JPH03181910A - Laser scanning microscope - Google Patents

Abstract

PURPOSE:To miniaturize the subject microscope and to improve the stability by providing a scan driving control means for two-dimensional scanning and a light emission control means which matches the phase of the output of laser beam with the phase of steps of stepwise scanning. CONSTITUTION:The microscope is equipped with the scan driving control means 13 which outputs short-width pulsating light from a laser beam source 1 and makes the two-dimensional scan of a fast X scan increasing stepwise and a Y scan increasing stepwise by galvanomirrors 3 and 4 constituting a deflection system, and the light emission control means 16 which puts the output of the laser beam in phase with the steps of the stepwise scanning. The laser beam source 1 makes the stepwise X-Y scan with its beam according to the scan driving control means 13 which outputs a driving control signal and the scan driving control means 13 generates a timing signal synchronized with the X-scan steps and outputs the signal to the light emission control means 16. The light emission control means 16 receives the signal and outputs the short-pulse light emission control signal to a laser driving means 15. Consequently, the short- wavelength laser beam source which is miniaturized and has the good stability is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a laser scanning microscope. In particular, it concerns technology for miniaturizing the light source of a laser scanning microscope, which scans a sample with a laser beam and displays the reflected light or transmitted light from the sample as an electrical signal as an image. ] In the case of an application in which a laser beam is focused into a spot and used for observing an object under a microscope, a laser light source with a shorter wavelength is desirable because the spot diameter is inversely proportional to the wavelength.

Similarly, in laser scanning microscopes, in order to increase their quantitative resolution, it is desirable to use a laser light source with a shorter wavelength, and in applications to the biological field, fluorescence and Raman spectroscopy are often used. However, in this case as well, a light source with a shorter wavelength is required to expand the measurable range.

On the other hand, argon ion lasers of 488 nm and 514 nm are commonly used as short wavelength laser light sources.
There are short wavelengths such as 5 nm and 441.6 nm for He-Cd lasers, but they require large equipment, require warm-up to start operation, have poor beam point stability, and require cooling. Therefore, there were drawbacks and problems such as the need for vibration prevention treatment.

The present invention has been made to solve the problems described in (-). In order to miniaturize the laser light source, a semiconductor laser is used, and in order to shorten the wavelength, a second harmonic generator (SH) is used.
G) converts the wavelength of the semiconductor laser to 172 wavelengths,
By using this as a laser light source, it is an object of the present invention to provide a laser scanning microscope having a small and stable laser light source that eliminates the above-mentioned drawbacks and problems.

[Means for Solving the Problems] In order to solve the above-mentioned technical problems, the present invention provides a laser light source, a beam deflection system that two-dimensionally deflects the mold 11 emitted from the laser light source, and In a laser scanning microscope that uses a focusing optical system that focuses beams on a sample, and an optical conversion system that converts light reflected or transmitted from the sample into an electrical signal, a laser light source is used to generate pulses of short width. The deflection system is a laser light source that outputs light in the form of a shape, and the deflection system has a scanning drive control means for performing two-dimensional scanning of high-speed X-scanning that increases and decreases in a stepwise manner and Y-scanning that increases and decreases in a stepwise manner; The laser scanning microscope described above has a light emission control means that matches the phase of the output of the laser beam and the step of the stepped scan. The laser light source includes a semiconductor laser, a second harmonic generator (SHG), and a laser drive means, and the semiconductor laser is driven by a short pulse drive current output by the laser drive means.
Preferably, the semiconductor laser is a gain-switched semiconductor laser that outputs short pulse light, and outputs this short pulse light as short pulse light of 1/2 optical wavelength via the second harmonic generator. The second harmonic generator also includes a first collimator lens that parallelizes the fundamental wave of the output beam of the semiconductor laser, a pair of cylindrical lenses that corrects astigmatism of the semiconductor laser, and converts the wavelength to 1/2. a second harmonic generation element that transmits the 1/2 wavelength light; a focusing lens that focuses the beam on the second harmonic generation element; a second collimator lens that parallelizes the 1/2 wavelength beam; , and an optical filter that does not transmit the fundamental wave is suitable.

In a laser scanning microscope, the optical output (power) required for the light source beam is several mW. The optical output of a semiconductor laser is around a wavelength of 840n-, and those with good phase characteristics have a maximum continuous output of about 50 mW. On the other hand, the converted output of the second harmonic generator is proportional to the square of the incident optical power density. LiNb0.840n is a second harmonic generation element with high conversion efficiency and high transmittance up to around 420nm. ,KNbO, ,LilO
Taking KNbO, which has the highest conversion efficiency, as an example, the conversion rate is only about 1% at a beam diameter of 50 μm and an input power of IW, but at an input power of 50 mW. , if the beam diameter is approximately the same as the wavelength, then approximately 100
Only a converted power of μW is obtained, which is a quarter of the required power and too weak for a light source.

Q-switch,
There is a short pulse operation method such as mode locking, which can increase the output power several hundred times.

Further, as methods for increasing the output of a semiconductor laser, there are an external resonance type in which a passive element or an active element is inserted into a resonator of the semiconductor laser, and a gain switch type in which the high gain is utilized.

Here, the latter gain-switch type semiconductor laser is used because of its simple configuration. In this case, the IW has a peak output with a pulse width of several topicoseconds, and there is an advantage that the pulse generation timing can be controlled with one timing of the drive current.

In other words, a semiconductor laser having a wavelength around 840 nm is operated with a gain swing, and a short pulse of light with a width of several tens of microseconds is generated. A light source with a wavelength (approximately 420 nm) is used as a laser light source, and the two-dimensional beam is deflected, but the deflection is increased or decreased in a stepwise manner by a scanning drive control means, and by a light emission control means. , performance can be improved by synchronizing the short pulse oscillation of the laser light source and the step-like deflection.

[Function] With the above configuration, the semiconductor laser is capable of repeating short pulse oscillation up to about 200 MHz, and in the operation of the gain switch, the repetition of the oscillation can be electrically controlled. That is, one step can correspond to step-like deflection of the beam at a high speed of about 5 nanoseconds.

Furthermore, it is compatible with any of the current deflection force formulas, and can range from real-time image acquisition to random scanning.

Furthermore, as a method of increasing the output of the semiconductor laser, in addition to the gain switching operation, the method of lowering the output as described above can also be used.

First, the laser scanning microscope according to the present invention will be specifically explained using Examples, but the present invention is not limited thereto.

[Example] The present invention will be explained using a reflection type laser scanning microscope as an example. It goes without saying that the present invention can also be applied to a transmission type.

FIG. 1 is a schematic diagram showing an example of the optical arrangement of the main parts of the scanning optical microscope of the present invention.

The scanning optical microscope of the present invention uses a semiconductor laser (LD) as a light source and pulse modulates the light source, which is a difference in configuration from a conventional scanning optical microscope. Reference numeral 1 denotes a short pulse laser light source, which has the configuration shown in FIG. 2A or 2B.

That is, a laser light source 1, a beam deflection system 3.4.5.6 for two-dimensionally deflecting the output beam from the laser light source 1, a focusing optical system 8 for focusing the deflected beam on the sample,
This laser scanning microscope includes photoelectric conversion systems 10, 11, and 12 that convert reflected or transmitted light from the sample 9 into electrical signals.

A laser light source 1 outputs pulsed light with a short width. The galvano mirrors 3 and 4 constituting the deflection system have a scanning drive control means 13 for performing two-dimensional scanning of high-speed X-scanning that increases and decreases in a stepwise manner and Y-scanning that increases and decreases in a stepwise manner. It has a light emission control means 16 that matches the phase of the light output and the stepped scanning step.

The laser light source 1 includes a semiconductor laser 19, a second harmonic generator (5HG) 40, and a laser driving means 15. The semiconductor laser 19 is a semiconductor laser whose gain is switched by a short pulse drive current outputted by the laser driving means 15 and outputs short pulse light. The light is output as short pulse light of 1/2 light wavelength.

In FIG. 2A, the second harmonic generator 40 includes a first collimator lens that makes the fundamental wave of the output beam of the semiconductor laser parallel, and a lens that corrects the astigmatism difference of the semiconductor laser=10. A lens group 20 consisting of a pair of cylindrical lenses, a second harmonic generation element 22 that converts the wavelength to 1/2, and a focusing lens 21.172 that focuses the beam on the second harmonic generation element 22. It consists of a second frimake lens 23 that parallelizes the wavelength beam, and an optical filter 24 that transmits the 1/2 wavelength light and does not transmit the fundamental wave.

In addition, in Figure 2B, 20.21 with the same function as above is shown.
22, reflecting mirrors 26 and 27 that constitute a resonator for the generated 1/2 wavelength, and a mirror 25.

In FIGS. 2A and 2B, the laser source 19 is a GaAffiAs semiconductor laser that oscillates at around 840 nm.
0 is composed of a -11 nometer and a pair of cylindrical lenses, and performs Jr point spacing correction, beam shaping, and collimation of the output of the single-conductor radar.

The beam a made parallel by the lens group 20 passes through the lens 2
1, the second harmonic generating element 22 is narrowed down to a diameter of several tens of μm. The second harmonic generation element 11-22 is made of KNbOs and is arranged at 90 degrees (57 phase matching). Phase matching is achieved by a temperature control device (not shown). Although critical angle matching may be used, The conversion efficiency decreases.The output becomes 1/2 wavelength by the second harmonic generation element 22, and its output is made parallel again by the lens 23, and the fundamental wave (approximately 840 nm) is converted by the filter 24.
Except for, only 1/2 wavelength (approximately 420na) waves are transmitted.

FIG. 2B is a schematic configuration diagram showing a different example of the laser light source 1. In this configuration, the second harmonic generation element 22 is arranged near the focal point of a confocal external resonator composed of concave mirrors 27, 26 and mirror 25, and a mirror that transmits the fundamental wave and reflects the 1/2 wavelength. 25, the output (original wave) of the semiconductor laser 19 is narrowed down to the second harmonic generation element 22 by the lens group 20 and the lens 21, as described above. The converted 1/2 wavelength light is 26.27
The new resonator mode oscillates, and the mirror 26, which also functions as a lens, converts the light into two parallel lines and outputs it.

FIG. 3 is a diagram showing the gain switching operation of the semiconductor laser, where a curve 92B shows the driving current pulse by the laser driving means 15, and a curve 29 shows the optical output.

As is well known, a drive current with a width of several hundred picoseconds increases the gain
A single light pulse with a width of several tens of picoseconds is obtained.

In the configuration diagram of FIG. 1, the parallel optical pulse beam a of approximately 420 nm obtained in the above manner passes through a polarizing beam splitter (PBS) or a semi-transmissive mirror 2, and then passes through a high-speed X-polarizing galvanometer mirror. Deflected by -3. Although a galvano mirror is used here, an acousto-optic device (AOM) or a polygon mirror may also be used.

Additionally, beam a is coupled to a low-velocity Y-polarized galvanometer mirror 4.
The beam is deflected in the Y direction and two-dimensional beam scanning is performed. The lens 5.6 is for matching the deflection planes in the pupil transmission optical system. The lens converges the beam at the rear image plane position of the objective lens 8. A two-dimensional scanning pattern on this surface is projected onto a sample 9 by an objective lens 8.

Further, the lens 7 has the function of making the deflection surface coincide with the entrance pupil of the objective lens 8.

The light beam reflected by the sample returns along the opposite optical path, and the separated light beam is separated by the deflection beam splitter 2 and is focused by the lens 10 onto the pinhole 11, and the transmitted light is converted into a photoelectric converter. The element 12 converts it into an electrical signal. Information on the sample is obtained as time-series electrical signals in the order of two-dimensional scanning.

The photoelectric conversion element 12 is preferably an avalanche element having a response speed on the order of one second.

13 is a drive control means for driving and controlling the galvano mirrors 3 and 4, and 16 is a light emission control means for matching the phases of laser emission and deflection.

4- FIG. 4C is a diagram showing two-dimensional step scanning, curve fi30 shows the drive waveform of high-speed X scanning, and curve 3
1 shows a drive waveform of a low-speed Y scan. As a result, a rask scan as shown by curve 31 is performed.

The advantage of this step scanning is that since the pixels to be sampled are fixed, the resolution is reduced by -L. The resolution limit can be increased by taking small steps of about 1/2 the diameter of the beat spot. Although harmonic drive is shown in FIG. 4, triangular wave drive is suitable for galvanometer mirrors in order to increase the scanning speed. In this case, by managing the address in the image memory 17, it is possible to treat the image as the same image as in normal Lasceux scanning.

The beam is step-scanned in the X-Y direction according to the scanning drive control means (3) which outputs the above-mentioned drive control signal, but the scanning drive control means 13 also generates a timing signal synchronized with the X-scanning block and controls the light emission. Output to control means 16. The light emission control means 16 receives the signal 5,- and outputs a light emission control signal of 15 pulses to the laser driving means 15.

The phase and pulse width of this output short pulse take different values depending on the semiconductor laser. The light emission control means 16 also performs integration or detection control of the integrating circuit or the peak detection circuit 14.

FIG. 5 is a graph showing a pulse curve showing this situation. Curve 30 shows the X-step emission, and curve 29 shows
It is laser emission. The output of the photoelectric conversion element 12 is also similar to the laser emission curve 29.

When the output of this photoelectric conversion element 12 is input to the integration/peak detection means 14 and integrated or peak held according to the control means 16, it becomes a waveform of a curve 33. This signal passes through an A/D conversion means (not shown) and is stored in the image memory 17.

Read out the image mail 17 sequentially and display it on a display device 1 such as a CRT.
8 is displayed.

[Effects of the Invention] The laser scanning microscope of the present invention has the following advantages: 6. Firstly, it is possible to obtain a short wavelength laser light source that is small and has good stability.Secondly, further, resolution can be improved by step scanning. Thirdly, it has brought about significant technical effects such as improving the laser scanning microscope with the following advantages: Thirdly, it can improve the S/N ratio of the received light signal.

[Brief explanation of drawings]

FIG. 1 is a schematic configuration diagram showing an optical system configuration of an example of a laser scanning microscope according to the present invention. Figures 2A and 2B show 1 used in the laser scanning microscope of the present invention.
- It is a schematic block diagram which shows the example of a light source. FIG. 3 is a diagram showing the output pulse of the laser light source of the laser scanning microscope of the present invention. FIGS. 4 and 5 are diagrams showing the relationship between scanning and laser output. [Explanation of the markings of the main parts... - Laser light source 7 3.401. Galvano mirror 8 , Objective lens 9 , Sample 12 , Photoelectric conversion element 13 , Scanning drive control means 14 , Integration/ Beak detection circuit 15,
, Laser drive means 16 , Light emission control means 17 , Image memory 19 , Semiconductor laser 20 , Lens group 22 , , , Second harmonic generation element 24 , , ,
,,,filter 26.27. ,,,,,,,mirror

Claims (1)

[Claims] 1. A laser light source; an output beam from this laser light source is 2.
In a laser scanning microscope having a beam deflection system that deflects the beam in three dimensions, a focusing optical system that focuses the deflected beam on a sample, and a photoelectric conversion system that converts reflected light or transmitted light from the sample into an electrical signal, the laser light source is A laser light source that outputs short-width pulsed light, and a scanning drive for the beam deflection system to perform two-dimensional scanning of high-speed X-scanning that increases and decreases in steps and Y-scan that increases and decreases in steps. The laser scanning microscope, comprising: a control means; and a light emission control means for matching the phase between the output of the laser beam and the step of the stepwise scanning. 2. The laser light source essentially consists of a semiconductor laser, a second harmonic generator (SHG), and a laser driving means, and the semiconductor laser has a gain that is increased by a short pulse driving current outputted by the laser driving means. A semiconductor laser that is switched and outputs short pulse light, and outputs this short pulse light as short pulse light of 1/2 optical wavelength via the second harmonic generator. 1. The laser scanning microscope according to 1. 3. The second harmonic generator includes a first collimator lens that makes the fundamental wave of the output beam of the semiconductor laser parallel, a pair of cylindrical lenses that corrects the astigmatism difference of the semiconductor laser, and a collimator lens that makes the fundamental wave of the output beam of the semiconductor laser parallel. a second harmonic generating element that converts the beam into a beam; a focusing lens that focuses the beam on the second harmonic generating element;
a second collimator lens for collimating the beam of wavelengths, 1
3. The laser scanning microscope according to claim 2, further comprising an optical filter that transmits light of /2 wavelength and does not transmit the fundamental wave.