DE102010047578A1 - Use of a combination of evaluation methods in a device for the detection of tumors and device for the detection of tumors - Google Patents

Abstract

The present invention relates to a combination of evaluation methods in a device for detecting tumors, the combination of the evaluation methods including the evaluation method of multiphoton excitation and / or the evaluation method of generating the second harmonic, as well as either the evaluation method of coherent anti-Stokes-Raman scattering or the stimulated Raman scattering evaluation method. In order to improve the security in the detection of tumors, it is proposed within the scope of the invention to combine the mentioned evaluation methods.

Description

The present invention relates to a use of a combination of evaluation methods in a device for detecting tumors according to claim 1 and a device for detecting tumors according to claim 2 or 3.

It is known in the art to use for detection of tumors one of the methods of multiphoton fluorescence, second harmonic generation or coherent anti-Stokes Raman scattering.

In contrast, the present invention is based on the object to improve the safety in the detection of tumors.

This is advantageously achieved according to the present invention by a combination of the mentioned evaluation methods.

Claim 1 relates to the use of a combination of evaluation methods in a device for detecting tumors, wherein the combination of the evaluation methods comprises the multiphoton excitation evaluation method and / or the second harmonic generation method, and furthermore either the evaluation method of the coherent anti-Stokes-Raman Scattering or the evaluation method of stimulated Raman scattering includes.

It has been shown that it is precisely through the combination that the evaluation reliability can be improved. Depending on whether the tumors to be examined have a multiphoton autofluorescence, this evaluation method may or may not be taken into account.

Claim 2 relates to a device for detecting tumors using a combination of evaluation methods, wherein the combination of the evaluation method comprises the multiphoton excitation evaluation method and / or the second harmonic generation evaluation method and furthermore the coherent anti-Stokes-Raman scattering evaluation method. wherein the device comprises a femtosecond oscillator or a picosecond oscillator whose output signals are supplied to an optical parametric oscillator, wherein the output signals of the optical parametric oscillator are projected onto the skin of a person to be examined by means of a focusing device, wherein scattered signals with a frequency , which results as a frequency due to the coherent anti-Stokes-Raman scattering from the two frequencies of the optical parametric oscillator, are analyzed in terms of their intensity, the Vorrichtun g further comprises means for blanking one of the output signals of the optical parametric oscillator for performing the evaluation method of multiphoton excitation and the evaluation method of generating the second harmonic.

It proves to be advantageous that several components can be used together to carry out the different evaluation methods. Said device is suitable for using the coherent anti-Stokes-Raman scattering evaluation method.

Claim 3 relates to a device for the detection of tumors using a combination of evaluation methods, wherein the combination of the evaluation method comprises the multiphoton excitation evaluation method and / or the second harmonic generation method and further the Raman scattering evaluation method; Femtosecond oscillator or a picosecond oscillator whose output signals are supplied to an optical parametric oscillator, wherein the output signals of the optical parametric oscillator are projected by means of a focusing on the skin of a person to be examined, wherein scattered signals of the frequency of Pumpfeldes and / or the Frequency of the Stokes E-field, are analyzed in terms of their intensity, the apparatus further comprising means for masking one of the output signals of the optical parametric oscillator for carrying out the multiphoton excitation evaluation method and the second harmonic generation evaluation method.

It also proves to be advantageous that several components can be used together to carry out the different evaluation methods. Said device is suitable when using the evaluation method of stimulated Raman scattering.

In the embodiment according to claim 4, the output signals of the optical parametric oscillator and the backscattered signals are distinguished by the polarization planes of the signals are rotated in the path of the signals.

This embodiment again relates to the stimulated Raman scattering, because in this case the frequencies of the scattered radiation coincide with the frequencies of the introduced radiation. The evaluation takes place in this case in that the scattered radiation amplifies the radiation introduced with respect to one frequency and with respect to the other Frequency is weakened. In order to distinguish the scattered radiation from the introduced radiation, these radiations are polarized differently.

The object of the present invention is therefore an apparatus for performing optical tomography of the skin, wherein the imaging signals by generation and detection of multiphoton fluorescence, coherent anti-Stokes Raman scattering (CARS) or the second harmonic (SHG) of the incident light arise. To generate the signals, one or more laser beams of pulsed lasers are focused on or into the skin and the focus volume is adjusted so that the area to be examined is scanned one after the other (pixel by pixel). The signals generated in the focus volume are collected and detected by the same focusing optics against the beam direction. By transferring the signal intensities into gray values of the corresponding pixels, an image is generated. In multiphoton fluorescence, two or more photons of naturally occurring molecules in the skin are absorbed per excitation process, and these are excited to autofluorescence, which is detected. In the generation of the second harmonic a special coherent scattering process takes place in which two simultaneously irradiated photons of the same energy are converted into a photon with twice the energy. The coherent anti-Stokes-Raman scattering is caused by simultaneously irradiated pump, Stokes and test photons and leads, when the energy difference of the injected pump and Stokes photons just a vibration transition of the molecule corresponds to a resonance-enhanced coherent molecule-specific signal. It also allows the detection of non-autofluresszierender or SHG-producing substances. In tissue, for example, this allows the detection of lipids and water.

Coherent anti-Stokes Raman microscopy allows imaging of chemical or biological samples using molecular vibration transitions as a contrasting mechanism. In CARS microscopy / tomography at least two laser fields, an electromagnetic pumping field with a central frequency ω _p and a Stokes electromagnetic field with central frequency ω _s are used. The interaction of pump and Stokes fields with the sample generates a coherent anti-Stokes field of frequency ω _AS = 2ω _p -ω _s in the direction dictated by the phase matching. In particular, when the frequency difference ω _p - ω _s corresponds to a certain vibration transition, a CARS signal is generated at the anti-Stokes frequency ω _AS . In contrast to fluorescence microscopy tomography, CARS microscopy (as well as SHG microscopy) does not require fluorophores because the imaging mechanism is based on vibrational excitation of chemical and biological samples. Due to the coherence properties of CARS microscopy, it provides significantly higher sensitivity than spontaneous Raman microscopy, which means that a significantly lower average excitation power can be used or a shorter measurement time is necessary.

Microscopy based on coherent anti-Stokes Raman scattering is already described in a number of patents. The U.S. Patent No. 4,405,237 deals with a CARS imaging device in which the sample is illuminated simultaneously by two laser pulse trains with different wavelengths, which are spatially and temporally superimposed. In combination with an area detector, the signal beam generates the spatial resolution in a phase-adapted direction.

The US Patent No. 6108081 deals with an apparatus and a method for the microscopic imaging of molecular vibrations with the help of CARS with a collinear excitation geometry. In the apparatus described there, collinear pump and Stokes beams are focused onto the sample with the aid of a high numerical aperture objective. Due to the non-linear dependence of the signal on the excitation intensity signals are generated only in the small focus volume, so that a three-dimensional scanning of the sample is possible. The signals are detected only in the beam direction.

In contrast to the detection of the signal in the beam direction treats this U.S. Patent No. 6934020 an apparatus in which the signal from the sample is detected in the reverse direction (opposite to the beam direction, Epi-CARS). This reverse signal typically has a higher signal-to-noise ratio, but is weaker in many applications than the signal simultaneously generated in the forward direction.

The U.S. Patent 7,414,729 describes an endoscopy system in which at least two stimulating electromagnetic fields and the generated signal are transmitted through an optical fiber system. The signal is again generated by the non-linear interaction of the exciting electromagnetic fields with the sample volume and passed through the same fiber as the exciting fields to the detector.

The subject matter of the present invention is a tomograph which generates and detects signals in a spatially resolved manner by non-linear optical excitation. The image signals are generated by pixel-by-pixel detection of the signal intensities. The spatial resolution is generated by minimizing the focus volume since only in this volume do signals arise. The signals to be detected are multiphoton autofluorescence, the second harmonics of the incident light and CARS light. For the generation of CARS signals, at least two collinear laser beams with different wavelengths must be focused by a microscope objective on the sample in a common focus volume. The signal light must be collected and (spectrally) separated from the incident radiation and detected. For generating the irradiated laser beams for generating the CARS signal, an oscillator in combination with an optical parametric oscillator (OPO) can be used. The laser beams can be moved over the skin with the aid of scanner mirrors and the collected signal can be detected with a photomultiplier. For the generation of multiphoton autofluorescence and SHG, the use of a single laser beam is sufficient, the second laser beam can be blocked in this mode of operation. For the generation of multiphoton autofluorescence. SHG and CARS generally require different excitation wavelengths and filters.

Exemplary embodiments of the tomograph for carrying out optical skin examinations on the basis of multiphoton excitations, the generation of the second harmonic and coherent anti-Stokes-Raman scattering are described below.

1 shows an embodiment of the combined multiphoton excitation CARS tomograph. The excitation is carried out by a femtosecond oscillator and an optical parametric oscillator. The pulses of a femtosecond oscillator (a) (eg, MaiTai HP Spectra Physics) are transformed into a Optical Parametric Oscillator (OPO) (c) (e.g., Chameleon The OPO generates sig- naled signal and idler pulses that emerge through an opening at the side, in addition to which the OPO has an exit opening for the oscillator pulses.) Oscillator and Stokes pulse trains are controlled by means of a delay line and a dichroic Spatially and temporally superimposed mirrors (d) The pulses are then focused on the skin to be examined via a scanner-detector module (e) and a mirror arm with focusing optics.

The (mirror) scanner is used for pixel-by-pixel scanning of the sample with the stimulating pulses. The generated signals (depending on the configuration CARS, autofluorescence, SHG) are collected by the same focusing optics and passed through the mirror arm (f) back into the scan detector module (e). Within the scan detector module (e), stimulating radiation and signal are separated by a dichroic mirror and the signal is detected.

The beam path of the exciting laser pulses and the generated signal is in 2 shown. To generate autofluorescence or SHG signals, a single beam is sufficient. For this purpose, either the oscillator, signal or Idlerpulse be used and blocked the other beams each. Depending on the signal to be measured z. B. by displaceable tabs suitable filter introduced into the signal beam path z. B. Color glass filters BG 39 for the detection of SHG and autofluorescence, and suitable bandpass filters for the exclusive detection of SHG or CARS signals. The superimposed Stokes and oscillator pulses are used for the excitation of CARS signals z. From water (eg, central wavelength of the oscillator pulses: 810 nm, signal pulses: 1105 nm, CARS signal at 639 nm, filter inserted: bandpass 624/40), or the CH ₂ stretch vibration used in lipid molecules (e.g. B. oscillator pulses: 810 nm, signal pulses: 1052 nm, CARS signal at 658 nm, bandpass filter 660/10). Instead of an oscillator, another laser is conceivable that can generate pulses in the fs range.

In another embodiment, a picosecond oscillator may be used in the described construction. The pulse duration of the OPO pulses is then also in the picosecond range. With these pulses, CARS can achieve higher spectral accuracy of excitation, but the pulse intensities available to excite autofluorescence are lower.

To generate the CARS signal, it is also possible to use two (or three) individual synchronized lasers (oscillators) (fs / fs, ps / ps, fs / ps). In the embodiment of 1 then the OPO c) is to be replaced by a second oscillator. In this case, an additional temporal synchronization of the pulses is necessary, with sufficiently high accuracy is then the use of a delay path to generate a temporal pulse overlap (in 1 with "(d)") no longer necessary. It is possible to use fs or ps oscillators or, for a high spectral accuracy of the CARS excitation (fs / ps) and high efficiency of the SHG / autofluorescence generation, a combination of ps- and fs-oscillators (multiplex-CARS).

To excite the CARS signals, a combination of oscillator pulses and supercontinuum, ie (oscillator) pulses whose spectrum is propagated through transmission through a fiber and the interaction process taking place (self-phase modulation), can be used. In this embodiment, (c) in Figure 1 is replaced by a medium for generation of a supercontinuum. The entire supercontinuum does not have to be used for the CARS process. B. with suitable filters - suitable spectral ranges are filtered out.

In another embodiment, one of the previously described possibilities is used to generate the exciting electromagnetic fields and to generate autofluorescence / SHG signals. However, instead of a CARS signal, another non-linear Raman technique is used. One such technique is stimulated Raman scattering (SRS). Two E-fields (pump and Stokes) of different wavelengths are simultaneously irradiated onto the sample and spatially superimposed.

If the energy difference of pump and Stokes E fields corresponds to the energy of a vibration transition, then the molecule can be excited to vibrate. In such a stimulated Raman process, the Stokes E field is amplified by stimulated emission and the pump field is attenuated. The intensity of this increase in intensity or attenuation is used as a signal for imaging. SRS measurement in epi geometry does not allow for spectral separation of exciting rays and backscattered signal.

In this case, according to the representation of the 3 For example, for a separation, a beam splitter may be used which either passes or reflects differently polarized light. For this purpose, a quarter-wave plate is placed in the excitation / signal beam path, which circularly polarizes the linearly polarized exciting radiation. The amplified or attenuated signal light passes through the quarter-wave plate a second time on the way to the detector and is thus linearly polarized with a plane of polarization shifted by 90 ° with respect to the exciting radiation. Exciting light and signal light are then spatially separated by a polarization beam splitter and the signal light is detected. To detect this small increase in Stokes pulses or decrease in pump pulses, the lock-in technique is used. This is z. B. modulates the intensity of the Stokes beam at high frequency and detects the intensity of the pump beam at the modulation frequency with a lock-in amplifier. Alternatively, the pump beam can also be modulated and the intensity of the Stokes beam can be detected. For the detection of SHG / autofluorescence signals, reference is made to the in 2 converted configuration by the detector and filters and beam splitters are mounted on slidable riders.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 4405237 [0015]
• US 6108081 [0016]
• US 6934020 [0017]
• US 7414729 [0018]

Claims (3)

Use of a combination of evaluation methods in a device for the detection of tumors, wherein the combination of the evaluation method comprises the multiphoton excitation evaluation method and / or the second harmonic generation method, and furthermore either the coherent anti-Stokes-Raman scattering evaluation method or the evaluation method stimulated Raman scattering. A device for the detection of tumors using a combination of evaluation methods, wherein the combination of the evaluation methods comprises the multiphoton excitation evaluation method and / or the second harmonic generation evaluation method and furthermore the coherent anti-Stokes-Raman scattering evaluation method, characterized in that the device comprises a femtosecond oscillator or a picosecond oscillator whose output signals are supplied to an optical parametric oscillator, the output signals of the optical parametric oscillator being projected onto the skin of a person to be examined by means of a focusing device, scattered signals having a frequency, which results in the frequency due to the coherent anti-Stokes-Raman scattering from the two frequencies of the optical parametric oscillator, are analyzed in terms of their intensity, the device Further comprises means for blanking one of the output signals of the optical parametric oscillator for performing the evaluation method of the multiphoton excitation and the evaluation method of generating the second harmonic. A device for detecting tumors using a combination of evaluation methods, wherein the combination of the evaluation methods comprises the multiphoton excitation evaluation method and / or the second harmonic generation evaluation method and furthermore the Raman scattering evaluation method.

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