JP2001159734A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To surely sample a fluorescent signal including a peak in a laser microscope using pulse laser oscillation. SOLUTION: A laser oscillation synchronizing signal synchronized with a laser oscillation signal from a laser device 21 is generated by a laser oscillation synchronizing signal generating circuit 35 within a laser control section. The laser oscillation synchronizing signal generated by the laser oscillation synchronizing signal generating circuit 35 is delayed at a prescribed rate by a delay circuit and is impressed as a sampling signal to an analog-to-digital conversion section 27. As a result, the sampling of the peak of the fluorescent signal is made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a fluorescence signal sampling mechanism when a pulse laser is used as an excitation light source in a laser scanning microscope.

[0002]

2. Description of the Related Art An outline of a laser scanning microscope is described in, for example, a foreign document "HANDBOOK OF BIOLOGICAL CONFOCAL MICROSCOPY".
(Edited by J. Pawley, PLENUM PRESS, 1990). Generally, a laser scanning microscope, as shown in FIG.
A light source 1 for oscillating laser light, and a half mirror 3 for separating laser light from the light source 1 and light from a sample 9 described later.
And a galvanomirror 5 that scans a laser beam in the X and Y directions.
An objective lens 7 for irradiating the sample 9 with laser light,
A pinhole 9 for extracting scattered light or the like from the light from the sample 9 to extract light only at the focal position, a light receiving element 11 for receiving light passing through the pinhole 9, and a photoelectric conversion by the light receiving element 11. And an image processing device 15 for supplying an electric signal. The image processing unit 15 converts the digital signal into a digital signal according to a sampling clock arbitrarily determined by an independent oscillator or the like, stores the digital signal in a line memory (not shown), and performs image processing in synchronization with laser beam scanning to perform a monitor display 17 is displayed.

[0003]

As a laser scanning microscope for irradiating a sample with a laser and detecting fluorescence from the sample, a continuous wave laser such as a helium neon laser is generally used. However, in recent years, lasers of various wavelengths have been used due to diversification of samples to be studied. Some of these lasers
In order to suppress the fluctuation of the average output due to competition between the oscillation modes, there are many types in which pulse oscillation is performed by a Q switch or a mode locking method.

Further, in order to irradiate a sample with an extremely high photon density and detect multiphoton fluorescence from the sample, a mode-locked ultrashort pulse laser has been used as a light source for a laser scanning microscope. Was. When these pulse lasers are used as the excitation light source, the fluorescence signal from the sample becomes a signal that attenuates with time.

At this time, since the pulse of the laser beam (hereinafter, referred to as a laser pulse) and the sampling pulse are generally not synchronized, the peak of the fluorescence is not always sampled. For this reason, it is not possible to generate an image corresponding to the obtained amount of fluorescent light, which may cause a problem that the image becomes dark. In particular, the occurrence rate of fluorescence in the multiphoton process is low, and it is difficult to efficiently sample the fluorescence signal because the laser pulse and the sampling pulse are not synchronized.

An object of the present invention is to provide a laser scanning microscope using pulsed laser excitation, which can reliably sample a fluorescence signal including its peak.

[0007]

According to a first aspect of the present invention, there is provided a laser scanning microscope comprising: a pulse laser oscillating means for oscillating a pulse laser for exciting a sample; and a light detecting means for detecting light from the sample and outputting an electric signal. And a memory for storing data from the sampling unit, wherein the sampling unit samples the electric signal from the light detection unit in synchronization with the oscillation of the pulse laser from the pulse laser transmission unit. And a unit.

A laser scanning microscope according to a second aspect of the present invention includes a synchronizing signal generating means for detecting the oscillation of the pulse laser from the pulse laser oscillating means and outputting a synchronizing signal synchronized with the oscillation of the pulse laser. An electric signal from the light detecting means is sampled by the sampling means in synchronization with a synchronizing signal from the signal generating means.

According to a third aspect of the present invention, the synchronization signal generating means of the laser scanning microscope has a delay circuit for outputting a trigger signal for delaying the synchronization signal by an arbitrary time, and synchronizes with the synchronization signal delayed by the delay circuit. And sampling an electric signal from the light detection unit.

A laser scanning microscope according to a fourth aspect of the present invention includes a pulse generator capable of controlling the oscillation of a pulse signal in synchronization with the synchronization signal delayed by the delay circuit, and responding to the pulse signal from the pulse generator. Sampling is performed.

According to the laser scanning microscope of the first aspect, the sampling means samples the electric signal from the light detecting section in synchronization with the oscillation of the pulse laser from the pulse laser transmitting means, and stores the data from the sampling means in the memory. Since the accumulation is performed in the section, the oscillation timing of the pulsed laser can be matched with the sampling timing, and the light from the sample can be sampled reliably.

According to the laser scanning microscope of the present invention, the synchronization signal generation means detects the oscillation of the pulse laser from the pulse laser transmission means and outputs a synchronization signal synchronized with the oscillation of the pulse laser. The electrical signal from the photodetector can be sampled by the sampling means in synchronization with the synchronization signal from the synchronization signal generating means.

According to the laser scanning microscope of the third aspect, since the delay circuit is provided in the synchronization signal generating means, a trigger signal for delaying the synchronization signal by an arbitrary time can be output. The electric signal from the light detection unit can be sampled in synchronization with the synchronization signal.

According to the laser scanning microscope of the present invention, the oscillation of the pulse signal can be controlled in synchronization with the synchronization signal delayed by the delay circuit by the pulse generator. Sampling can be performed accordingly.

[0015]

Embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment] FIG. 1 is a system block diagram relating to a sampling mechanism of a laser scanning microscope. The sampling mechanism shown in FIG. 1 is a pulse laser device (hereinafter, referred to as a laser device) 21 that emits a pulse laser beam for sample excitation (hereinafter, referred to as a laser beam).
A photoelectric conversion unit 25 including a photodetector or a photomultiplier for photoelectrically converting fluorescence emitted from the sample 23 by irradiating the sample 23 with laser light from the laser device 21;
5 is provided with a function of converting an analog signal from the digital camera 5 into a digital signal, and sampling a signal from the photoelectric conversion unit 25 in synchronization with laser oscillation from the laser device 21.
D converter 27 (sampling means) and A / D converter 2
And a digital data image (confocal image) extracted from the memory 29.
And a sampling controller (synchronization signal generator) 33 that supplies a clock signal for sampling to the A / D converter 27. Here, the sampling control unit 33 includes a laser oscillation synchronization signal generation circuit unit 35 and a delay circuit unit 37. The laser oscillation synchronizing signal generation circuit section 35 has a detection section 36 that detects laser oscillation from the laser device 21. The detection unit 36
For example, the oscillation of the laser device 21 is detected by using a trigger output signal output from the laser device 21 as an electric signal synchronized with the laser oscillation, or an electric signal obtained by photoelectrically converting a part of the laser light by, for example, a photodetector or a photomultiplier. Then, a laser oscillation signal is output. The laser oscillation synchronizing signal generator 35 outputs a laser oscillation synchronizing signal to the delay circuit 37 in response to the laser oscillation signal detected by the detector 36. The delay circuit section 37 delays the laser oscillation synchronizing signal output from the laser oscillation synchronizing signal generation circuit section 35 by a delay time (Δt1) given from the external input circuit 39 to generate a trigger signal. / D
It is input to the converter 27.

The operation of the embodiment of the present invention configured as described above will be described with reference to FIGS.

In FIG. 1, the laser device 21 is
The pulse laser shown in (a) is oscillated with respect to the sample 23, and outputs an electric signal synchronized with the laser oscillation from the laser device 21 to the laser oscillation synchronization signal generation circuit unit 35 of the sampling control unit 33. The laser oscillation synchronizing signal generation circuit section 35 detects the laser oscillation signal of the laser beam by the detecting section 36, generates a laser oscillation synchronizing signal as shown in FIG. 2C, and outputs it to the delay circuit section 37. The laser beam from the laser device 21 is applied to the sample 23, and a fluorescence signal as shown in FIG.
And converted into an electric signal.

On the other hand, the delay circuit section 37 delays the input laser oscillation synchronizing signal by a delay time (Δt1) given via the external input circuit 39, and changes the trigger signal 38 shown in FIG. / D conversion unit 27. The A / D converter 27 converts the electric signal (analog signal) from the photoelectric converter 25 into a digital signal using the trigger signal as a sampling clock. At this time, FIG.
The delay time (Δt1) at which the peak of the fluorescence signal can be sampled by matching the peak of the fluorescence signal shown in FIG. 2 with the sampling timing shown in FIG.
Compares the trigger signal with the fluorescent signal after photoelectric conversion, and adjusts the timing of the trigger signal with the peak of the fluorescent light, or adjusts appropriately using the external input circuit 39 so that the image becomes the brightest. be able to. The A / D converter 27 outputs sampling data as shown in FIG. The image display unit 31 outputs a control signal for reading data stored in the memory 29 to the memory 29, similarly to a general laser scanning microscope, and outputs sampling data (digital data) stored in the memory 29. And the address are read and displayed on the screen in synchronization with the scanning position of the laser beam.

As described above, by using the trigger signal as the sampling clock, the timing of the peak of the fluorescent signal and the timing of the sampling can be matched, and the peak of the fluorescent signal can be reliably sampled.

Further, by reliably sampling the peak of the fluorescent signal, it is possible to make the image brighter than when the image is not synchronized with the laser pulse.

Furthermore, even if the frequency of fluorescence generation is low and a fluorescence signal is not necessarily generated every time a laser pulse is generated,
By sampling in synchronization with laser oscillation, a fluorescent signal can be efficiently obtained.

Next, a second embodiment of the present invention will be described with reference to FIGS. In FIG. 3, the same parts as those in FIG. 1 are denoted by the same reference numerals, and the description thereof will be omitted.

In this embodiment, a pulse generator 45 is used as a sampling clock of the A / D converter 27.
Is used. The pulse generator 45 synchronizes with the trigger signal 38 output from the delay circuit 37, as shown in FIG.
The pulse signal shown in (e) is generated. The pulse repetition frequency fp and the output time (Δt2) can be arbitrarily set by the external input circuit 39. That is, the A / D conversion unit 27 performs sampling using the trigger signal from the delay circuit 37 as a sampling clock, and performs sampling only during the output time (Δt2) (FIG. 4E). Therefore, the trigger signal is transmitted to the delay circuit unit 37.
By adjusting the delay time (.DELTA.t1) and the output time (.DELTA.t2) of the pulse of the pulse generator 45, the change with time of the fluorescence signal attenuating with time can be reliably sampled including the peak. Where no fluorescent signal is generated, the timing can be adjusted so that sampling is not performed. For example, when the above-described configuration is used for a laser scanning microscope using a two-photon process, laser light having a pulse repetition frequency of 80 [MHz] having a pulse width of 100 [fs] is used in the two-photon process. Assuming that sampling is performed 100 times during one pulse repetition, the pulse output time (Δt2) is 10
[Ns], the pulse signal of the pulse generator 45 is
It can be applied if it is set to about [GHz].

Thus, by sampling the peak of the fluorescence signal without fail, it is possible to make the image brighter than an image obtained when the image is not synchronized with the laser light pulse. Even if the frequency of fluorescence is low, a fluorescence signal is not always generated every time a laser pulse is generated. Even in such a case, by continuing sampling in synchronization with laser oscillation, a fluorescent signal can be obtained efficiently. In addition, since the data including the peak of the fluorescent signal is reliably sampled and stored in the memory 29, digital data, for example, digital integration of the fluorescent signal, analysis of the maximum value,
Digital processing such as analysis of the time constant of the fluorescence signal can be performed. Also, by not sampling unnecessary portions where no fluorescent signal is generated, the memory can be used effectively.

The present invention is not limited to the above-described embodiment, but can be variously modified without departing from the gist of the invention. For example, the laser oscillation from the laser device can be performed without interposing a delay circuit. The fluorescent signal may be sampled in synchronization with the above. Alternatively, the fluorescence signal emitted from the photoelectric conversion unit may be used as a synchronization signal to sample the fluorescence signal in synchronization with the timing at which fluorescence is emitted from the sample. The laser device of the present invention may be a mode-locked ultrashort pulse laser for two-photon excitation or a pulse laser for one-photon excitation.

[0027]

According to the present invention, in a multiphoton laser scanning microscope using pulsed laser excitation, a fluorescence signal including its peak can be reliably sampled.

[Brief description of the drawings]

FIG. 1 is a block diagram showing one embodiment of a laser scanning microscope of the present invention.

FIG. 2 is a diagram showing a timing chart of the laser scanning microscope shown in FIG.

FIG. 3 is a block diagram showing another embodiment of the laser scanning microscope of the present invention.

FIG. 4 is a diagram showing a timing chart of another embodiment of the laser scanning microscope shown in FIG. 3;

FIG. 5 is a system block diagram of a conventional laser scanning microscope.

[Explanation of symbols]

 Reference Signs List 23 sample 25 photoelectric conversion unit 27 A / D conversion unit 29 memory 31 image display unit 33 sampling control unit 35 detection unit 37 delay circuit 39 external input circuit 41 control signal 43 data 45 Pulse generator

Claims (5)

[Claims] 1. A laser scanning microscope comprising: a pulse laser oscillation unit that oscillates a pulse laser for exciting a sample; and a light detection unit that detects light from the sample and outputs an electric signal. 1. A laser scanning microscope comprising: a sampling unit that samples an electric signal from a sampling unit in synchronization with a pulse laser oscillation from the pulse laser oscillation unit; and a memory unit that stores data from the sampling unit. 2. The laser scanning microscope according to claim 1, further comprising: a synchronizing signal generating unit that detects oscillation of the pulse laser from the pulse laser transmitting unit and outputs a synchronizing signal synchronized with the oscillation of the pulse laser. 2. A laser scanning microscope according to claim 1, wherein said sampling means samples an electric signal from said photodetector in synchronization with a synchronization signal from said means. 3. The synchronizing signal generating means has a delay circuit section for outputting a trigger signal for delaying the synchronizing signal by an arbitrary time, and the light detecting section is synchronized with the synchronizing signal delayed by the delay circuit section. 3. The laser scanning microscope according to claim 2, wherein an electric signal from the laser scanning device is sampled. 4. A laser scanning microscope, comprising: a pulse generator capable of controlling oscillation of a pulse signal in synchronization with a synchronization signal delayed by the delay circuit unit; and sampling according to the pulse signal from the pulse generator. The laser scanning microscope according to claim 3, wherein: 5. The laser scanning microscope according to claim 1, wherein said pulse laser generating means is a mode-locked ultrashort pulse laser, and detects fluorescence from a sample by multiphoton excitation. .