JPH11271626A - Scanning type laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide images without distortion at high speed even when any phase delay between the driving signals of the mirror and an actual scanning position occurs in the case that the microscope is provided with a scanner for scanning a sample by a laser beam and a detector for sampling data from the sample at an optional scanning position. SOLUTION: This microscope is provided with a memory 30 for preserving driving waveform data for driving the scanner and a sampling clock pattern for sampling sample data at the optional scanning position in the same address, and a digital delay circuit 34 for delaying and outputting the sampling clock pattern by optional time against the driving waveform data preserved in the memory 30. In this case, the scanner is driven based on the driving waveform data in the memory 30 and a sampling clock is delayed by the optional time corresponding to the driving waveform data.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 1. Field of the Invention [0002] The present invention relates to a scanning laser microscope using a galvanometer mirror.

[0002]

2. Description of the Related Art In a scanning laser microscope, a laser beam, which is a point light source, is irradiated with an X-axis and a Y-axis of a sample through an objective lens.
Irradiation while scanning in the axial direction, fluorescence or reflected light from the sample is detected again by a detector via an objective lens and an optical system, so as to obtain density information of a two-dimensional image,
The two-dimensional distribution of the grayscale information is displayed on an image output device such as a CRT monitor or a color printer in correspondence with the XY scanning position as a distribution of bright spots, so that the image can be formed and observed.

Further, by providing a stop having a diameter approximately equal to the diffraction limit of the illumination light or the light to be measured at a position conjugate with the sample of the detection optical system, only the information on the in-focus plane is detected. The result is a confocal scanning laser microscope.

In such a scanning laser microscope,
As means for two-dimensionally scanning the laser beam from the light source, a galvanometer mirror has been mainly used. When the galvanometer mirror is used as a two-dimensional scanning means, as disclosed in Japanese Patent Publication No. Hei 6-27905, the drive signal waveform is a triangular wave or a sawtooth wave,
In some parts, where the displacement of the mirror moves linearly with time, data is sampled with a sampling clock having a constant period, thereby capturing an image without distortion.

[0005] In addition, rather than driving a galvanometer mirror with a drive signal having a high-frequency component at the apexes of peaks and valleys in a waveform, such as a triangular wave or a sawtooth wave, a drive signal having a single frequency, For example, it is also known that an image can be acquired at high speed while suppressing distortion of an obtained image by modulating the period of the sampling clock so as to compensate for the fluctuation of the scanning speed of the mirror by making the mirror a sinusoidal wave. is there.

The following two methods are generally known as methods for modulating and generating the period of the sampling clock so as to compensate for the above-mentioned fluctuation in the scanning speed of the mirror.

The first method is disclosed in JP-A-5-2135, JP-A-5-136954, and JP-A-6-148.
No. 525, for example, a sample is scanned by a laser beam, and at the same time, a linear scale pattern is scanned, and a sampling clock is generated from the reflected or transmitted light intensity from this pattern. Is the way.

The second method is a method in which a sample clock pattern is created on a memory so that data can be sampled at a linear scanning position for a sine wave of a drive signal waveform.

[0009]

However, these compensation methods have the following problems. That is, in the first method, it is necessary to provide an optical system for scanning a linear scale separately from the optical system for scanning the sample, and further, an optical system for scanning the linear scale in the optical system for scanning the sample. , There is a problem that the loss of light amount is large.

In the second method, there is a problem that a phase delay occurs between the drive signal waveform and the actual scanning position by the mirror due to the phase characteristics of the mirror itself and the drive circuit.

As shown in FIG. 8, the phase delay changes with the change of the driving frequency, and the amount of the phase delay changes with the change of the amplitude. If the amount of phase delay between the drive signal waveform and the sample clock pattern is not appropriately changed in response to such a change in phase delay, there is a problem in that the obtained image will be distorted.

Further, when data is sampled on both the forward path and the return path, since the timing of the synchronization signal indicating the sampling start position must be finely adjusted, it is extremely difficult to sample the same position on the forward path and the return path. There was also a problem that it was difficult.

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and has as its object to provide a scanner using a galvanometer mirror that causes a phase delay between a drive signal of the mirror and an actual scanning position. However, it is possible to acquire images without distortion at high speed, and to collect images with good image quality without distortion and pixel shift even when sampling data using both the forward and return paths of reciprocal scanning. It is an object of the present invention to provide a scanning laser microscope capable of performing the above.

[0014]

According to a first aspect of the present invention, there is provided a scanning laser having a scanner for scanning a sample with a laser beam, and a detector for sampling data from the sample at an arbitrary scanning position. In a microscope,
Storage means for storing the drive waveform data for driving the scanner and a sampling clock pattern for sampling the sample data at an arbitrary scanning position at the same address; and a drive waveform data stored in the storage means. Delay means for delaying the sampling clock pattern by an arbitrary time and outputting the same, and driving the scanner based on the drive waveform data in the storage means, and performing sampling by the detector by the delay means. It is characterized in that the sampling clock is delayed for an arbitrary time in accordance with the drive waveform data.

As a result of such a configuration, a phase delay occurs between the driving signal for driving the scanner and the actual scanning position,
Even if the delay amount changes depending on the frequency and amplitude of the drive signal, the time delay between the drive waveform data and the sampling clock pattern is adjusted without rewriting the stored drive waveform data, so that the distortion is always reduced. It will be possible to obtain no images.

According to a second aspect of the present invention, in the first aspect of the present invention, an address control means for controlling an address of the storage means and a comparison address to be compared with an address output from the address control means are set. And a setting unit for comparing the address output from the address control means with the set comparison address, and outputting a match signal when they match, indicating that the match signal indicates the start of sampling. It is characterized in that it is a synchronization signal.

As a result of such a configuration, the above-mentioned claim 1 is obtained.
In addition to the operation of the described invention, when adjusting the sampling start position, fine timing adjustment can be performed without rewriting the stored driving waveform data and without increasing the circuit scale of the digital delay circuit. It becomes possible.

According to a third aspect of the present invention, in the second aspect of the present invention, the address comparing means outputs a coincidence signal for a plurality of addresses. As a result of such a configuration, in addition to the effect of the invention described in claim 2, when sampling is performed on both the forward path and the return path of the reciprocal scanning, both sampling ranges are precisely matched to prevent distortion and pixel shift. It is possible to obtain a high quality image.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A confocal scanning laser microscope according to one embodiment of the present invention will be described below with reference to the drawings. FIG. 1 shows the basic configuration of the laser beam generator, wherein 1 is a laser oscillator as a light source, 2 is a beam expander, 3 is an optical path splitting element, and 4 and 5 are galvanometer mirrors.
Direction scanner and Y direction scanner, 6 is a pupil projection lens,
7 is an imaging lens, 8 is an objective lens, 9 is a sample to be observed, 14 is a confocal optical system, 15 is a wavelength selection element, 16 is a photoelectric conversion element as a detector, 17 is a data processing device, and 18 is CRT monitor as image display device,
19 is a drive waveform generation circuit.

Illumination light from the laser beam emitted from the laser oscillator 1 is applied to a beam expander 2 and an optical path splitting device 3 such as a beam splitter or a dichroic mirror.
, And is incident on an X-direction scanner 4 and a Y-direction scanner 5 using a galvanometer mirror or the like, and is deflected to scan in two dimensions.

X direction scanner 4 and Y direction scanner 5
Are driven by scanner drive circuits 21 and 22, respectively.
The second drive signal is supplied from a drive waveform generation circuit 19 described later.

The illumination light passing through the X-direction scanner 4 and the Y-direction scanner 5 enters the objective lens 8 via the pupil projection lens 6 and the imaging lens 7, and scans the sample 9 two-dimensionally. From the light reaches the optical path splitting element 3 along the same optical path as the illumination light, passes through the optical path splitting element 3 and the confocal optical system 14, and passes through the wavelength selecting element 15 including a dichroic mirror, an interference filter, and the like. To the photoelectric conversion element 16.

Although the case where one photoelectric conversion element 16 is used is described here for ease of explanation, the present invention is not limited to this case, and a plurality of wavelength selection elements can be permanently combined with the photoelectric conversion element. The present invention is also applicable to a case where a plurality of fluorescence wavelengths are measured.

The fluorescence luminance information converted into an electric signal corresponding to the luminance by the photoelectric conversion element 16 is digitized by the A / D conversion circuit 23 in synchronization with the sampling clock supplied from the drive waveform generation circuit 19. After the necessary processing is performed by the data processing device 17, the CRT monitor 1
At 8, it is displayed and output as a two-dimensional image.

FIG. 2 shows a detailed circuit configuration of the driving waveform generating circuit 19. The driving waveform generating circuit 19 actually corresponds to the two scanners of the X direction scanner 4 and the Y direction scanner 5. Scanner drive circuits 21 and 22
Although the drive signal is generated and output, the configuration and operation are the same, so that the configuration and operation for one system of scanner will be described here.

As shown in FIG.
A memory 30 as storage means in which driving waveform data for driving the two scanners and a sampling clock pattern for sampling sample data at an arbitrary scanning position to be described later are written and stored, and a data bus from the memory 30. A D / A conversion circuit 32 connected to the digital bus 31 for generating an actual drive waveform from the transmitted digital data, and a bit line 33 of the data bus 31 to which sampling clock pattern data is output, A digital delay circuit 34 capable of variably setting a delay amount of the pattern data to be sent to the D conversion circuit 23,
A memory address control circuit 36 connected to the address bus 35 of the memory 30 for controlling the address of the memory 30; an address comparison circuit 37 also connected to the address bus 35 for generating a synchronizing signal; It is composed of a CPU 38 as a setting section for writing data and performing various settings.

The operation of the above configuration will be described. When driving the scanner, the CPU 38 first stores the D / A in the memory 30 as shown in FIG.
A bit width corresponding to the resolution of the conversion circuit 32, here, n-bit drive waveform data, and single-bit sampling clock pattern data for determining whether or not to perform sampling at a scanner position driven based on this waveform data. The indicated clock bit is written to the same memory address.

Here, the driving waveform data D for outputting a sine wave includes an address A, an effective address range N, and an amplitude G.
Can be expressed by the following equation. That is, D (A) = G · sin (2πA / N) (1) The sampling clock pattern data generated in accordance with such waveform data is the driving waveform data D as shown in FIG. 1 bit "0" so that a clock is output at each position where the variation amount of
Consecutive pattern data of “1” is created and written into the memory 30 together with the writing of the drive waveform data D. The write operation to the memory 30 is performed by setting the drive waveform data D to at least 1
The number of pattern data constituting the cycle, here, is repeated N times.

Next, the CPU 38 sets the start address of the memory 30, the effective address range, the address output speed, and the like to the memory address control circuit 36, and then starts outputting the memory address.

The memory address control circuit 36 counts up the addresses sequentially from the set start address, outputs an address value within the effective address range, returns to the start address, and repeats the address count-up. According to the address thus output, the waveform data is output from the memory 30 to the D / A conversion circuit 32, and the waveform of FIG. 4 is output from the D / A conversion circuit 32 as a voltage waveform.

FIG. 5 shows the relationship between the driving waveform and the actual position of the mirror when the galvanometer mirror constituting the X-direction scanner 4 or the Y-direction scanner 5 is driven by the driving waveform data D created as described above. The solid line indicates the driving waveform, and the broken line indicates the actual position of the mirror.

As can be seen from the figure, the actual position of the mirror has a delay Δt with respect to the driving waveform. This is due to the phase characteristics of the scanner drive circuits 21, 22 and the X, Y direction scanners 4, 5 themselves. If the data is sampled using the sampling clock pattern as it is in the state where the delay has occurred, the obtained image is greatly distorted.

Therefore, the CPU 38 operates the digital delay circuit 3
4 is set so that the sampling clock pattern is delayed and output by the delay amount Δt at the actual position, and the signal output from the digital delay circuit 34 is used to set the A / D conversion circuit 2.
By sampling the data in step 3, it is possible to obtain a distortion-free image in which the delay at the actual position is compensated.

Here, when the drive frequency is changed in order to change the time until one image is obtained, FIG.
The delay amount changes as shown by. Further, when the amplitude range of the galvanometer mirrors constituting the X-direction scanner 4 and the Y-direction scanner 5 is changed in order to change the scanning range, the generated delay amount increases as the amplitude increases, as shown in FIG. become.

Therefore, the digital delay circuit 34 is configured by a FIFO memory or the like so that the delay amount can be varied, so that an appropriate sampling clock can be generated even when the amplitude of the galvanometer mirror is changed.

As for the above-mentioned delay amount, the drive frequency and the drive amplitude may be used as parameters, and the delay amount may be stored as a data table or an approximate expression, or the actual scanning position can be detected. Such a detecting means may be separately provided.

On the other hand, in a normal configuration, the synchronization signal indicating the sampling start position within one scanning line is provided with a dedicated bit in the memory 30 similarly to the sampling clock pattern, and a synchronization signal pulse is output at an arbitrary position. If the same configuration is adopted in the drive waveform generation circuit 19 of the present invention, the circuit scale of the digital delay circuit 34, specifically, when the delay circuit is configured by a FIFO memory, the memory capacity is reduced. There is a disadvantage that it is required twice.

Therefore, in this embodiment, an address comparison circuit 37 is provided. The address comparison circuit 37 generates a synchronization signal when the address value output from the memory address control circuit 36 to the address bus matches the comparison address value set by the CPU 38, and sends the synchronization signal to the A / D conversion circuit 23. Output.

With this configuration, it is possible to arbitrarily set the output timing of the synchronization signal only by changing the comparison address in accordance with the delay amount of the digital delay circuit 34. Here, as for the setting of the comparison address, similarly to the above-described delay amount of the sampling clock pattern data, a data table stored in advance is used or a means for detecting the actual scanning position is separately provided. As a result, an optimal setting value can be obtained.

By using the driving waveform generating circuit 19 as described above, the delay between the driving signal of the galvanometer mirror constituting the X-direction scanner 4 and the Y-direction scanner 5 and the actual scanning position is compensated, and the driving frequency and amplitude are compensated. In this case, the amount of delay can be appropriately adjusted even for the change of .times., So that it is possible to acquire a two-dimensional image at high speed and without distortion.

Next, consider a case where data in the same range is obtained on both the forward and backward scan paths. In this case, the address comparison circuit 37 is configured so that a plurality of comparison addresses can be set.
If the synchronizing signal can be output to the / D conversion circuit 23, a plurality of synchronizing signals can be obtained within one cycle of the sine wave driving waveform as shown in FIG. It can be adjustable.

That is, in the driving waveform of FIG.
Assuming that the sampling of the outward path is started from the position> and the position where the sampling of the data of n pixels ends is <3>,
The sampling start position on the return path must be set exactly at the position <2>.

In such a case, if the above-described configuration is used, the two synchronization signal positions can be set to arbitrary positions, respectively, so that the positions corresponding to the positions <1> and <2> can be set. The synchronization signal can be easily set.

As described above, according to the present embodiment, even when reciprocating scanning is performed in which data is sampled using both the forward path and the return path, distortion and pixel shift do not occur, and high image quality can be obtained. A clear image can be obtained.

It should be noted that the present invention is not limited to the above-described embodiment, and can be variously modified and implemented without departing from the gist thereof. In the above-described embodiment, a sine wave is described as an example of the drive signal of the galvanometer mirror. However, the present invention is not limited to this. For example, the correspondence between the sampling clock and the drive waveform data is set according to various waveforms. Accordingly, even if a triangular wave or a sawtooth wave is used as the drive signal waveform of the galvanometer mirror, it is possible to compensate for the delay between the drive and the scanning position.

[0046]

As described above, according to the present invention, the delay between the drive signal of the galvanometer mirror and the actual scanning position is compensated, and the delay amount is appropriately adjusted even when the drive frequency and the amplitude are changed. As a result, it is possible to acquire images at high speed and without distortion. Furthermore, even when data is sampled using both the forward path and the return path of the reciprocating scanning, it is possible to collect an image without distortion or pixel shift.

[Brief description of the drawings]

FIG. 1 is a diagram showing a basic configuration of a scanning laser microscope according to an embodiment of the present invention.

FIG. 2 is a block diagram showing a detailed circuit configuration mainly in a drive waveform generation circuit of FIG. 1;

FIG. 3 is a diagram showing a format configuration of waveform data according to the embodiment;

FIG. 4 is a diagram for explaining an operation according to the embodiment;

FIG. 5 is a diagram illustrating an operation according to the embodiment.

FIG. 6 is a diagram illustrating an operation according to the embodiment.

FIG. 7 is a diagram illustrating an operation according to the embodiment.

FIG. 8 is a view for explaining a delay amount between a drive signal given to a galvanometer mirror and an actual position of the mirror.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Laser oscillator 2 ... Beam expander 3 ... Optical path splitting element 4 ... X direction scanner 5 ... Y direction scanner 6 ... Pupil projection lens 7 ... Imaging lens 8 ... Objective lens 9 ... Sample 14 ... Confocal optical system 15 ... Dichroic Mirror 16 Photoelectric conversion circuit 17 Data processing device 18 CRT monitor 19 Drive waveform generation circuit 21, 22 Scan drive circuit 23 A / D conversion circuit 30 Memory 32 D / A conversion circuit 34 Digital delay circuit 36 memory address control circuit 37 address comparison circuit 38 CPU

Claims (3)

[Claims] 1. A scanning laser microscope comprising: a scanner for scanning a sample with a laser beam; and a detector for sampling data from the sample at an arbitrary scanning position, wherein drive waveform data for driving the scanner is provided. Storage means for storing, at the same address, a sampling clock pattern for sampling sample data at an arbitrary scanning position; and storing the sampling clock pattern at an arbitrary time with respect to the drive waveform data stored in the storage means. Delay means for delaying and outputting the data, driving the scanner based on the drive waveform data in the storage means, and causing the delay means to cause a sampling clock for sampling by the detector to correspond to the drive waveform data. Running characterized by delaying any time Type laser microscope. 2. An address control unit for controlling an address of the storage unit, a setting unit for setting a comparison address to be compared with an address output from the address control unit, and an address output from the address control unit. 2. The scanning device according to claim 1, further comprising address comparing means for comparing the set comparison address and outputting a coincidence signal when they coincide with each other, wherein the coincidence signal is a synchronization signal indicating the start of sampling. Type laser microscope. 3. The scanning laser microscope according to claim 2, wherein said address comparing means outputs a coincidence signal for a plurality of addresses.