DE102005044422A1 - Coherent anti-stroke Raman scattering-microscope for detecting symmetry of e.g. protein, has illuminating device polarizing pump and stroke radiation, and polarization analyzer executing ellipsometric analysis of scattering radiation - Google Patents

Abstract

The microscope has a pump-laser (2) delivering pump-radiation (3), and a stroke-laser (4) delivering stroke-radiation. A sample illuminating device focuses the pump-and stroke-radiation in a sample area, and a spectral analyzer (25) detects coherent anti-stroke Raman scattering (CARS)-radiation emitted by a CARS-process in the area. The illuminating device polarizes the pump and stroke radiation linear and parallel to one another. A polarization analyzer (22) is arranged before the analyzer (25) and executes an ellipsometric analysis of the CARS-radiation.

Description

The The invention relates to a CARS microscope with at least one Pumping radiation emitting pump laser source and a Stokes radiation emitting Stokes laser source, a sample illumination device, the pump and Stokes radiation focused in a sample space, and a detector that emitted through a CARS process in the sample space CARS radiation detected.

developments in nonlinear laser microscopy have made it possible molecular species by their characteristic vibrational states the use of coherent anti-Stokes Raman scattering (CARS = coherent anti-Stokes Raman scattering) with high spatial resolution too identify. The publication Volkmer A. et al., J. Phys. D, 2005, 38, R59-R81, describes a corresponding CARS microscope.

The physical process of the CARS process is schematic in 2 shown. By means of a pump radiation P, a molecule is excited from a ground state G into an intermediate state Z. By coherent Wellenenzumischung Stokes radiation S is simultaneously a de-excitation to an excited state V, so that in the end a resonant excitation of a vibration state corresponding to the excited state V is given. From this vibration state takes place by means of a further pump radiation P, which is identical in most cases to the aforementioned pump radiation P, an excitation to an excited state A. From this excited state A, the molecule passes under emission of CARS radiation C back to the ground state G. Four photons are involved in this process: pump, Stokes, probe and anti-Stokes CARS photon. The pump, Stokes and probe photons can be from a spectrally broadband or from two or three synchronized pulsed laser sources. In order to achieve the required light intensities, one usually starts with short pulsed lasers, for example with femtosecond or picosecond pulses. Thus, there is overall a coherent generation of an anti-Stokes photon by three-wave mixing. The wavelength of the anti-Stokes photon is shorter than the wavelengths of Stokes and pump radiation. It is possible to excite in the near-infrared spectral range.

At the CARS process is the Stokes radiation spectrally red-shifted relative to the pump radiation. By the interaction of a medium with the pump and Stokes wave a microscopic eigenstate is coherently populated in the medium, its energy the difference of pump and Stokes photon corresponds. This condition may be of any nature, e.g. a molecular vibration, Rotation or an electronic resonance correspond. The state in the animated ensemble is coherent and whose phase is determined by the phase relationship between the pumping and Stokes wave determined. Due to the inelastic scattering of the sample photon at excited ensemble, the corresponding spectral blue-shifted, anti-Stokes CARS photon. The CARS signal is coherent and the phase matching aligned accordingly. The system returns throughout the process Initial state back. It is possible to excite in all spectral ranges. The in the following described method for microscopic imaging may also be four-wave mixing processes, e.g. the degenerate four-wave mixing or CSRS (coherent Stokes Raman scattering) become. In these processes, the photons involved have different Frequencies relative to each other, but analogous to the CARS process leads the Interplay of two photons for the coherent occupation of a microscopic one Eigenstate, from which the scattering of another sample photon the coherent one Signal generated.

Due to the conservation of energy and momentum during in 2 As shown, the anti-Stokes photon is emitted in a certain spatial direction, whereby emission may occur in a forward or reverse direction.

By the non-linear relationship between the intensity of the incoming laser radiation and the CARS radiation is created only in one of the laser focus narrowed area, whereby a high spatial resolution is achieved. A spatial scanning of the sample volume then provides desired two- or three-dimensional Images.

The Molecular recognition is thus based on the intrinsic Raman resonance amplification of a CARS process. It takes place when those involved in the CARS process Lasers on the Raman shift a characteristic, molecular vibration to be tuned. Due to the optical nonlinearity, the high spatial resolution is possible, and the process can be done without synthetic marker, which otherwise interferes with the Structure and function of the system under investigation. CARS microscopy is therefore especially for biological Issues of great Interest.

However, investigations on biological systems show problems in the contrast between different substances, since the relevant regions in the Raman spectrum have a high spectral density and the bands of these systems often show a considerable inhomogeneous broadening. Thus, the selective interrogation of a particular Raman resonance is difficult due to the superposition of different bands and limits the differentiation of molecular species. In addition, there is the limited spectral resolution of the laser due to the pulsed operation. Further degradation of signal quality is caused by the high background of the non-resonant CARS signal.

The WO 03/004983 A1, which is a CARS microscope of the type mentioned discloses a polarization-sensitive detector, the a rotatable polarization analyzer. Continue to be the polarizations of the pump beam and the Stokes beam on each other voted so that they both are linearly polarized, being between the polarization directions a certain angle> 0 is set.

Of the The invention is therefore based on the object, a CARS microscope to create that non-resonant CARS signal background better suppressed and improved differentiation enable molecular species.

These Task is according to the invention with a Microscope of the type mentioned, in which the sample illumination device having a polarizer unit which pump and Stokes radiation polarized linearly and parallel to each other, and in which the detector a polarization analyzer unit is arranged upstream, which is an ellipsometric Analysis of CARS radiation performs.

These Analysis can e.g. be done by the unit elliptically polarized Shares of CARS radiation filters and suppresses non-elliptical portions.

A significant improvement in contrast, in molecular recognition and in the structural assignment is so through the analysis and Selection of different polarization states in the CARS signal achieved. Therefore, the polarization analysis unit is a means for analysis elliptical part of a signal at a nonlinear, microscopic Method provided. The optical design allows the nonlinear Raman optical activity (NL-ROA) from the Isolate and detect the total signal. To which can the non-resonant Proportion of CARS radiation is decisive repressed or for the heterodyne amplification of significantly weaker NL-ROA share are used.

The Detection of NL-ROA is particularly useful in biological systems of Relevance, because chirality or chiral structural elements in almost all important biological classes of molecules, how proteins, DNA, RNA, carbohydrates, are present. This attribute plays an increasingly important role in synthetic materials. The comparison between linear ROA and Raman spectra of complex biological systems shows contrasting, characteristic bands in the case of ROA, while the Raman spectra only partially definite structures and good contrast exhibit. This significant increase in information makes it possible e.g. in proteins, a characteristic secondary structure clearly differentiate. The now achieved detection of NL-ROA in CARS microscopy combines the selective detection of certain molecular systems based on Raman resonance amplification classical CARS microscopy with the identification of characteristic chiral structural elements by means of the Raman optical activity. Consequently becomes the non-linear Raman optical activity introduced as a new contrast agent in addition to the Raman resonance in the CARS microscopy and created a novel method in microscopic imaging.

The Analysis of ellipticity in the polarization of the CARS radiation, e.g. by filtering out the elliptically polarized portions for detection and suppression of not elliptic-polarized Shares, leads ultimately a chiral-sensitive CARS microscopy. The ellipsometric Polarization analysis of the CARS signal after previous parallel Polarization of pumping and Stokes radiation contrasts in the microscopic Imaging the nonlinear Raman optical activity. In order to are molecular structures like Proteins, DNA, RNA, polysaccharides, etc. selectively detectable. non-resonant Underground is also suppressed.

The Microscope according to the invention is suitable for non-point-symmetric molecules, which is why chirality, So a preference for circular polarization states with show specific direction of rotation in CARS activity.

Essential For this Approach is that pumping and Stokes radiation with the same linear polarization in the sample space or invade the sample volume. It is therefore preferred that the sample illumination device brings the pumping and Stokes radiation in superposition and then the Polarizer unit the linear polarization of the superimposed Radiation causes. This approach ensures that the two Radiations are identically linearly polarized.

The ellipsometric analysis of the CARS radiation can in principle be carried out by any suitable means. Advantageously, the Pola Risk analysis unit on a lambda / 4-plate.

Especially simple is the analysis when the polarization analyzer unit the elliptically polarized portions in linearly polarized radiation The polarization direction is independent of the elliptical polarization is adjustable. This adjustability ensures that the subsequent analysis the converted in linear polarization elliptical polarization without possibly disturbing polarization dependent Effects in the detector or the detector optics can be done. Otherwise could one possible polarization dependence superimpose or suppress the effect to be measured on the subsequent optics.

A especially functional and easy to implement variant of the polarization analyzer unit uses a series combination of a lambda / 4 element and a linear polarization filter. If you switch between a lambda / 2 element, is the mentioned one independence the linear polarization direction of the elliptical polarization given that by turning the lambda / 2 element a constant Orientation of the polarization direction of the converted radiation can ensure. Polarization-dependent reflections of the following Optics, as they occur for example in optical reflection gratings can, then do not work for different elliptical polarization states different, but can always minimized evenly become.

Around the CARS microscope for Apply different elliptic polarization states particularly convenient to be able to it is advantageous, the polarizer unit, the linear polarizing filter and, if present, the lambda / 2 element in terms of their for the Polarization effective direction adjustable to design, for example rotatably in the beam path to keep.

A preferably Fully automatic microscope can be reached by using a control unit, that the Polarization unit, the linear polarizing filter and, as far as present, also controls the lambda / 2 element and this regard her for sets the polarization effective direction and / or reads.

The introduction of the lambda / 2 element a polarization-invariant detection in the analysis of NL-ROA content in the signal, e.g. by the rotation of the element and a fixed analyzer setting. The elliptical polarization will not be affected by the rotation of the Measure analyzer. By this approach, the polarization remains at the detector constant in their orientation. This is extremely cheap to polarization dependent Reflections on the subsequent optics, e.g. on optical reflection gratings, to avoid. The polarization dependence of the following Optics could otherwise overlay the NL-ROA effect or suppress.

The Lambda / 2 element allows at rotation a variable setting a heterodyne gain of NL-ROA by the orthogonally polarized CARS portion from the electrical Dipole moment. Turning the element results in a variable ratio in the projection of these two signal components onto the analyzer level, without this must be turned. Thus, a variable heterodyne gain is without rotation in the polarization the detected signal possible. This is as discussed above due to the polarization dependence the subsequent optics of advantage.

The Lambda / 2 element allows further polarization methods from the classical CARS spectroscopy / microscopy without the utilization of chiral information, so that a suppression of the non-resonant background or selection of different tensor components CARS process is realized. For this purpose, a microscope is favorable, the a rotation of the polarization of the Stokes radiation with respect to Pump radiation e.g. effected by means of a lambda / 2 plate. For analysis the signal will not be delayed set between orthogonal polarization components, and the polarization analyzer unit selects the desired polarization components. Thus, this polarization method established in CARS microscopy is in Combination with the for chiral-sensitive NL-ROA measurements be modified.

The Microscope according to the invention achieves the necessary sensitivity for the detection of NL-ROA components in the CARS radiation. Both the complete information from polarized CARS measurements in achiral systems as well as the information from NL-ROA and / or Optical Rotation Dispersion (ORD) in chiral samples is accessible. The invention clearly shows advantages over other chiral-sensitive ones Techniques. Methods that examined optical activity are based on Bulk up a difference signal. This must be left and right circular polarized input radiation, the signal can be measured, or the proportion of left- and right-circularly polarized light in the signal be determined. Difference procedures, however, lead to a clear slower data recording. Direct measurement of NL-ROA content achieves the high speed required for a laser scanning process. additionally the difficulties associated with switching between left and right and right circularly polarized light occur and especially at the elevated Bandwidth of pulsed sources are to be avoided.

Of the Invention is further based on the object, a CARS microscope specify that an improved spatial resolution, especially in the depth direction having. To the solution This object is inventively a microscope provided of the type mentioned, which is a device for Generation of a reference radiation, the pump and the Stokes radiation phase-locked, and means for interferometric superposition the reference radiation with the CARS radiation at the detector has.

Of the Use of phase-coupled pump and Stokes lasers in a CARS process as well as phase-locked heterodyne detection of the CARS signal with reference radiation provides an improvement in the axial resolution of up to two orders of magnitude across from classical diffraction-limited imaging methods. The exact resolution is dependent from the stability the phase coupling and the spatial stability the involved laser pulses and the signal as well as the wavelength.

The Microscope according to the invention realizes an interferometric mapping to increase the axial resolution combined with a laser scanning method for lateral imaging and also allows a time resolution in the imaging, which only by repetition rate and detector speed or signal strength and detector sensitivity is limited. Thus, a single-shot signal detection and simultaneous real-time imaging possible.

Thus, according to the invention a CARS microscope with an interferometric detection method Mistake. The CARS microscope leads So by means of an interferometer a holographic, microscopic imaging, combining this with the intrinsic marker-free molecular recognition and nonlinearity of the CARS process. essential Feature is the phase coupling between pump, Stokes and Reference radiation, or corresponding laser pulses. in principle it is possible, the phase-sensitive invention CARS microscope in two different optical arrangements too realize. So is a confocal radiation arrangement with a Laser scanning approach possible for imaging. Also, a non-collinear beam arrangement may be used as described, for example, in the publication Heinrich C. et al., Appl. Phys. Lett., 2005, 84, 812-818.

The Reference radiation may i.a. in two different ways be won. On the one hand, an optically parametric process by a suitable optical parametric Oscillator used to pump and Stokes radiation and so that it is phase-locked to the CARS radiation. On the other hand, the Device for generating the reference radiation the reference radiation from the superimposed Generate pump and Stokes radiation. She has suitable means for this to overlay the Pump and Stokes radiation on. Preferably, for the production The reference radiation uses a separate CARS process that provides the desired reference radiation as CARS radiation of a CARS process independent of the sample measurement provides. Such generated reference radiation is then automatically phase-locked for pumping and Stokes radiation.

As mentioned, For example, a scanner device may be used for imaging be superimposed with the sample space Scanned pump and Stokes radiation and absorbs the CARS radiation abscannend. This delivers the desired Imaging.

alternative can also the mentioned non-collinear arrangement can be used as mentioned in the publication is described. Then a phase grating of two arbitrarily expanded Pump laser radiation pulses with a non-collinear Stokes laser radiation pulse sampled. The pump laser radiation then does not have to be scanned. The combination with the phase coupling to the reference radiation reaches then an interferometrically increased axial resolution and at the same time a high time resolution, since no scanning process is necessary for image acquisition, if as a detector an imaging detector is used. Pump, Stokes and reference radiation are then appropriately expanded to cover the image as a whole.

The The invention will be described below with reference to the drawing by way of example explained in more detail. In the drawings shows:

1 a schematic representation of a CARS microscope for chiral-sensitive CARS microscopy,

2 5 is a schematic illustration illustrating the state of the art CARS principle;

3 a schematic representation of a CARS microscope for phase-sensitive CARS microscopy, wherein the reference radiation is generated by an optical-parametric process,

4 a representation similar to the 3 wherein the reference radiation is generated from the pump and Stokes radiation of the CARS microscope,

5 a CARS microscope for phase-sensitive CARS microscopy with a non-collinear Arrangement of pump and Stokes radiation, wherein the reference radiation is generated by an optical parametric oscillator, and

6 a CARS microscope similar to the one of 5 wherein the reference radiation is generated from the pump and Stokes radiation.

In 1 is a microscope 1 which follows the principle of collinear forward CARS microscopy, ie the generated CARS radiation is obtained in the transmitted light mode of a sample. Alternatively, however, backscatter can also be evaluated in the sense of the known epi-CARS process. The forward CARS microscope arrangement shown here has the advantage that a signal with significantly higher intensity is obtained. This is advantageous in view of the low intensities to be expected in the non-linear Raman optical activity (NL-ROA) to be detected. This in 1 The microscope shown accomplishes a polarization analysis of the CARS radiation to suppress the non-resonant CARS signal or to heterodyne amplification of the NL-ROA portion. In addition to the collinear arrangement shown, another CARS setup can be used.

This in 1 shown CARS microscope 1 has a pump laser 2 , the pump radiation 3 and a Stokes laser 4 , the Stokes radiation 5 gives up. The rays from pump laser 2 and Stokes lasers 4 become by means of a dichroic mirror 5 superimposed so that they run collinear. The superimposed radiation falls through a polarizer 7 that the superimposed pump radiation 3 and Stokes radiation 5 linearly polarized. The thus polarized radiation 8th is from a lens 9 focused. The polarizer 7 and the lens 9 together form a lighting device 10 that a sample 11 with focused, superimposed pump radiation 3 and Stokes radiation 5 illuminated. In focus 12 So then lies a superimposed, in the same direction linearly polarized mixture of pump radiation 3 and Stokes radiation 5 before, wherein the polarization directions preferably vary only by the different angles of incidence in the focus.

The radiations are pulsed so controlled that in focus 12 a CARS process is triggered. The resulting sample radiation 13 is received in transmissive mode (in an epi-CARS array in reflective mode) at the angle predefined by the CARS process and by a lens 14 collected. A downstream high-pass filter 15 separates any transmitted pump or Stokes radiation 3 . 5 off, so that after the high pass filter 15 only CARS radiation 16 is present. The CARS radiation 16 then passes through a lambda / 4 plate, the elliptically polarized portions of the CARS radiation 16 linearly polarized.

A downstream lambda / 2 plate 19 which is rotatable, allows the generated linear polarized radiation to rotate in the direction of polarization. After the lambda / 2 plate is linearly polarized, rotated radiation 20 in front. An analyzer 21 analyzes the polarization direction and leaves only the CARS radiation originating from a certain elliptical polarization state 16 happen. After the analyzer 21 is therefore filtered radiation 23 in front. The lambda / 4 plate, the lambda / 2 plate and the analyzer 21 Together they represent a polarization analysis unit that provides an ellipsometric analysis of the CARS radiation 16 performs. Of course, other implementations of the polarization analysis unit are also 22 possible.

The filtered radiation 23 is from a detector optics 24 on a spectral analyzer 25 which acts as a detector and the filtered radiation 23 spectrally analyzed to identify specific components of the CARS process in the sample 11 take place, to select. The lambda / 4 plate 17 compensates for a phase shift between orthogonally polarized radiation components of the CARS radiation 16 , The rotation in the linear polarization can then be determined by the angle to the lambda / 2 plate 19 must be turned so that the radiation is the analyzer 21 can happen.

The polarization analysis unit 22 analyzes the elliptical portion of CARS radiation 16 , which correlates directly with the NL-ROA fraction of the total radiation. The CARS microscope 1 In addition, it can analyze the linear polarization and thus select different tensor components of the CARS signals that do not have NL-ROA contribution, thus achieving selective detection according to the symmetry of the molecular system (s). In addition, the non-resonant part of classical CARS microscopy is suppressed.

To analyze the polarization of CARS radiation 16 is determined by means of the polarization analysis unit 22 made a differentiation between two signal components. The total signal has a contribution from electrical dipole portion of a CARS process, this dipole portion carries no information about an optical activity of the sample. There is also a contribution in the form of the NL-ROA signal, which originates from an electric quadrupole and a magnetic dipole component and carries information about the chirality of the sample.

The analysis makes use of the fact that the NL-ROA content in the CARS radiation 16 with a phase shift of 90 degrees to the optically inactive portion of the CARS radiation 16 and that further the NL-ROA moiety has an orthogonally polarized moiety to the optically inactive moiety. This is possible with the CARS microscope 1 different polarization analyzes.

To determine if the sample 12 an NL-ROA content in the CARS radiation 16 caused, one can first the rotation angle of the lambda / 4-plate 17 opposite the lambda / 2 plate 19 and the analyzer 21 firmly set so that a phase shift of 90 degrees between the dipolar, ie optically inactive portion of the CARS radiation 16 and the NL-ROA component is compensated. The superposition of these orthogonally polarized portions becomes a rotation in the linear polarization of the total signal. The exact rotational value can be adjusted by means of suitable adjustment of the lambda / 2 plate 19 and the analyzer 21 be determined. For this purpose, the analyzer 21 90 degrees to the polarizer 7 aligned so that linear polarization is blocked from the dipolar portion of the CARS radiation.

The intensity of the signal after the analyzer 9 is then recorded as a function of the angle of rotation of the lambda / 2 plate. For an achiral material in the sample 11 the minimum would be expected at a rotation angle of 0 degrees. If the minimum lies at a different angle of rotation, there is an NL-ROA component. In this way, the polarization analysis unit analyzes 22 the elliptical portion of the CARS radiation 16 which correlates directly with the NL-ROA fraction and confirms the presence of an NL-ROA fraction.

The sample is imaged for NL-ROA content 11 using pump and Stokes laser radiation 3 . 5 spatially scanned by suitable means. This is for example in the lighting device 10 a scanner (not shown) is provided. The NL-ROA fraction in the entire CARS signal is determined and as a function of the position in the sample 11 recorded.

This is done by adjusting the Lambda / 4 plate as described above and turning the analyzer 90 degrees into a polarizer 7 aligned to block the dipolar, ie linearly polarized portion of the CARS radiation. Further, the lambda / 2 plate is set to a rotation angle that matches the contrast between NL-ROA fraction and optically inactive CARS signal. At high contrast, an angle of almost 0 degrees can be set to largely suppress the non-resonant ground. An angle >> 0 degrees is chosen to contrast the NL-ROA component as much as possible. The angle selection thus allows a variable adjustment of the contrast between NL-ROA content and optically inactive content coupled with the background suppression and heterodyne enhancement of the NL-ROA content by the linear-polarized component.

As a result, the plot of the detected signal intensity as a function of the location coordinate is automatically an image of the molecular components of the sample with the selected Raman resonance, ie, in the selected spectral analysis in the spectral analyzer 21 , wherein the imaged components have a chiral structure, that are optically active.

The detection of the NL-ROA moiety can now preferably with the much more intense, non-linear portion of the CARS radiation 16 , which has a maximum of 10 ⁶ to 10 ⁸ times the intensity value, be heterodyne amplified. For this purpose, the lambda / 2 plate is adjusted by a small value of 0 degrees. At this setting, both the NL-ROA content and the linear portion of the CARS radiation have 16 a proportion in the projection on the polarization plane of the analyzer 21 , The two signals are thus at a suitable adjustment of the lambda / 4 plate to 90 ° phase shift of the orthogenically polarized CARS radiation in phase at the spectral analyzer 25 So mixed at the detector. The NL-ROA component is thus heterodyne-enhanced with the dipolar, linear-polarized radiation component.

The Polarization analysis also offers the possibility of optical rotation dispersion (ORD) in a sample and analogous to the NL-ROA as Contrast in imaging to use. For this purpose, the lambda / 4 plate removed from the beam path. ORD in an optically active sample causes a dispersion in the rotation of the linear polarization of pump and Stokes radiation and thus leads to a rotation in the linear polarization of the entire CARS signal. This rotation can be determined by the analyzer 90 ° to the polarizer adjusted and the signal as a function of the rotation angle of the lambda / 2 plate is recorded. In achiral substances no rotation of the Total polarization of the CARS signal instead, while optically active or chiral Sample a rotation angle ≠ 0 exhibit.

Imaging of ORD becomes the sample 11 using pump and Stokes laser radiation 3 . 5 spatially scanned by suitable means. For this purpose z. B. in the lighting device 10 a scanner (not shown) is provided. ORD is determined by the rotation in the linear polarization of the CARS signal and as a function of the position in the sample 11 recorded.

For this purpose, the lambda / 4 plate, as described above, removed from the beam path, and the analyzer is 90 ° to the polarizer 7 aligned. The lambda / 2 plate is set at a rotation angle that provides good contrast between the rotation angles of the linear polarization of the CARS signal of a sample with ORD and an optically inactive sample. With a high contrast ratio, an angle of almost 0 ° can be set, and an angle> 0 is chosen to characterize ORD with the highest possible contrast, but coupled with an increased background signal for large angles of the lambda / 2 plate.

hereby is the plot of the detected signal intensity as a function The coordinate of the place automatically maps the molecular components of the Sample which are optically active and thus chiral structural elements have.

The detection of the filtered radiation 23 takes place by means of the spectral analyzer 25 through a monochromator and a multichannel detector. As a result, different frequency components in the filtered radiation can be detected simultaneously. As a result, contributions from different Raman resonances, which in their overlay could lead to the suppression of the NL-ROA fraction, are detected separately. Moreover, in the case of a spectrally resolved signal, such as the spectral analyzer 25 takes into account the dispersion of the lambda / 4 and lambda / 2 plate delay.

3 shows a CARS microscope 1 additionally or independently for the polarization analysis according to 1 , which performs a chiral-sensitive CARS microscopy, allows a phase-sensitive CARS microscopy. The microscope 1 of the 3 Of course, schematically illustrated, therefore, combines an interferometer arrangement to increase the resolution in the axial direction with a Raman resonance enhancement of a CARS process for molecular recognition. The basis 1 Pictured microscope corresponding components are in the representation of 3 (as well as in the 4 to 6 ) provided with the same reference numerals, so that can be dispensed with a repeated description of these elements here.

The CARS microscope 1 of the 3 is contrary to the microscope 1 further formed as an epi-CARS arrangement, ie the pump and Stokes radiation 3 . 5 is called superimposed radiation 27 in a sample 11 focused and the CARS radiation 16 is opposite to the irradiation direction by using a dichroic beam splitter 28 through the same lens 9 collected, which also causes the lighting. Essential for the phase-sensitive CARS microscope 1 of the 3 is a reference source 26 that contribute to the superimposed radiation 27 and thus also to CARS radiation 16 provides phase-locked reference radiation. Their importance will be discussed later. Essentially, the principle is used as described in the publication, Dubois F. et al., Applied Optics, 1 December 1999, Vol. 38, No. 34, pp. 7085-7094.

For imaging, the superimposed radiation 27 ie the collinear combination of pump radiation 3 and Stokes radiation 5 by means of a scanner 29 , the two scanning mirrors 31 . 32 has, distracted, so that the focus 12 scanned over the sample. In return, the scanner causes 29 automatically de-scanning, so that the beam splitter 28 a static beam of CARS radiation 16 is transmitted. This CARS radiation 16 becomes at another dichroic mirror 33 Reference radiation 34 from the reference source 26 superimposed, so that then an interfering radiation 35 present, by means of the detector optics 24 on one as a CCD detector 47 trained detector is directed.

The reference radiation 34 is from the reference source 26 in the embodiment of the 3 provided by an optical parametric process so that the radiation of the pulsed pump laser 2 , also operated pulsed Stokes laser 4 and therefore also the pulsed CARS radiation 16 phase-coupled to the likewise pulsed reference radiation 34 is.

The phase coupling between CARS radiation 16 and reference radiation 34 realizes a heterodyne interferometric detection of the two fields. The detected intensity with a collinear superposition of the two beams is a measure of the phase shift between the CARS signal and reference radiation and thereby reflects a change in the refractive index in the lateral image plane with an interferometric resolution in the axial direction. Thus, the combination of an imaging of the lateral plane of a sample with interferometric resolution along with molecular recognition based on the Raman resonance enhancement of a CARS process is achieved.

To generate the reference radiation 34 can instead of an external, phase-locked reference source 26 also the pump and Stokes radiation 3 . 5 be used. This shows exemplarily the 4 , This is done at the beam splitter 28 the superimposed radiation 27 not just partially towards the sample 11 but a part passes the beam splitter 28 and falls through a lens 36 in a Raman medium 37 , In this Raman medium 37 a separate CARS process is initiated with its CARS radiation from a lens 38 taken and a retroreflector 39 with me chanical measuring table as a delay unit and a variable attenuator 40 as reference radiation 34 again the dichroic mirror 33 fed. Because the reference radiation 34 Thus comes from another CARS process, is automatically phase coupling to CARS radiation 16 ensured. By scanning the delay unit, the interference pattern between reference radiation and CARS radiation in the time domain can be recorded and obtained by Fourier transformation. As a result, the fluctuation in the phase relationship of the laser and reference can be compensated, which would otherwise lead to a reduction of the spatial resolution.

The 5 and 6 show other interferometric structures, but here in a non-collinear arrangement, as described in the already mentioned in the introduction and here in its disclosure content publication of Heinrich et al. is described. In deviation to the construction of the 3 and 4 here is the pump radiation 3 via a deflection mirror 41 and a darkfield condenser 42 from below into the sample 11 irradiated. Two non-collinear pump radiation pulses thus generate a phase grating, that of one through the lens 9 extended pulse of Stokes radiation 5 is scanned. The result is an expanded CARS radiation 16 , 5 shows as well as the 6 only the main beam axes. The superposition with phase-locked reference radiation 34 from a reference source 26 by means of a dichroic mirror 33 again leads to interfeteormetric imaging at the CCD detector 47 , The difference between the 5 and 6 is analogous to the 3 and 4 again to see that in 5 an external optical parametric oscillator as a reference source 26 is used, whereas in 6 (as well as in 4 ) the reference radiation 34 by another CARS process directly from the pump radiation 3 and the Stokes radiation 5 is produced.

Claims (10)

CARS microscope with at least one pump radiation ( 3 ) emitting pump laser source ( 2 ) and a Stokes radiation ( 5 ) emitting Stokes laser source ( 4 ), a sample illumination device ( 6 . 10 ), pumping and Stokes radiation ( 3 . 5 ) in a sample room ( 11 ) and a detector ( 25 ) by a CARS process in the sample room ( 11 ) emitted CARS radiation ( 16 ), characterized in that the sample illumination device ( 6 . 10 ) a polarizer unit ( 7 ), which pump and Stokes radiation ( 3 . 5 ) polarized linearly and parallel to each other, and that the detector ( 25 ) a polarization analyzer unit ( 22 ), which is an ellipsometric analysis of the CARS radiation ( 16 ). Microscope according to claim 1, characterized in that the sample illumination device ( 6 . 10 ) Pump and Stokes radiation ( 3 . 5 ) and then the polarizer unit ( 7 ) causes the linear polarization of the superimposed radiation. Microscope according to one of the preceding claims, characterized in that the polarization analyzer unit ( 22 ) elliptically polarized portions of CARS radiation ( 16 ) into linearly polarized radiation ( 23 ), wherein the polarization direction is adjustable independently of the elliptical polarization. Microscope according to one of the preceding claims, characterized in that the polarization analyzer unit ( 22 ) a series connection of lambda / 4-element ( 17 ) and linear polarization filter ( 21 ), preferably with an interposed lambda / 2 element ( 19 ). Microscope according to claim 4, characterized in that the polarizer unit ( 7 ), the linear polarizing filter ( 21 ) and, if present, the lambda / 2 element ( 19 ) are adjustable in terms of their direction of polarization effective. Microscope according to claim 5, characterized by a control unit which controls the polarization unit ( 7 ), the linear polarizing filter ( 21 ) and, if present, the lambda / 2 element ( 19 ) and adjusts them with respect to their direction of polarization effective. CARS microscope with at least one pump radiation ( 3 ) emitting pump laser source ( 2 ) and a Stokes radiation ( 5 ) emitting Stokes laser source ( 4 ), a sample illumination device ( 6 . 10 ), pumping and Stokes radiation ( 3 . 5 ) in a sample room ( 11 ) and a detector ( 25 ) by a CARS process in the sample room ( 11 ) emitted CARS radiation ( 16 ), characterized by a device ( 26 ) for generating a reference radiation ( 34 ), which contribute to the pumping and Stokes radiation ( 3 . 5 ) is phase-locked, and means ( 33 ) for the interferometric superimposition of the reference radiation ( 34 ) with the CARS radiation ( 16 ) at the detector ( 25 ). Microscope according to claim 7, characterized in that the device ( 26 ) for generating the reference radiation ( 34 ) the reference radiation ( 34 ) has an optical parametric oscillator. Microscope according to claim 7, characterized in that the device ( 26 ) for generating the reference radiation ( 34 ) from the superimposed pump and Stokes radiation ( 3 . 5 ) generated. Microscope according to one of the preceding claims, characterized by a scanner device ( 29 ), the sample space ( 11 ) with superimposed pump and Stokes radiation ( 3 . 5 ) and CARS radiation ( 16 ) absorbs.