal of the photoelectric conversion element 251 in synchronization with a sampling clock, and outputs the converted signal. It is assumed that the period of the sampling clock is equal.

[0022] The control device 30 controls the laser light source device 10 and the microscope body 20. In addition, the control device 30 performs operations such as generating pixel data (brightness data) of each pixel constituting the image of the sample S from the output signal of the photodetector 25 (output signal of the photodetection circuit 252), as will be described in detail later.

[0023] A PC (Personal Computer) 40 to which a monitor 50 such as a liquid crystal display is connected is connected to the control device 30. The PC 40 generates image data for the sample S based on pixel data for each pixel generated by the control device 30 and image synchronization signals such as a horizontal synchronization signal and a vertical synchronization signal generated based on the movement of the scanner 13 that determines the scanning position on the sample S, and causes the monitor 50 to display an image of the sample S related to the generated image data.

[0024] The process by which the control device 30 generates pixel data for each pixel from the output signal of the photodetector 25 will now be described in more detail.

[0025] The control device 30 receives scanning conditions such as the scanning speed and resolution of the scanner 13 from the PC 40, calculates a pixel period indicating the period of one pixel based on the received scanning conditions, and generates a pixel synchronization signal that determines the start and end timings of the calculated pixel period. Note that the pixel period corresponds to the time during which the scanning position of the laser light on the sample S by the scanner 13 remains at a position equivalent to one pixel.

[0026] 2, the control device 30 sets the sampling time per pixel to an integer multiple (positive integer multiple) of the pulse period of the pulsed laser light emitted from the laser light source 11, and synchronizes the sampling start timing of each pixel with the pixel synchronization signal to generate pixel data for each pixel from the output signal of the photodetector 25. However, the sampling time per pixel is set to a time equal to or shorter than the pixel period (the period of the pixel synchronization signal).

[0027] 2 is a timing chart illustrating the pulsed laser light, the excited fluorescence, and each signal. From the top, the pulsed laser light emitted from the laser light source 11, the fluorescence excited in the sample S (light from the sample S), the pixel synchronization signal, the data sampling timing A, and the data sampling timing B are illustrated.

[0028] Data sampling timing A is the sampling time per pixel (T S ) is the pulse period of the pulsed laser light (T LD ) times the time (1T LD ), the sampling time for each pixel (T S ) (Note that in FIG. 2, the sampling clock for one pixel (T S (Only the sampling clock at ) is shown. The data sampling timing B is the sampling time per pixel (T S ) is the pulse period of the pulsed laser light (T LD ) times the time (3T LD ), the sampling time for each pixel (T S) (Note that in FIG. 2, the sampling clock for one pixel (T S In each data sampling timing, the sampling start timing (sampling time (T S The start timing of the sampling clock (T) is synchronized with the pixel synchronization signal (specifically, the rising edge of the pixel synchronization signal). Therefore, the sampling start timing of each pixel is determined based only on the pixel synchronization signal, without taking into consideration the emission timing of the pulsed laser light, etc. The sampling clock is a sampling clock for A / D conversion performed by the photodetection circuit 252, and its cycle is, for example, a sampling time (T S The period is set to be sufficiently short so as not to miss (miss sampling) the peak of the fluorescence generated at the sampling time (T S ), the signals A / D converted by the photodetection circuit 252 are captured and integrated (or averaged, etc.) to generate pixel data for each pixel.

[0029] For example, assume that the laser light source 11 is an ultrashort pulse laser that emits ultrashort pulse laser light of 80 MHz, and the pixel period is 100 ns. In this case, the pulse period (T LD ) is 12.5ns, so the optimal sampling time per pixel (T S ) is the pulse period of the ultrashort pulse laser light (T LD ) is 8 times the sampling time per pixel (T S ) is the pulse period of the ultrashort pulse laser light (T LD ), but to obtain more brightness, it is desirable to have a longer time. Therefore, here, the optimal sampling time per pixel (T S ) is the pulse period of the ultrashort pulse laser light (T LD ) and is the same as the pixel period of 100ns. S ) is, for example, the pixel period as the pulse period of the ultrashort pulse laser light (TLD ) is divided by the integer part of the result, the pulse period of the ultrashort pulse laser light (T LD ) multiplied by N.

[0030] In this case, if the frequency of the sampling clock is 960 MHz, the number of sampling times per pixel is 96. Therefore, in this case, the sampling time (T S ) 96 samplings (A / D conversions) are performed in the sampling time (T S The number of samplings in is the sampling time per pixel (T S ) and the sampling clock frequency.

[0031] In this case, the sampling clock frequency of 960 MHz may be, for example, a result of making the period of the original oscillation of the sampling clock the same as the pulse period (12.5 ns) of the ultrashort pulse laser light, and multiplying the original oscillation frequency (80 MHz) by 12. Alternatively, the period of the original oscillation of the sampling clock may be an integer multiple (e.g., 2 times) of the pulse period (12.5 ns) of the ultrashort pulse laser light, and multiplying (e.g., 24 times) the original oscillation frequency (e.g., 40 MHz).

[0032] 3 is a timing chart illustrating each signal when the scanner is a resonant scanner. From the top, the timing chart illustrates a sampling clock, a laser pulse signal, a pixel synchronization signal, and data sampling timing for A / D conversion performed by the light detection circuit 252.

[0033] As shown in FIG. 3, when the scanner 13 is a resonant scanner, the period of the pixel synchronization signal is irregular, so the sampling time per pixel (T S ) is the minimum period of the pixel synchronization signal (T min ) or less. However, even in this case, the sampling time per pixel (T S) is an integer multiple of the pulse period of the pulse laser light (the pulse period of the laser pulse signal), and the sampling start timing of each pixel (sampling time (T S The start timing of the pixel synchronization signal is still synchronized with the pixel synchronization signal. S ) is six times the pulse period of the pulsed laser light, and the minimum period of the pixel synchronization signal (T min ) time. The frequency of the sampling clock is three times the frequency of the pulsed laser light, so that the number of samplings (number of A / D conversions) per pixel is 18. Therefore, the sampling time (T S ) 18 samplings (A / D conversions) are performed.

[0034] The timing t shown in FIG. 3 is the sampling time for each pixel (T S ) shows the timing at which the laser pulse signal (more specifically, the rising edge of the laser pulse signal) is first detected. In this way, the sampling start timing of each pixel is independent of the laser pulse signal.

[0035] As described above, the sampling time per pixel may be an integer multiple of the pulse period of the pulsed laser light. This will be further explained with reference to Fig. 4. Fig. 4 is a timing chart illustrating the pulsed laser light and excited fluorescence.

[0036] As shown in FIG. 4, the generation of fluorescence from the sample S in response to the irradiation of the sample S with the pulsed laser light is a periodic phenomenon. For this reason, the sampling time per pixel is set to the pulse period (T LD ) (for example, 2T LD), then whatever the sampling start timing, the integrated value of the fluorescence generated at the sampling time will not differ, and even if it does differ, the difference will be minute. Therefore, it is possible to eliminate or reduce uneven brightness that may occur in the generated image, and there is no need to synchronize the sampling start timing with the pulse period of the pulsed laser light. However, as described above, the period of the sampling clock needs to be set to a sufficiently short period (e.g., about 1 ns for an 80 MHz pulsed laser light) so as not to miss the peak of the fluorescence generated at the sampling time.

[0037] As described above, according to this embodiment, by setting the sampling time per pixel to an integer multiple of the pulse period of the pulsed laser light, it is possible to eliminate or reduce brightness unevenness that may occur in a generated image. This is true even when the sampling time per pixel is shortened as the resolution and speed of a scanning microscope increases. In addition, since it is sufficient to synchronize the sampling start timing only with the pixel synchronization signal, there is no need to synchronize the sampling start timing with the pulse period of the pulsed laser light, and phase adjustment or the like for synchronizing the sampling start timing is also unnecessary.

[0038] In this embodiment, the sampling time per pixel is an integer multiple of the pulse period of the pulsed laser light, but in reality, the pulse period of the pulsed laser light varies slightly depending on the surrounding environment (e.g., ambient temperature) of the laser light source 11, and so the sampling time per pixel is approximately an integer multiple of the pulse period of the pulsed laser light. In detail, since the amount of variation in the pulse period of the pulsed laser light is 2 ns or less, the actual sampling time per pixel is an integer multiple of the pulse period of the pulsed laser light with an error of 2 ns or less. However, even if the actual sampling time per pixel is such a time, since the time from when the laser light is irradiated on the sample S to when the fluorescence is generated originally varies within a range of 2 ns or less, the above-mentioned effect of being able to eliminate or reduce uneven brightness that may occur in the generated image is not affected, and such an effect can still be obtained.

[0039] In this embodiment, pixel data is generated by integrating the signal sampled (A / D converted) during the sampling time per pixel. Alternatively, pixel data may be generated by integrating the output signal of the photoelectric conversion element 251 over the sampling time per pixel and A / D converting the result. In this case, the photodetection circuit 252 integrates the output signal of the photoelectric conversion element 251 over the sampling time per pixel and outputs a signal obtained by A / D converting the integration result. Then, the control device 30 uses the signal output from the photodetection circuit 252 as pixel data. Note that, in this case as well, the sampling start timing (integration start timing) of each pixel is synchronized with the pixel synchronization signal. In this way, it is possible to sample all the fluorescence generated during the sampling time per pixel.

[0040] In this embodiment, the sampling time per pixel is set to be equal to or less than the pixel period, but in the following cases, the sampling time per pixel may be set to be greater than the pixel period. For example, when the pulse period of the pulsed laser light is 12 ns and the pixel period is 35 ns, if the sampling time per pixel is set to 24 ns, which is twice the pulse period of the pulsed laser light, the time is less than the pixel period, but the obtained brightness is reduced. On the other hand, if the sampling time per pixel is set to 36 ns, which is three times the pulse period of the pulsed laser light, the time exceeds the pixel period, but the obtained brightness is increased. Therefore, when the sampling time per pixel is slightly longer than the pixel period, the sampling time per pixel may be set to be greater than the pixel period, taking into account the obtained brightness. However, in this case, since it is necessary to start processing the subsequent pixel while processing the previous pixel, parallel processing is performed.

[0041] In this embodiment, the pulse period of the pulsed laser light is in principle equal to or shorter than the pixel period, but if the pulse period of the pulsed laser light is longer than the pixel period, pixel data may be generated as follows. The sampling time per pixel is set to one time the pulse period of the pulsed laser light, and the sampling start timing of each pixel is synchronized with a pixel synchronization signal, and pixel data (provisional pixel data) is generated once. The provisional pixel data is then further multiplied by the ratio of the pixel period to the pulse period of the pulsed laser light (pixel period / pulse period of the pulsed laser light), and the multiplication result is used as the final pixel data. However, even in this case, since it is necessary to start processing a subsequent pixel while processing an earlier pixel, parallel processing is performed.

[0042] In the present embodiment, the control device 30 may detect the pulse period of the pulsed laser light emitted from the laser light source 11 based on the laser pulse signal output from the laser light source 11 .

[0043] In this embodiment, the control device 30 may be realized by a computer as exemplified in Fig. 5. Fig. 5 is a diagram illustrating a hardware configuration of a computer 100.

[0044] The computer 100 illustrated in FIG. 5 includes a processor 101, a memory 102, a storage device 103, a portable storage medium drive device 104, a communication interface 105, and an input / output interface 106, each of which is connected to a bus 107 and capable of transmitting and receiving data to and from each other.

[0045] The processor 101 may be, for example, a single processor, a multi-processor, or a multi-core processor. The processor 101 executes programs such as an OS (Operating System) and applications to perform various processes (for example, the process of generating pixel data for each pixel described above).

[0046] The memory 102 includes a RAM (Random Access Memory) and a ROM (Read Only Memory). The RAM temporarily stores a part of the program executed by the processor 101. The RAM is also used as a working storage area for the processor 101. The ROM stores the program executed by the processor 101 and various data required for executing the program.

[0047] The storage device 103 is a device for storing data, and is a hard disk drive (HDD), a solid state drive (SSD), or the like.

[0048] The portable storage medium drive device 104 drives the portable storage medium 104a, accesses the stored contents, and reads and writes data. The portable storage medium 104a is a memory device, a flexible disk, an optical disk, a magneto-optical disk, etc. The portable storage medium 104a also includes a CD-ROM (Compact Disc Read Only Memory), a DVD (Digital Versatile Disc), a Blu-ray disk, a USB (Universal Serial Bus) memory, an SD card memory, etc.

[0049] The communication interface 105 is connected to a network by wire or wirelessly, and communicates with external devices connected to the network.

[0050] The input / output interface 106 is connected to an external device and performs input / output of data between the external device and the input / output interface 106. The external devices connected to the input / output interface 106 are, for example, the laser light source device 10, the microscope body 20, and the PC 40, and may further include an input device and an output device. The input device is a keyboard, a mouse, a joystick, a touch panel, etc., and the output device is a liquid crystal display, etc.

[0051] In such a computer 100, the programs executed by the processor 101 and various data required for the execution of the programs may be stored in the storage device 103 or the portable storage medium 104a, not limited to the memory 102. Furthermore, the programs executed by the processor 101 and various data required for the execution of the programs may be stored in the storage device 103 or the portable storage medium 104a via the communication interface 105 from an external device connected to the network.

[0052] Furthermore, computer 100 is not limited to the one illustrated in FIG. 5, and may be configured to include multiple components that are part of the components illustrated in FIG. 5, or may be configured to omit some of the components that are illustrated in FIG. 5.

[0053] Furthermore, the computer 100 may be configured to include hardware such as a microprocessor, a DSP, an ASIC (Application Specific Integrated Circuit), a PLD (Programmable Logic Device), an FPGA (Field-Programmable Gate Array), etc. For example, the processor 101 may be implemented using at least one of these pieces of hardware.

[0054] Although the embodiment of the present invention has been described above, the present invention is not limited to the above-described embodiment, and various improvements and modifications can be made without departing from the gist of the present invention. [Explanation of symbols]

[0055] 1. Scanning microscope 10 Laser light source device 11 Laser light source 12 Dimming section 13. Scanner 13a, 13b Galvano mirror 20 Microscope body 21, 23 Lens 22 Dichroic mirror 24 Objective Lens 25 Photodetector 30 Control device 40 PC 50 Monitors 100 Computers 101 Processor 102 Memory 103 Storage device 104 Portable storage medium drive device 104a Portable storage media 105 Communication Interface 106 Input / Output Interface 107 Bus 251 Photoelectric conversion element 252 Photodetector circuit S Sample

Claims

1. A scanning microscope that samples light from a sample irradiated with pulsed light and converts the light into data for each pixel, a sampling time per pixel is approximately an integer multiple of a pulse period of the pulsed light, The sampling start timing of each pixel is synchronized with the pixel synchronization signal.

1. A scanning microscope comprising:

2. The sampling time per pixel is an integer multiple of the pulse period, the error of which is 2 ns or less.

2. The scanning microscope according to claim 1.

3. 1. A scanning microscope comprising: A laser light source that emits pulsed light; a scanner that scans the pulsed light two-dimensionally on a sample; a photodetector for detecting light from the sample; a control device that generates pixel data for each pixel constituting the image of the sample based on an output signal from the photodetector and a pixel synchronization signal synchronized with a scanning position of the scanner, sets a sampling time per pixel to an approximately integer multiple of a pulse period of the pulsed light, and synchronizes a sampling start timing of each pixel with the pixel synchronization signal; A scanning microscope comprising:

Images