DE19719344A1 - Arrangement for optical micromanipulation, analysis and processing of objects - Google Patents

Abstract

the arrangement has only one laser system (1) used in the continuously emitting mode or in the pulsed mode with ultra short laser pulses in the nano-, pico- or femto-second range to produce a focussed beam of variable intensity in the spectral range from 400 to 1200 nm. A switching device (2) for the laser system can consist of an intracavity switching element or of an extracavity switching element.

Description

The invention relates to an arrangement for optical micromanipulation, analysis and processing of objects in the nanometer and micrometer range (10 ^-9 -10 ^-3 m) by means of focused radiation in the spectral range from 400 nm to 1200 nm of a laser. Such a laser works either in the continuously emitting (cw) mode or in the pulsed mode with ultra-short laser pulses in the nanosecond, picosecond or femtosecond range. The arrangement is suitable for transporting objects to a specific location by optical micromanipulation, analysis and localized laser processing by nonlinear fluorescence excitation, the processing products being able to be transported away by means of optical micromanipulation.

Previous arrangements for optical micromanipulation include one Laser in cw mode in combination with a microscope. So for the optical micromanipulation of biological objects prefers a laser in cw mode with emission in the near infrared (NIR) spectral range used. Suitable lasers are the Nd: YAG laser with a typical one Emission at 1064 nm, the tunable cw-Ti: sapphire laser with a typical emission in the range 700-1000 nm and the diode laser with a typical emission in the range 670-1000 nm. Such an arrangement is also called light trap or laser tweezers. Instructions for Optical micromanipulation has already been patented (US 4,887,721,  US 4,893,886, US 5,212,382, US 5,308,976, US 5,363,190, US 5,512,745). US Pat. No. 4,887,721 describes a two-laser arrangement, wherein one laser for optical micromanipulation and a second for one Analysis is used.

Nonlinear fluorescence excitations as well as the release of substances that enclosed by photolabile chemical cages ("caged compounds "), in biological systems are generally associated with Femtosecond pulses from a mode-locked MR laser (typically a pulsed Ti: sapphire laser). Through simultaneous Absorption of two long-wave photons (two-photon excitation) visible fluorescence can be induced, which otherwise only with short-wave radiation, e.g. B. in the ultraviolet (UV) range, would have stimulated can be. These methods for nonlinear fluorescence diagnostics and substance release by means of laser pulses in the femtosecond range and in red / NIR spectral range are also patented (US 5.034.613, EP 500.717) and z. B. in Science 248 (1990) pp. 73-76 have been described. Damage to or processing of cells Structures or biomolecules are not provided. On the contrary, destructive effects in the cell or on the biomolecule should be avoided will. The average power typically used is in the range < 10 mW. These services are often used for optical micromanipulation too low. An increase in the average power of the pulsed However, laser radiation would induce destructive effects [König et al. J. Microscopy 183 (1996) pp. 197-204]. Therefore z. B. none Femtosecond pulses used for optical micromanipulation. By doing European patent EP 706.671 uses a scanning microscope for analysis based on the two-photon excited fluorescence  cw or pulsed laser radiation with pulse lengths in the picosecond range or uses longer pulse lengths.

The processing of intracellular structures using light is already off Tschachotin in 1912. He edited individual ones Cellular organelles using a conventional radiation UV source (lamp). With the Construction of the laser can be achieved. So z. B. 1965 by Amy and Storb [Science 150 (1965) pp. 756-757] and 1969 by Berns et al. [Nature 221 (1969) pp. 74-75] on the laser-induced destruction of individuals Mitochondria and chromosomes have been reported. 1981 was published in Science 213 (1981) pp. 505-513 Laser-generated lesions in chromosomes, organelles and nerve cells using a dye laser in the nanosecond range and an Nd: YAG laser in the picosecond range. This Lesions made it possible to draw conclusions about the functioning of the cellular Areas. EP 182.320 describes a method for making cuts of biological material pumped using an Eximer laser Dye laser presented in which diffraction phenomena to manufacture used by cuts. EP 52.409 describes a method for Fusion of biological cells using a pulsed laser beam of pulse energy 0.1-100 µJ described and embodiments using pulsed UV laser specified.

The optical micromanipulation as well as the laser processing smaller Structures by means of an arrangement have been described several times. The disadvantage of this arrangement is the use of two different ones Lasers. The publications Labor 2000 (1989) p. 36 and Nature 368 (1994) p. 667 from a laser microscope with a cw Nd: YAG laser for optical micromanipulation and another 337 nm nitrogen laser reported for microsurgical use. In EP 679.325 a  Device and a method for handling, processing and Observation using two lasers with emissions in a different one Spectral range described. In US 5,393,957 an arrangement for a Micromachining with a laser and optical manipulation with a other lasers, in particular microcapsules. A two- Laser arrangement for micromanipulation and for triggering Photoreactions is published in US 5,308,976.

The disadvantage of these arrangements is the high expenditure in use different lasers for processing and optical Micromanipulation. In addition to complicated different Beam guidance systems and separate controls for the Applications, moreover, are generally lasers different emission wavelength used. This has the other Disadvantage of different beam paths in the microscope as a result chromatic aberration and the use of complex dichroic Optics.

It is an object of the invention in an arrangement for processing, analysis and optical micromanipulation of objects in the nanometer and Micrometer range by means of focused laser radiation Generation of the laser radiation and the effort for the optical Significantly reduce the beam path.

According to the invention for processing, analysis and optical Micromanipulation uses only a single laser system, the focused and variable radiation in the spectral range 400 nm to 1200 nm optionally in the continuously emitting (cw) Mode or in the pulsed mode with ultra-short laser pulses in nano,  Pico or femtosecond range is switched. Depending on the application laser radiation (processing, analysis or optical micro manipulation) the laser system while maintaining the beam path in the microscope either in cw mode or in pulse mode switched.

The switching element serves to interrupt the mode synchronization (Mode coupling) in the pulsed laser system and the transformation into one Laser system in cw mode or the transfer of a laser system in cw mode in a pulsed system with ultra-short pulses. In cw mode the individual longitudinal modes of the laser system operate independently, while in a laser system in pulse mode, the individual modes there is a fixed phase coupling "synchronized".

The switching element can use the relatively narrow spectral bandwidth cw laser radiation (typically less than or equal to 1 nm) compared to Femtosecond radiation (e.g. spectral bandwidth of a 50 fs pulse ≈ 20 nm) for its mode of action. So the switching element e.g. B. represent an intracavity wavelength selective element, e.g. B. a Etalon, Lyot filter, absorption or interference filter, an AOTF ("acousto optical tunable filter "), an absorbent liquid or gas switchable liquid crystal filter, a prism or grid arrangement or a mechanical device (e.g. aperture, slot) for spatial Separation or addition of spectral components (e.g. by changing the slot width).

The switching element can also be based on the principle of the fault or Restore mode synchronization based. So one can Amplitude or frequency modulating intracavity element (e.g. Chopper; acousto-optical modulator at two different frequencies, where a frequency corresponding to the pulse repetition frequency / resonator length  ), an element that changes the resonator length or a mechanical element (e.g. a slot) to influence different Losses of cw and pulsed radiation in the resonator, e.g. More colorful Exploitation of beam self-focusing on the basis of the optical Kerr effect. An extracavity- Element that is used with a feedback mechanism the resonator specifically the mode synchronization when introduced into the Can cancel the beam path or restore it when removed. Such a In the simplest case, the mode synchronization can be influenced by the Appropriate insertion / removal of a flat glass plate in the extracavity beam path can be realized, the feedback Mechanism through the appropriate reflection of the beam on the glass plate given is. The mode synchronization can also be influenced by suitable modulation of the pump laser.

The radiation of a mode-locked laser system with ultra-short Pulses can also contain cw shares, which depend on performance, among other things depend on the pump laser. The mode synchronization of a laser system can thus be prevented with certain pump laser outputs. In this respect, the switching element can also vary the pump laser power cause.

The switching element can be used to implement the switching process mechanically inserted or removed in the beam path or represent an electro-optical switching element.

This can be used to switch from cw mode to pulsed mode rapid reaching of the stable mode-synchronized state by a suitable modulation with a by the pulse repetition frequency or by the Resonator length certain frequency, z. B. with an acousto-optical Modulator or by means of mechanical vibrations.

The invention is intended to be described in the following on the basis of three exemplary embodiments are explained in more detail.

In the drawing is a basic arrangement with a switchable intracavity birefringent liquid crystal filter as a switching element shown.

A cw argon ion laser pumped, pulsed mode-synchronized titanium: Sapphire Laser 1 with an output pulse repetition frequency of 80 MHz, an output pulse duration of 100 fs, an average output power of 1.5 W and a wavelength of 800 nm serves as an excitation source for the non-linear fluorescence analysis and the processing of particles in the micrometer range. A switching element in the form of an intracavity birefringent liquid crystal filter 2 which can be switched in the microsecond range enables the rapid changeover to the cw mode for optical micromanipulation. The output radiation of the laser 1 is coupled into the external port of an inverse laser scanning microscope 5 via a 1: 4 beam expander 3 and a beam attenuator 4 and with a lens 6 having a high numerical aperture (100 ×, NA = 1.3, oil immersion) focused on a sample 7 almost diffraction limited. The sample areas to be analyzed and processed are marked with a fluorescent dye with a high molecular two-photon absorption coefficient in the NIR range. First, with the cw laser beam of 100 mW power (liquid crystal filter "on"), the sample 7 is checked by a video camera 8 (a halogen lamp 9 is used as the illumination source) and transported to the desired location by means of optical micromanipulation. The laser 1 is then switched to the femtosecond mode (liquid crystal filter "off"), the laser beam being simultaneously attenuated to an average power of 2 mW on the sample 7 by means of an electrical control 10 . By means of two-photon fluorescence excitation, the sample area to be processed is first analyzed in the scanning mode with an attenuated laser beam and the microscope-internal fluorescence detector 11 . The laser beam is then adjusted by means of a scanning unit 12 and a motorized sample table 13 to the area of the sample 7 to be processed for a point irradiation (scanning mirror fixed). The irradiation time is determined with the microscope's own software. The processing takes place with the unattenuated laser beam with high pulse power. After changing back to the cw mode (liquid crystal filter "on"), the processing products are removed by means of optical micromanipulation.

In principle, the same basic structure shown in the drawing is provided as the second exemplary embodiment, with the difference that the Ti: sapphire laser 1 already internally has an acousto-optical modulator (AOM), not shown, of an operating frequency f ₁ corresponding to the pulse repetition frequency or the resonator length of the laser 1 contains for the implementation of mode synchronization. This AOM serves as a switching element such that the mode synchronization is disturbed by changing to a second operating frequency f ₂ . Switching to the operating frequency f ₁ in turn causes laser operation in the pulsed mode. The switching element liquid crystal filter 2 from the first embodiment example in the drawing is omitted.

As a third exemplary embodiment, the basic structure of the first exemplary embodiment shown in the drawing is again provided, with the difference that an absorption filter (not shown) that can be mechanically inserted and executed in the intracavity beam path is provided as the switching element. Here too, the liquid crystal filter 2 from the first exemplary embodiment shown in the drawing is omitted.

Reference list

1

laser

2nd

Liquid crystal filter

3rd

Beam expander

4th

Beam attenuator

5

Laser scanning microscope

6

lens

7

sample

8th

Video camera

9

Halogen lamp

10th

electrical control

11

Fluorescence detector

12th

Scanning unit

13

Rehearsal table
f ₁

, f ₂

Operating frequency

Claims (17)

1. Arrangement for optical micromanipulation, analysis and processing of objects in the nanometer and micrometer range with a laser system, characterized in that only a single laser system ( 1 ) is used and that means ( 2 ) are provided which the laser system ( 1 ) with switch its focused and variable-intensity radiation in the spectral range 400 nm to 1200 nm depending on the use of the laser radiation either in the continuously emitting (cw) mode or in the pulsed mode with ultra-short laser pulses in the nano, pico or femtosecond range. 2. Arrangement according to claim 1, characterized in that the means can be realized by an intracavity switching element. 3. Arrangement according to claim 1, characterized in that the means can be realized by an extracavity switching element. 4. Arrangement according to claim 1, characterized in that the means consist of a switching element for changing the spectral bandwidth of the laser system ( 1 ). 5. Arrangement according to claim 4, characterized in that the switching element for changing the spectral bandwidth of the laser system ( 1 ) is based on known birefringent liquid filters ( 2 ). 6. Arrangement according to claim 4, characterized in that the switching element for changing the spectral bandwidth of the laser system ( 1 ) is based on known interference or absorption filters. 7. Arrangement according to claim 4, characterized in that the switching element for changing the spectral bandwidth of the laser system ( 1 ) is based on absorbing liquids or gases known per se. 8. Arrangement according to claim 4, characterized in that the switching element for changing the spectral bandwidth of the laser system ( 1 ) on optical filters known per se, for. B. Lyot filters, based on interferometric arrangements, on AOTF elements ("acousto-optical tunable filter") or on prism and grating arrangements. 9. Arrangement according to claim 4, characterized in that the switching element for changing the spectral bandwidth of the laser system ( 1 ) on known mechanical arrangements for spatially influencing spectral components, such as. B. pinhole and column based. 10. The arrangement according to claim 1, characterized in that the means a switching element to influence the mode synchronization. 11. The arrangement according to claim 10, characterized in that the Switching element for influencing mode synchronization on elements to vary the resonator length.   12. Arrangement according to claims 3 and 10, characterized in that the Switching element for influencing mode synchronization from one extracavity element with a feedback mechanism. 13. Arrangement according to claims 2 and 10, characterized in that the Switching element for influencing mode synchronization from one intracavity modulator, e.g. B. consists of an acousto-optical modulator. 14. Arrangement according to claim 10, characterized in that the switching element for influencing the mode synchronization consists of a modulator for the pump laser of the laser system ( 1 ). 15. The arrangement according to claim 1, characterized in that the means consist of known elements for changing the pump laser power of the laser system ( 1 ). 16. The arrangement according to claim 1, characterized in that the means an electro-optical switch. 17. The arrangement according to claim 1, characterized in that the means for the purpose of their introduction and removal in or out of the beam path of the laser system ( 1 ) are arranged movably.