JPH11149045A - High compact laser scanning type microscope with integrated short pulse laser - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high compact laser scanning type microscope with a short pulse laser incorporated into a scanning module by arranging the short pulse laser being the component of a scanning part and a connecting means for connecting the short pulse laser radiation into a scanning means. SOLUTION: The microscope is provided with a scanning head part S which is connected to a microscopic beam process through an opening part for emitting light. The housing of the short pulse laser KPL32 with which the scanning head part S forms a compact unit is incorporated in the housing. The scanning head part S is effectively connected to the photoelectric tube of the erect microscope and also to the side exit of the inverted microscope. And by directly connecting the short pulse laser 32 in the scanning module of the laser scanning type microscope or connecting the laser 32 through a fiber, the laser is arranged in a compact state, then, the microscope is efficiently used, for example, in a multiple photon microscopic inspection method for performing, for example, the 3D resolution microscopic inspection of an organism sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Laser scanning microscopes using short pulses (for example, picosecond or femtosecond lasers) have heretofore been known, in particular, time-resolution microscopy and multiphoton microscopy.

[0002]

2. Description of the Related Art A two-photon scanning microscope is known from WO 91/07651, which has an excitation by a laser pulse in the sub-picosecond range with an excitation wavelength in the red or infrared region. EP666473A1, WO95 / 30166,
DE 44 14 940 A1 describes excitations in the picosecond or higher region with pulsed or continuous emission.
Procedures for optically exciting a sample via two-photon excitation are described in DEC 24331570. Female applicant DE 29609850 describes the coupling of short-pulse laser radiation through a light guide fiber into the microscope beam path.

[0003]

The short pulse laser used (wavelength adjustable laser or constant wavelength laser)
Are generally very bulky, very expensive due to their technical complexity, and are only very cumbersome for the user. Generally, the laser is coupled directly optically into the scanning module of a laser scanning microscope with the aid of a beam delivery system (eg, a mirror or glass prism). This generally requires a long optical beam travel, which makes the system adjustment-sensitive and very wide. Generally, when laser adjustment is often required,
In particular, when the laser wavelength is changed or adjusted, the laser beam emitted from the laser generally moves spatially. As a result, the laser beam inside the laser scanning microscope can no longer be adjusted optically. Finally, fiber coupling of short pulse lasers (eg, when using a single mode polarization maintaining fiber) introduces the problem that the laser and laser scanning microscope must be adjusted separately.

[0004] Such a fiber coupling of a short pulse laser to a laser scanning microscope is possible in principle, but for short pulses, because of the optical dispersion of the glass fibers and also with high intensity lasers. However, nonlinear optical effects such as self-phase modulation, Brillouin scattering, and Raman scattering that can appear in the glass fiber nucleus appear, and are generally very problematic.

[0005]

SUMMARY OF THE INVENTION The present invention relates to a high compact laser scanning microscope with a short pulse laser incorporated in a scanning module. This arrangement advantageously allows short pulse lasers to be directly or optically coupled to the laser scanning microscope.

For example, short pulse lasers that can be produced in a technically compact form of manufacture are ion-donating fiber lasers pumped by diode lasers, such as diode-pumped lasers that emit laser light at a wavelength of about 1550 nm from a laser resonator. Er ³⁺ fiber laser. This laser radiation can be doubled in frequency to a wavelength of about 790 nm based on high efficiency and high laser intensity (resonant, non-resonant, quasi-phase matching) via a nonlinear optical crystal outside the laser cavity. Some of the radiation is in the crystal, typically at the third harmonic of about 515 nm,
The fourth harmonic is also converted to about 387 nm. Similarly,
All other possible non-linear conversion processes emerge with a fixed conversion efficiency in the crystal. Along with that, exemplary high-compact short-pulse lasers are available for a variety of microscopy applications, with several simultaneously present various fixed output wavelengths.

The laser can be provided, for example, in a compact housing, which is fixed to the chassis in the scanning module and in which the laser beam can be adjusted, possibly in an adjustable manner, through an opening in the compact housing. (Figure 1). The laser beam can pass through an optical system arranged outside the compact housing, which converts the laser beam into a suitable beam diameter and a suitable beam divergence. The optics may be made variable and may be advantageous for the beam to match the color characteristics of the microscope optics (eg, to a variable beam width adjuster). Similarly, to optimize the relationship between transmission and spatial resolution, the beam diameter can be tailored to fit the objective pupil diameter (variable zoom).

[0008] A laser shutter (beam breaker) for maintaining the laser safety of the device must be incorporated into the scanning module of the laser scanning microscope which is in the laser beam path. This can be made as a paradigm for mechanical beam breakers. The laser beam can be introduced through a wavelength selective optic to select the required laser wavelength for the application. The element can be made as a dielectric filter, acousto-optic, electro-optic, refractive, or scattering element, or a combination thereof.

[0009] The laser beam can be introduced through a weakening element to adjust the laser power required for the application. This element can be a neutral filter, acousto-optic, electro-optic, refraction, or scattering element,
Or they can be made as a combination of these. In the case of acousto-optic devices, this can also be used effectively as a pulse picker for fluctuations in the pulse repetition rate of the laser (one of the consecutive laser pulses is isolated in time). it can.

A pre-hirp unit, such as one comprising a series of gratings or prisms, or a combination thereof,
It can be introduced into the laser beam path (illumination beam path) to provide a negative dispersion that compensates for the positive dispersion commonly found in scanning modules, microscopes, and sample optics. At the same time, it is advantageous to provide a laser pulse with the conversion limited to the maximum at the sample location.

The laser radiation then enters a main beam splitter (eg, a dielectric color splitter), which directs the laser radiation toward the sample under examination. This main beam splitter
In the main beam splitter revolver, it can be made as one of many color splitters, and at the same time can be made as a wavelength selective optic to select the laser wavelength needed for the application. Non-optical verification technology (eg OBIC or
In the case of LIVA), the main beam splitter can also be made as a full mirror.

As the laser beam scanner, a galvanometer scanner for deflecting a beam in the x direction and the y direction can be used. In the case of a multiphoton microscope, the fluorescence in the sample is excited at one or several available laser wavelengths. The detection of fluorescence emission is generally illustrated in FIG.
The use of a confocal stop in such a multi-channel arrangement is not necessary to obtain a 3D resolution. However, as a variant, confocal pinholes are used to increase the depth resolution to far beyond that of a simple multiphoton microscope (FIG. 3).

Detection of an optical signal or a non-optical signal depending on whether the detector configuration is out of scan or not. Depending on the button relationship of the pulsed laser beam, a Lock-In or Box-Car amplifier is used for phase-sensitive verification of the detection signal synchronized to the laser pulse repetition rate . A greatly improved signal-to-noise relationship is thereby obtained through a reduction in the bandwidth of the certification system, which on detection involves noise.

The use of a wavelength of 1550 nm is, for example, for semiconductor,
This is particularly interesting when examining structured silicon wafers under a microscope. The wavelength of 1550 nm is well transmitted through silicon. Only in the direct region of the objective lens focus, and thus deep discrimination, leads to excitation by non-linear optical processes such as multi-photon excitation (eg also OBIC and LIVA as non-optical detection techniques) or by the production of higher harmonics . In addition, this non-linear process can be used as a microscopic contrast procedure, even in the context of thick silicon materials, in the field of 3D resolution microscopy, for example non-destructive wafer inspection.

The wavelength of 790 nm is, for example, particularly suitable for the general excitation of the two-photon process of dyes usually used for fluorescent labeling of biological samples. 517nm of green or ultraviolet wavelength
Alternatively, 387 nm can be used for time resolution microscopy of, for example, reflection contrast, fluorescence contrast samples. The green emission of 517 nm can be used as a safe pilot emission for coordination during optical montage. In particular, the system is suitable for use in a physiological problem setting, for example for release from a "birdcage camp". Here, the 790 nm beam is 3 for “release”.
Through the photon process, even deep layers of thick preparations are available, while observation of the released ions is made by two-photon excitation of the fluorophore used for labeling.

The present invention has particularly advantageous handling features for the following difficulties. -Incorporation of a short pulse laser into the scanning module of the laser scanning microscope to prepare a high-compact microscope system. -Frequency doubling, connected to a laser resonator to prepare several wavelengths for various microscope applications,
An array of optics consisting of one or several nonlinear optical crystals that convert the frequency of the laser beam through frequency doubling, frequency addition, or frequency subtraction, other optical parameterization processes, or any of these Incorporation of short pulse lasers (fixable frequency adjustable or wavelength adjustable) using a combination. • Use in laser scanning microscopy with time resolution. • Use in confocal or non-confocal laser scanning microscopy using optical detection signals. -Use in laser scanning microscopy using non-optical detection signals. -Use in laser scanning two-photon microscopy.・ Use of frequency doubling (SHG) using a laser scanning microscope, especially on the surface, in material inspection. -Use in material inspection, especially for 2D-OBIC or 3D-OBIC. The combined use of two or more techniques as described in the preceding paragraph.

[0017]

FIG. 1 shows a scanning head S coupled to a microscope beam path through a light exit aperture L, schematically showing a housing of the scanning head described in detail in the following figures. Show. The housing of the short pulse laser KPL32 which forms a compact unit together with the scanning head S is incorporated in the housing. A cover (not shown) is provided to cover the laser 32 together to effect the scanning head.

FIG. 2 schematically shows a microscope unit M and a scanning head S having a common optical contact. The scanning head S can be effectively connected both to the phototube of an upright microscope and to the side exit of an inverted microscope (DE 4323129 A1). FIG. 2 shows a microscope beam path that can be switched between illumination light scanning and transmitted light scanning via a rotating mirror 14, and includes a light source 1, an illumination lens system 2, a beam splitter 3, an objective lens 4, and a sample. 5, condenser 6, light source 7, light receiving array 8, first cylindrical lens 9, observation beam path at second cylindrical lens 10, eyepiece 1
1. A microscope beam path with a beam splitter 12 for combining scanning beams is shown. According to the invention, the components of the scanning head are, according to the invention, a short-pulse laser 32, a subsequently arranged shutter 34, and a filter 33 for selecting the wavelength, or as shown schematically in FIG. One or several non-linear optical crystals 43 arranged in succession to convert the frequency through multiple multiplication, addition or subtraction, into the housing as shown in FIG. During the scanning beam travel, through a suitable beam deflecting element, in particular a mirror 38 as shown in FIG.
Here, it is a non-linear optical crystal coupled through a split mirror 24.

To be effective for setting and / or changing the focus during the preparation,
A lens system 38 movable along the optical axis is provided. Only then can the separate laser unit be particularly effective in that it is extremely compact and can be adjusted tightly.
Becomes effective. In the case of fiber coupling, somehow it is a matter of coordination or coupling. Furthermore, the means 35 for non-optical detection, in particular for current measurement during semiconductor inspection, and other detection means, PMTs with pre-prepared optics and filters, are partially passed. It is arranged behind the mirror 12 without the light of the sample by the scanning means passing through it.

The scanning unit further comprises a scanning objective 2
2, a scanner 23, a main beam splitter 24, and a common mapping lens system 25 for the detection channel. The deflecting prism 27 behind the imaging lens system 25 moves the imaging lens system 25 toward the dichroic beam splitter 28.
The beam coming from the sample 5 is imaged in the convergent beam path of the radiation filter 30 arranged behind and the corresponding light receiving element 31 (PMT).

Via a mirror 18 which passes through a part, a monitor diode 19 and a line filter 21 mounted on a rotatable filter carriage, not shown here, arranged in advance for this purpose, and a neutral filter 20 are connected. In the direction, the monitoring beam stroke is faded out. In order to be effective for multiphoton excitation, a spatially high resolution allows a pinhole arranged in front of the receiver 31 to be placed, which is used for coupling with another laser as shown in FIG. The position can be spatially adjusted in three directions, and can be replaced with the aperture 37 as shown in FIG.

In FIG. 4, an additional replaceable external laser 42 is connected by laser coupling with the scanning head S via an optical fiber 41 and is movable via a movable collimating lens system 40 and a beam deflecting element 39. And into the scanning beam path. Here, several single-wavelength lasers and multi-wavelength lasers that are coupled to one or several fibers, either alone or common, can also be handled. Furthermore, the beam emission to the microscope side is mixed by a color aggregator after passing through a matching lens system not shown here,
At the same time, coupling through several fibers is also possible. It is also possible to mix the beam radiation of the various lasers in the fiber path by means of a split mirror not shown here. The short-pulse laser 32 incorporated in the scanning head in accordance with the invention now very well passes through the deflection element 38 in the direction of its geometrically wide scanning beam path and mirror 3.
9 positions.

[0023]

Industrial Applicability The present invention provides a compact arrangement of lasers by directly or fiber-coupled short-pulse lasers in a scanning module of a laser scanning microscope, for example, for 3D resolution microscopy of biological samples. For example in multiphoton microscopy. Due to the deep discrimination power of multiphoton technology, there is no need to use a confocal stop (pinhole) during the inspection beam stroke. In doing so, the microscope system is very easy to implement technically and is particularly easy to handle in application. By using a short pulse laser system that utilizes several wavelengths at the same time, various applications can be realized simultaneously with only one compact microscope system.

[Brief description of the drawings]

FIG. 1 is an exemplary completed view of incorporating a compact short pulse laser into a scanning module of a laser scanning microscope.

FIG. 2 shows the optical beam path when implementing a compact short pulse laser into the scanning module of a laser scanning microscope without using a confocal stop in the detection beam.

FIG. 3 shows the optical beam path when implementing a compact short pulse laser into the scanning module of a laser scanning microscope using a confocal stop in the detection beam.

FIG. 4 shows the beam path when combining a laser coupled through an optical fiber externally according to the invention for standard operation with a laser scanning microscope and an integrated short pulse laser.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Light source 2 Illumination lens system 3 Beam splitter 4 Objective lens 5 Sample 6 Condenser 7 Light source 8 Light receiving array 9 First cylindrical lens 10 Second cylindrical lens 11 Eyepiece 12 Beam splitter 14 Rotating mirror 19 Monitor diode 20 Neutral filter Reference Signs List 21 line filter 22 scanning objective lens 23 scanner 24 main beam splitter 25 imaging lens system 27 deflecting prism 28 dichroic beam splitter 30 radiation filter 31 light receiving element 31 (PMT) 32 short pulse laser 33 filter 34 shutter 35 non-optical detection means 37 aperture 37 37 deflecting element 39 beam deflecting element 40 collimating lens system 41 optical fiber 42 external laser 43 nonlinear optical crystal

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Ulrich Simon (Original notation) Ulrich Simon Germany D-07751 Rotenstein Burgstrasse 35 (Address or residence Original notation) Burgstr. 35, D-7551 Rothenstein, Germany (72) Inventor Ralph Woleschensky (Name in original language) Ralf Wolleschen sky Germany D-99510 Shetten and der Promenade 3 (Address or address in original language) Ander Perme D-99510 Schoen, Germany

Claims (12)

[Claims] 1. A microscope section having a microscope beam path, a transmission lens system for mapping radiation from a sample to be inspected in the direction of an observation means and / or an inspection means, and a deflection section for deflecting a laser beam. A high-compact laser scanning microscope comprising a scanning section having scanning means, wherein the microscope section and the scanning section show optical contacts for coupling laser light into a microscope beam path, and the components of the scanning section A high-compact laser scanning microscope equipped with a short-pulse laser for producing lasers, in particular a short-pulse laser for multiphoton excitation, and coupling means for coupling the short-pulse laser radiation into the scanning means. 2. The method according to claim 1, further comprising the steps of:
Alternatively, a high-compact laser scanning microscope comprising a first housing having at least one transfer (cylindrical) lens for mapping in the direction of the inspection means and a second housing mountable on the first housing. And the first
And the second housing show optical contacts for coupling the laser light into the microscope beam path;
In the second housing, scanning means for deflecting the laser beam, a short pulse laser incorporated in the second housing, particularly a short pulse laser for multiphoton excitation, and scanning of the short pulse laser radiation High compact laser scanning microscope with coupling means for coupling into the means 3. A high-compact laser scanning microscope according to claim 1, wherein the detection means is incorporated in the second housing / scanning section for detecting radiation from the sample. 4. A high-compact laser scanning microscope according to claim 1, wherein the coupling means converts the laser beam into an appropriate beam diameter and an appropriate beam divergence. 5. The high-compact laser scanning microscope according to claim 4, wherein the coupling means is variable. 6. The high-compact laser scanning microscope according to claim 5, wherein the coupling means is a coupling lens movable along the optical axis. 7. A high-compact laser scanning microscope according to claim 1, wherein a laser shutter (beam breaker) for maintaining the laser safety of the apparatus is arranged at a laser exit. 8. A high-compact laser scanning microscope according to claim 1, wherein a filter for selectively adjusting a laser wavelength is arranged at a laser exit. 9. A high-compact laser scanning microscope according to claim 1, wherein an element for reducing the intensity for adjusting the laser power is arranged at the laser exit. 10. A high-compact laser scanning microscope according to claim 1, wherein an optical system comprising a non-linear optical crystal for frequency conversion by frequency doubling, tripling, addition or subtraction is arranged. 11. A high-compact laser scanning microscope according to claim 1, wherein the fiber laser has a wavelength of about 1550 nm, wavelength: diode-pumped Er3 + donation. 12. The high-compact laser scanning microscope according to claim 10, wherein the frequency is doubled to about 790 nm via a nonlinear optical crystal.