JP5100461B2 - LIGHT SOURCE DEVICE FOR NONLINEAR SPECTROSCOPY MEASUREMENT SYSTEM - Google Patents

Description

The present invention relates to a light source device for a nonlinear spectroscopic measurement system. In the present specification, a biological sample including living cells is mainly described as a target for nonlinear spectroscopic measurement. However, a target for nonlinear spectroscopic measurement using the light source device of the present invention is not limited to a biological sample.

In studying life phenomena, the method using light has the excellent feature of being able to approach the object in a non-destructive and non-contact manner. For this reason, the dynamic behavior of molecules that function in living cells has been studied in various ways using light. Of these, the most widely used technique is fluorescence imaging using fluorescent dyes or fluorescent proteins. However, in this method, it is necessary to introduce a foreign substance called a fluorescent substance into the sample itself.

On the other hand, Raman spectroscopy is a very powerful method that can observe the molecular distribution and dynamics in living cells without staining. Raman spectra, also called “molecular fingerprints”, have the characteristic of reflecting the individuality of the molecule, so that detailed information on the intracellular molecular species, molecular structure and their dynamics is extracted from “as is” samples. It is possible. Raman microspectroscopy, which decomposes time and space and can be analyzed at the molecular level, can track the distribution of molecules at various points in a living cell. Therefore, Raman microspectroscopy can be a very powerful weapon in life science. It has become clear that "reading" a Raman spectrum can selectively monitor not only the molecular species and molecular structure in the cell, but also the vital activity of the cell.

Raman microspectroscopy is a very good method, but there are weak points. In general, since the scattering cross section of Raman scattering is very small, an exposure time of several seconds to several minutes is required to obtain a good Raman spectrum from living cells. Coherent anti-Stokes Raman scattering (hereinafter referred to as CARS) microspectroscopy is a method that can amplify a weak Raman signal and obtain a Raman image at high speed in order to overcome this weak point. As one of the next generation Raman microspectroscopy, it is one of the methods that are attracting particular attention.

The CARS process generally requires two light pulses (pump light and Stokes light) having different wavelengths. When the frequency difference of this light pulse coincides with the molecular vibration, very strong CARS light is generated. The power of the CARS microscope that can visualize an object as it is at a high speed at the molecular level without requiring tagging of a fluorescent probe on the sample has been shown in various forms.

However, the CARS microscope can generally only obtain a monochrome image using a specific vibration mode. Spectroscopic measurement is essential to effectively use abundant information on molecular vibrations. In order to obtain a CARS spectrum, a method called a multiplex CARS process is generally used. In this process, narrow-band pump light and wide-band Stokes light are used, but generation of supercontinuum light used as a Stokes light source requires a picosecond or femtosecond light pulse. Since femtosecond lasers are expensive, a complicated and expensive light source system is required.

In addition to this, in order to superimpose two narrow-band and wide-band light pulses in time, a high degree of adjustment on the scale of a hundred micrometers is essential, and a high level of expertise is required. More specifically, on the optical path of the pump light, a moving stage that can translate what is on the stage with a minimum step of typically 2 micrometers (40/3 femtoseconds for reciprocation) is arranged. By installing a folding mirror (a mirror placed opposite to each other at an angle of 90 degrees, etc.) on this stage, the stage is moved and the mirror is accurately adjusted to maintain the light propagation direction while maintaining the optical path. The length was adjusted.

For these reasons, CARS microspectroscopy devices are not widely used in life science in general.

The apparatus disclosed in Patent Document 1 uses a laser light source that generates a short pulse of femtoseconds to picoseconds, and adjusts the optical path length from the light source unit of the first pulse laser light to the analysis target. Is an essential component. The apparatus disclosed in Patent Document 2 uses a laser light source that generates a short pulse of femtoseconds to picoseconds, and a phase adjustment unit that adjusts the phase of the divided pulsed light (this adjusts the optical path length) Including an optical path length adjusting unit) is an essential component. Further, as shown in Patent Document 2, in an optical system using a conventional picosecond laser or femtosecond laser, if an optical isolator (which is relatively expensive) is not introduced, the return light from the fiber causes femto There was a problem that the second or picosecond oscillation (mode synchronization) stopped, and its introduction was essential.

Non-Patent Document 1 discloses a sub-nanosecond microchip laser light source. The light emitted from the laser light source is passed through an LBO crystal to generate a second harmonic, and the fundamental wave and the second harmonic are photoed. Super continuum light is generated by adopting a double pumping method of injecting into a nick crystal fiber, and a delay line using a moving stage is provided. Non-Patent Document 2 discloses a sub-nanosecond fiber laser, but does not disclose any application to CARS.
JP2007-278768 JP2007-271783 M. Okuno, H. Kano, P. Leproux, V. Conderc, and H. Hamaguchi, “Ultrabroadband (> 2000cm-1) multiplex coherentanti-Stokes Raman scattering spectroscopy using a subnanosecond supercontinuumlight source,” Opt. Lett. 32, 3050- 3052 (2007). Laroche, M .; Leproux, P .; Couderc, V .; Lesvigne, C .; Gilles, H .; Girard, S., “Compact sub-nanosecond wideband laser source forbiological applications”, Applied Physics B, Volume 86, Number 4, March 2007, pp. 601-604.

An object of the present invention is to provide a light source device for a nonlinear spectroscopic measurement system which is simple, compact and inexpensive and has high performance.

The technical means adopted by the present invention in order to solve such a problem includes a laser light source that emits a light pulse having a pulse width of 0.1 to 10 nanoseconds, and a light pulse emitted from the laser light source. A non-linear spectroscopic measurement system comprising a photonic crystal fiber that generates and emits supercontinuum light by broadening an optical pulse, and performs non-linear spectroscopic measurement using supercontinuum light emitted from the photonic crystal fiber Light source device.

If the pulse width is smaller than 0.1 nanoseconds, there is a problem that “It is necessary to introduce a special, large and complex oscillation laser such as mode synchronization”. If the pulse width is larger than 10 nanoseconds, “Supercontinue” There is a problem that "no light is generated". In one aspect, the pulse width is 1 nanosecond or less. In the experiment described below, in one aspect, the pulse width is 800 ps. In one aspect, the spectral width of the light pulse is 1 cm ⁻¹ or less. On the other hand, the pulse width and spectral width of a femtosecond laser (for example, mode-locked titanium sapphire laser) used for conventional SC light generation are, for example, on the order of 100 fs and 300 cm ⁻¹ . The pulse width and spectral width of the picosecond laser are, for example, on the order of 3 ps and 3 cm ⁻¹ .

It is known that supercontinuum light, which is a coherent light source having a very broad spectrum, is generated by introducing femtosecond light pulses into a photonic crystal fiber. Photonic crystal fiber (PCF) has a microstructure structure with a clad in which a large number of holes are arranged in a honeycomb shape and an ultrafine core made of glass. Due to the large refractive index difference between the glass core and the air-hole cladding, the light introduced into the PCF is strongly confined in the core. Further, the chromatic dispersion characteristics of the PCF can be adjusted by the size of the holes surrounding the core and the arrangement method thereof. As a result, femtosecond optical pulses having a wide spectrum component can propagate through the PCF without increasing the pulse width. As described above, since a high photon number density in time and space can be realized in the PCF, various nonlinear optical effects can be generated in the PCF. Recently, it has been found that supercontinuum light can also be obtained by injecting a sub-nanosecond pulse into a photonic crystal fiber. By selecting a sub-nanosecond pulse light source and a photonic crystal fiber, supercontinuum light in a desired wavelength band can be obtained.

In one aspect, the laser light source and the photonic crystal fiber are selected such that a wavelength band of supercontinuum light emitted from the photonic crystal fiber is 1900 nm or less, and the light source device further includes Band limiting means for narrowing the wavelength band to a predetermined band is provided. In one aspect, the band limiting means blocks at least a spectral component having a wavelength shorter than the wavelength of the laser light source. In one aspect, the wavelength of the laser light source is 1064 nm, and in this case, spectral components having a wavelength band shorter than 1064 nm of supercontinuum light emitted from the photonic crystal fiber are blocked, and at least 1064 nm or more. Thus, at least a wavelength band of 1900 nm or less, that is, a supercontinuum light in any band within a wavelength band of 1064 nm to 1900 nm can be obtained. Accordingly, when the light source device of the present invention is applied to CARS, CARS measurement in the wavelength band 1064 to 1900 nm can be performed. This not only enables detection of all vibration modes necessary for vibrational spectroscopy, but also allows a longer penetration length into a biological sample than existing reports. The wavelength band in the conventional CARS is 800 to 1200 nm, whereas the wavelength band of the present invention is about 1064 to 1900 nm in the near infrared.

In one aspect, the nonlinear spectroscopic measurement system uses coherent anti-Stokes Raman scattering, and the supercontinuum light is used as broadband Stokes light.

In one aspect, the light source device includes an optical element (beam splitter) that divides a light pulse emitted from the laser light source into a first light pulse and a second light pulse, and the first light pulse includes: The light enters the photonic crystal fiber and is emitted as supercontinuum light, and the second light pulse is used as narrow-band pump light.

In one aspect, the light source device includes a laser light source that emits a light pulse having a pulse width of 0.1 to 100 nanoseconds that generates pump light. If the pulse width is smaller than 0.1 seconds, there is a problem that “It is necessary to introduce a special large-sized complex and expensive oscillation laser such as mode synchronization”. If the pulse width is larger than 100 nanoseconds, “CARS is generated. There is a problem that there is not enough peak output to make it happen.

In one aspect, the nonlinear spectroscopic measurement system is a multiphoton microscope. In one aspect, the multi-photon microscope includes a two-photon excitation fluorescence microscope. The light source device according to the present invention can also be used as an excitation light source that generates nonlinear optical effects (two-photon fluorescence, second harmonic, etc.) other than CARS.

In one preferable aspect, the light source device according to the present invention is applied to a CARS microscope. In order to solve the problem of the conventional CARS microscope, a breakthrough in the light source portion is required. In this regard, it is suitable for mass production, and it is possible to generate subnanosecond supercontinuum light using a subnanosecond microchip laser that can be manufactured at a relatively low cost as a CARS light source, while being simple, compact and inexpensive. Invented a completely new CARS spectroscopy and imaging method with high performance.

That is, another technical means adopted by the present invention includes a laser light source that emits a light pulse having a pulse width of 0.1 to 10 nanoseconds, a light pulse emitted from the light source, a first light pulse, and a second light pulse. An optical element that divides into optical pulses, a photonic crystal fiber that generates and emits supercontinuum light by making the first optical pulse incident, broadens the first optical pulse, and broadbands the supercontinuum light. Stokes light, the second light pulse as narrow-band pump light, a means for superimposing these and condensing them on the analysis object, a detection unit for detecting coherent anti-Stokes Raman scattered light emitted from the analysis object, A non-linear spectroscopic measurement system comprising:

In constructing the CARS microscope, a laser light source that generates pump light and Stokes light may be provided. In this case, the technical means employed by the present invention is a first pulse width of 0.1 to 10 nanoseconds. A first laser light source that emits a light pulse; a second laser light source that emits a second light pulse having a pulse width of 0.1 to 100 nanoseconds; and the first light pulse is incident, and the first light pulse is A photonic crystal fiber that generates and emits supercontinuum light by broadening the band, and the supercontinuum light is used as broadband Stokes light, and the second optical pulse is used as narrowband pump light. A non-linear spectrometer comprising: means for condensing light; and a detection unit for detecting coherent anti-Stokes Raman scattering light emitted from the analysis target System, it is.

This method does not require a complicated laser light source system, and has an advantage that a wide-band CARS spectrum over> 2000 cm ⁻¹ as a wave number bandwidth can be obtained at a time. In addition to this, the time width of the sub-nanosecond pulse has a characteristic that it is robust against a timing shift of about several tens of centimeters. Therefore, when irradiating the sample with two light pulses in synchronization, It is less susceptible to timing jitter and the construction of the device can be innovatively simplified. Therefore, in one aspect, the means for superimposing the Stokes light and the pump light and condensing them on the analysis object does not include the optical path length adjusting device for the pump light. Here, the optical path length adjusting device of the pump light is a device composed of one or a plurality of elements different from optical elements such as a beam splitter that divides the optical pulse and a mirror that directs the optical path of the optical pulse in different directions, For example, it is composed of a moving stage and a mirror as described above. In the present invention, it is not necessary to use such an optical path length adjusting device. That is, as a means for superimposing the pump light and the Stokes light, an optical delay path is not required as in the prior art. The superimposing means may be an edge filter, a notch filter, or an optical element such as a dichroic mirror or a beam splitter.

In one aspect, the supercontinuum light is used as a light source for two-photon excitation, and fluorescence generated by the two-photon excitation can be measured. By using a part of the CARS apparatus, a two-photon fluorescence image by near-infrared two-photon excitation can be obtained. As shown in FIG. 11 and FIG. 12, a CARS microscope and a two-photon excitation fluorescence microscope can be constructed using the light source device (including a laser light source and a photonic crystal fiber) according to the present invention. FIG. 11 is a schematic diagram of a CARS microscope, in which a light pulse emitted from a laser light source is divided into two light pulses by a beam splitter, and one of the light pulses is injected into a photonic crystal fiber to increase the bandwidth and supercontinue. The sample light is generated, the other light pulse and supercontinuum light are superimposed and irradiated onto the sample, and the coherent anti-Stokes Raman scattered light emitted from the sample is acquired with the objective lens of the microscope, and then dispersed with the spectroscope to obtain the CCD. Get it with the camera. FIG. 12 is a schematic diagram of a two-photon excitation fluorescence microscope. A light pulse emitted from a laser light source is divided into two light pulses by a beam splitter, and one of the light pulses is injected into a photonic crystal fiber to increase the bandwidth. The supercontinuum light is generated, and the sample is irradiated through the galvano scanner, and the excitation fluorescence emitted from the sample is acquired by the objective lens of the microscope and acquired by the detector.

The present invention is also provided as a nonlinear spectroscopic measurement method. Therefore, another technical means adopted by the present invention is to use an optical pulse emitted from a laser light source emitting an optical pulse having a pulse width of 0.1 to 10 nanoseconds. A step of dividing the first light pulse into a second light pulse, and a step of making the first light pulse incident on a photonic crystal fiber, generating a supercontinuum light by broadening the first light pulse, and emitting the supercontinuum light. And the supercontinuum light as a wide-band Stokes light and the second optical pulse as a narrow-band pump light without superimposing the optical path length of the first pulse light using an optical path length adjusting device. And collecting the light onto the analysis object, and detecting the coherent anti-Stokes Raman scattering light emitted from the analysis object. Form spectroscopic measurement method is,.

Another technical means employed by the present invention is that a first light pulse is emitted from a first laser light source that emits a light pulse having a pulse width of 0.1 to 10 nanoseconds, and a pulse width of 0.1 to 100 nanoseconds. Emitting a second light pulse from a second laser light source that emits the light pulse, and entering the first light pulse into a photonic crystal fiber to broaden the band of the first light pulse, thereby producing supercontinuum light. Generating and emitting the supercontinuum light as a wide-band Stokes light and the second optical pulse as a narrow-band pump light without using an optical path length adjusting device for the first pulsed light. And superposing and condensing on the analysis target, and detecting the coherent anti-Stokes Raman scattering light emitted from the analysis target. Linear spectroscopic measurement method,
It is.

In the present invention, a laser light source that emits an optical pulse having a pulse width of 0.1 to 10 nanoseconds is used as a light source of an optical pulse that generates supercontinuum light. A sub-nano that emits such an optical pulse is used. The second microchip laser is suitable for mass production and can be manufactured at a relatively low cost.

Because the light source is small and compact, it can be easily integrated into a microscope.

In CARS, it is necessary to superimpose pump light and Stokes light temporally and spatially (overlapping at least when the sample is irradiated), but by using the light source device according to the present invention, both light pulses are superimposed. It can be done very simply. The present invention does not require micrometer-scale optical adjustment unlike the conventional (picosecond or femtosecond mode-locked laser), and can easily realize the CARS process. Therefore, the system is robust against mechanical fluctuations. Conventionally, it was necessary to match the pump light and Stokes light with an accuracy of 100 micrometers. However, according to the present invention, since the accuracy of 10 cm is sufficient, there is no need for a moving stage for delaying the pump light required for the conventional apparatus. It does not require advanced adjustments on the scale of a hundred micrometers or advanced expertise.

The present invention using a sub-nanosecond laser light source does not require an optical isolator that is necessary for an optical system using a conventional mode-locked picosecond laser or femtosecond laser.

When a (sub) nanosecond light source is used, the spectral width of the pump laser (for example, about 10 cm ^{-1 in} the prior art, but <1 cm ^{-1 in} the light source device of the present invention) is narrow, so the spectral resolution is low. Greatly improved. This also contributes to a clear capture of protein signals and information on secondary structures.

The CARS microscope according to the present invention, (or amide I near 1650 cm ^-1, amide III near 1300 cm ^-1) protein bands and, a band of the lipid (CH bending around 1450 cm ^-1), the fingerprint region (500 Compared with the conventional system, a signal having a very different spectral pattern depending on the structure of the molecule, which is called -1700 cm ⁻¹ ), appears sharply.

Combined with the above effects, by applying sub-nanosecond supercontinuum light, it is possible to provide a completely new CARS spectroscopy and imaging method with high performance while being simple, compact and inexpensive. It can be expected to spread to various fields including science, medicine and material / material science.

[A] Nonlinear Raman Spectroscopic Imaging Method The background art of the present invention will be described mainly for the purpose of helping understanding of the present invention.

[A-1] What is CARS? Coherent anti-Stokes Raman scattering (CARS) is one of nonlinear Raman processes and is one of the most advanced techniques of imaging at present. FIG. 1 (a) shows a scheme of the CARS process. In CARS, two laser beams (ω ₁ , ω ₂ light, pump light, Stokes light) having different wavelengths are generally used. When the angular frequency difference ω ₁ −ω ₂ between the two incident lights matches the angular frequency Ω of the vibration mode of the sample molecule, the vibration modes of many sample molecules are resonantly aligned in phase (coherently). As a result, it is possible to obtain Raman scattered light that is very strong and has good directivity.

Compared with normal spontaneous Raman scattering, CARS has the following excellent features. First, the wavelength of the CARS light is shorter than the wavelength of the incident light, and the signal light is emitted in the form of a beam, so that it is not easily disturbed by one-photon fluorescence. Second, since the CARS process is a non-linear optical process, it occurs only from the portion where the laser beam is strongly focused. As a result, it is not necessary to introduce a pinhole as in the confocal microscope, and the three-dimensional spatial resolution is high.

[A-2] Multiplex CARS Process In order to realize a CARS microscopic “spectroscopy” method capable of acquiring images in multiple colors using various vibration modes, it is necessary to improve the light source portion. One of the most direct methods is the multiplex CARS process shown below. Fig. 1 (b) shows the energy diagram of the multiplex CARS process. In the multiplex CARS process, a broadband light source is used as Stokes light (ω ₂ light). Therefore, a wide variation occurs in the angular frequency difference ω ₁ −ω ₂ with the narrow-band pump light (ω ₁ light). As a result, a combination that can resonate with a plurality of vibration modes of the sample molecule is generated. When the vibration polarization generated in this way is probed with ω ₁ light, CARS light having various angular frequencies 2ω ₁ −ω ₂ is finally generated. When this is spectroscopically measured, a multiplex CARS spectrum having a sharp structure at each vibration resonance wave number position can be obtained. With such a scheme, it is possible to obtain an ultra-wideband multiplex CARS microspectroscopic device capable of almost covering the region of the molecular vibration fundamental tone, where the measurable wavenumber band is> 3500 cm ⁻¹ . It is a photonic crystal fiber (PCF) and supercontinuum (SC) light obtained thereby that enables ultra-wideband multiplex CARS spectrum measurement.

[A-3] Multiplex CARS microspectroscope using SC light FIG. 2 shows an experimental apparatus for a conventional ultra-wideband multiplex CARS microscope. A mode-locked titanium sapphire laser oscillator (Coherent; Vitesse) was used as the light source, and a part of the output was introduced into PCF (Crystal Fiber; NL-PM-750) to generate SC light. SC light has a wide spectrum from visible to near infrared, but only the near infrared component was used as broadband Stokes light (ω ₂ light). On the other hand, the remaining fundamental wave from the oscillator is narrowed by a band-pass filter to be pump light (ω ₁ ) (wave number width of about 20 cm ⁻¹ ), passed through an optical delay path, and then coaxial with Stokes light by a notch filter. Introduced into a microscope. Two light pulses are focused on the sample by the objective lens. The optical delay path is arranged on the optical path of the pump light with a moving stage that can translate the stage on the stage, typically with a minimum of 2 micrometers (40/3 femtoseconds for round trip), It is configured by installing a folding mirror (a mirror placed at an angle of 90 degrees, etc.) on this stage. By moving the stage and accurately adjusting the mirror, the light propagation direction is maintained. While adjusting the optical path length.

Normally, the phase matching condition needs to be satisfied for CARS generation. However, because of the high NA value of the objective lens, this condition is relaxed, and CARS light can be generated in a wide wave number region. The CARS light generated from the sample was collected by an opposing objective lens, passed through various filters, and spectroscopically measured with a spectroscope (Acton; SpectraPro 300i) and a CCD camera (Roper Scientific; PIXIS 100B). The sample is placed on a triaxial piezo stage (MadCity; Nano-LP-100), and three-dimensional scanning is possible. Since the CARS process is a non-linear optical process, signal light is generated only from the portion where the laser light is strongly condensed. As a result, it is not necessary to introduce a pinhole unlike a confocal microscope, and an essentially high three-dimensional spatial resolution can be realized.

The measurement of the multiplex CARS spectrum makes it possible to simultaneously measure a plurality of vibration resonance components, but in addition to this, there is another important advantage. A signal generally called non-resonant background is superimposed on CARS. Non-resonant background is one of the third-order nonlinear optical processes that occur in the path of FIG. 1 (c), for example. This signal is “non-resonant” at the vibration level, but particularly when it occurs in the process of FIG. 1 (c), when 2w ₁ approaches the electronic level, it increases due to the electronic resonance effect. This non-resonant background is often treated as a contamination that lowers the vibration contrast. On the contrary, we have devised and realized a method for efficiently extracting the vibration resonance component by using this signal. This utilizes the property that both the vibrationally-resonated CARS light and the non-resonant background are coherent. This method will be described below.

The signal intensity obtained by the CARS microscope / CARS microspectroscope is generally expressed by the following equation.
Here, I (omega) is the intensity spectrum of the whole signal observed, A _NR and φ is non-resonant background amplitude and phase, A _R is the amplitude of the vibration resonance CARS light, omega _R the vibration resonance angle frequency, Γ _R is a coefficient proportional to the line width. Here, since the non-resonant background of the first term and the vibrational resonance component of the second term interfere with each other, in the wave number region where both coexist, the asymmetrical state where the higher wave number is the valley and the lower wave number is the mountain. Giving a "dispersed" spectral shape. Therefore, by analyzing this spectrum pattern, it is possible to extract only vibration resonance components. This corresponds to heterodyne detection of vibration resonance CARS light by a non-resonant background. In this analysis, only the spectral information is used to improve the contrast, so no processing is performed for the spatial direction. Therefore, the spatial resolution at the time of measurement is maintained even after spectral analysis. This is one of the unique features of the method that measures another dimension of spectrum, not the measurement of “monochrome” images.

[A-4] Nonlinear Raman Spectroscopic Imaging of Living Cells FIG. 3 shows the results of multiplex CARS imaging of living cells of Schizosaccharomyces pombe (S. pombe). FIG. 3 (a) shows a multiplex CARS spectrum when the yeast is irradiated with laser light. The peak observed in the vicinity of 2850 cm ⁻¹ is a signal derived from CH stretching vibrations contained in phospholipids, proteins, polysaccharides and the like. The result of constructing an image with this CARS signal is shown in FIG. A plurality of yeast cells in various division cycles are clearly visualized by molecular vibration. In yeast, there are a plurality of portions with strong signal intensity, which are thought to originate from membrane organelles such as mitochondria and the like, which are rich in phospholipids, and partition walls composed of polysaccharides.

SC light can be used not only as a light source for a wide range of Raman excitation but also as a light source for resonant two-photon excitation. When fluorescence generated by two-photon excitation can be spectrally separated from CARS, it is possible to simultaneously measure CARS and two-photon fluorescence using the same optical system. As an example, FIG. 4 shows the result of CARS / two-photon fluorescence multi-imaging of viable fission yeast cells stained with a green fluorescent protein (GFP). Since the fluorescence of GFP appears in the vicinity of 510 nm, it can be spectrally separated from the currently detected CARS light. In this way, by using SC light, two-photon excitation can be performed in a wide wavelength range, and therefore, it is expected that high-efficiency imaging that resonates with an electronic state that allows two-photons is possible. The exposure time was 50 ms per point, and the time required to acquire one image was 3.8 minutes (at the time of measurement). This result clearly captures the state of yeast cell division. Looking at the CARS image in Fig. 4 (a), it is observed that there are multiple spots with high signal intensity, derived from membrane organelles such as mitochondria, and moving inside the cell during division. it can. In addition, it is also visualized that a partition wall composed of a polysaccharide appears near the center of the cell before division. FIG. 4 (b) shows a two-photon fluorescence image of a nucleus dyed with GFP. The contrast of the fluorescent image deteriorates with time, which is considered to be caused by the fading accompanying laser irradiation. On the other hand, the CARS image does not fade, and the cell division process can be visualized while maintaining good contrast. As described above, it was shown that the dynamics of living cells can be traced with high contrast by using CARS.

[B] Nonlinear Raman spectroscopic imaging method using sub-nanosecond laser
Generation of SC light can also be obtained by a light source device combining a sub-nanosecond microchip laser and a photonic crystal fiber. This approach provides a low-cost, compact, ultra-broadband, phase-coherent white light source. The SC light can be obtained based only on the 1064 nm laser pulse emitted from the Sananosecond microchip laser, and the entire system can be made compact.

In the embodiment described below, the ultra-wide multiplex CARS microspectroscopy was applied to biological cells in the near infrared region. Deep near-infrared CARS excitation minimizes light damage to biological samples. Further, deep near-infrared light is absorbed or / and scattered by a biological medium, so that deeper light penetration is possible than visible light.

FIG. 5 shows a schematic diagram of a near-infrared ultra-wide area multiplex CARS microscope. A Q-switched Nd: YAG microchip laser (JDS Uniphase, NP-10820-GM1; center wavelength 1064 nm; spectral width <1 cm ⁻¹ ; pulse width <1 ns; pulse energy 8 μJ; repetition rate 6.6 kHz) was used as the laser light source.

The laser beam is transmitted through the half-wave plate and then split into two light pulses by a beam splitter. The half-wave plate may be disposed between the beam splitter and the photonic crystal fiber in place of the laser light source and the beam splitter. The present invention using a sub-nanosecond laser light source does not require an optical isolator that is necessary for an optical system using a conventional mode-locked picosecond laser or femtosecond laser.

About 10% of the emitted light is used as pump light (ω ₁ ) in the CARS process. By introducing a bandpass filter, unwanted spectral elements in the light emitted from the microchip laser are blocked.

The remaining 90% of the emitted light is used to generate near infrared SC light in the photonic crystal fiber. The length of the optical fiber is 1.6 m. The SC light is used as Stokes light (ω ₂ ) in the CARS process. Several band pass filters and long wavelength pass filters are used to block spectral components with wavelengths shorter than 1064 nm in SC light.

The pulse energy of the pump light and the ultra-wide Stokes light whose spectral components are shaped are 800 nJ and 150 nJ, respectively. Two laser pulses are superimposed on the same axis using a 1064 nm edge filter and focused on the sample using a 40 × 0.9 NA microscope objective. Under this condition, the CARS phase matching condition is greatly relaxed due to the large angular dispersion and the small interference length. The CARS light in the forward detection is collected by another 40 × 0.6 NA microscope objective. After passing through the two short wavelength pass filters, CARS light is introduced into the optical fiber.

The CARS signal is dispersed by a spectrometer (Acton, SpectraPro-300i) and detected by a CCD detector (Roper Scientific, PIXIS-100B).

The sample is scanned with a piezo stage (Madcity, Nano-LP-100) for CARS imaging. In the piezo stage, position control on the nanometer scale is possible.

From the edge of the signal intensity profile of the polyethylene film, the lateral resolution is estimated to be 0.9 ± 0.1 micrometers. From the rise of the signal at the air-film interface, the depth resolution is estimated to be 4.6 ± 0.3 micrometers.

FIG. 6 is a typical spectral profile of SC light in the near infrared region. The spectrum was measured with an InGaAs spectrometer (Ocean Optics, NIR 512). As shown in FIG. 6, an ultra wide-area SC having an extremely wide spectral profile in the range of 1100 nm to 1700 nm was obtained. Non-resonant background was used to correct the distorted spectral profile of the CARS signal.

FIG. 6 (a) shows a deep near-infrared multiplex CARS spectrum with corrected intensity of polystyrene beads. The bead diameter is 3 micrometers. The exposure time is 1 second. The signal intensity was corrected using a non-resonant background signal of the cover glass. It is clear that CARS signals are obtained in the spectral range> 2000 cm ⁻¹ . As shown in FIG. 7 (a), several peaks are observed due to a plurality of Raman resonances. The intensity peaks at 3052, 2890, 2844, 1595, 1572, 1169, 1141, 1019 and 992cm ^-1 are aromatic CH stretch, aliphatic CH stretch, aliphatic CH stretch, aromatic C = C stretch, aromatic C = It is derived from C stretching, aromatic in-plane CH variation, aromatic in-plane CH variation, ring deformation, and ring breathing vibration mode.

FIG. 7 (b) shows the detailed spectral profile of the CARS signal from 900 to 1700 cm ⁻¹ . It can be seen that the vibrational resonance component is observed in the fingerprint region. In particular, vibrational resonance components are spectrally resolved at Raman shifts 1595, 1572, 1169, 1141, and 1019 cm ⁻¹ . Due to interference with non-resonant background, the spectrum shows a dispersive shape.

It is noted that the simultaneous measurement of the CARS signal was performed in the spectral range of 1000 to 3000 cm- ¹ without changing the delay time between the pump light and the Stokes light. This means that group delay dispersion is not a problem in the CARS process using sub-nanosecond optical pulses, compared to SC based on femtosecond and picosecond optical pulses.

FIGS. 7 (c), (d), (e) show three CARS images of polystyrene beads with a diameter of 3 micrometers at three different Raman shifts 992, 1572 and 1900 cm ⁻¹ . The exposure time is 100 ms at each point. As shown in FIGS. 7 (c) and (d), vibration resonance CARS images were observed at 992 and 1572 cm ⁻¹ . On the other hand, no clear vibration contrast was obtained at 1900 cm ⁻¹ where only non-resonant background signals were observed. 7C and 7D, the vibration contrast is higher than that in FIG. 7E.

Fig. 8 (a) shows Nicotiana tabacum L. cv. Bright Yellow
2 shows CARS spectra and images obtained from 2 (BY2) cells. In the experiment, BY2 cells were diffused in water and sandwiched between a glass slide and a cover glass. All measurements were performed at room temperature.

FIG. 8B shows a CARS image at a Raman shift of 2902 cm ⁻¹ in the CH stretching mode. The exposure time for each pixel is 300 ms. Although non-resonant background is dominant in the signal, it is possible to improve vibration contrast by using spectral analysis. In FIG. 8 (b), a positive peak (average of 2860 to 2870 cm ⁻¹ ) and a negative peak (2960 to 2970) of the CH stretch CARS band are dispersed.
The signal intensity difference between (mean cm ^-1 ) was mapped. It is emphasized that this task cannot be done with single wave number detection in a CARS microscope. In FIG. 8 (b), BY2 cells are visualized as high vibration contrast. In BY2 cells, strong CARS signals were observed in the nucleus and cell wall. The cytoplasm was also visualized with CARS signals at moderate intensity. At the vacuole position, the CARS signal is very weak.

The present invention can be used for CARS microspectroscopy. In CARS microspectroscopy, various molecular species constituting a biological sample can be variously “color-coded” based on a Raman spectrum that is a fingerprint of the molecule. CARS microspectroscopy has never been known in living cells because it enables analysis and detection of unknown molecules through spectra compared to fluorescence imaging, which is good at visualizing known molecules. The molecules and molecular structures can also be studied. Nonlinear Raman spectroscopic imaging methods represented by CARS microspectroscopy have applicability in a wide range of fields including medicine, life science, and materials / materials science.

(a) Coherent anti-Stokes Raman scattering (CARS) energy diagram, (b) Multiplex CARS process, (c) Non-resonant background energy diagram. It is an experimental apparatus diagram of a multiplex CARS microspectroscope; BS: beam splitter; LF: long wavelength transmission filter; NF: 800-nm notch filter; SF: short wavelength transmission filter. (a) CARS spectrum (uncorrected intensity) of viable fission yeast (S. pombe), (b) CARS image obtained by extracting C—H stretching vibration resonance components. The scale bar is 2 micrometers. (a) CARS image obtained by extracting the CH stretching vibration resonance component of viable fission yeast (S. pombe) introduced with green fluorescent protein (GFP) into the nucleus, and (b) GFP two-photon fluorescence image. . The actual image is color. The scale bar is 2 micrometers. The numbers on the upper left indicate the time order. The measurement time for one image is 3.8 minutes. It is a multiplex CARS microspectroscope according to the present invention. It is a typical spectral profile of SC light in the near infrared region. (a) shows the near-infrared multiplex CARS spectrum of polystyrene beads with corrected intensity. (b) shows the detailed spectral profile of the CARS signal from 900 to 1700 cm ⁻¹ . (c), (d), (e) show three CARS images of polystyrene beads with a diameter of 3 micrometers at three different Raman shifts 992, 1572 and 1900 cm ⁻¹ . (a) shows CARS spectrum and image obtained from Nicotiana tabacum L. cv. Bright Yellow 2 (BY2) cells. (b) shows a CARS image at a Raman shift of 2902 cm ⁻¹ in the CH stretch mode. (And amide I near 1650 cm ^-1, amide III near 1300 cm ^-1) bands of proteins and, (CH bending around 1450 cm ^-1) bands of lipids such as, called fingerprint region (500-1700Cm around ^-1) It is observed that signals showing very different spectral patterns depending on the molecular structure are sharp. 10 shows a conventional spectral pattern for comparison with FIG. It is a schematic diagram of a nonlinear spectroscopic measurement system using coherent anti-Stokes Raman scattering light. 1 is a schematic diagram of a nonlinear spectroscopic measurement system using two-photon fluorescence. FIG.

Claims (7)

A laser light source emitting a light pulse having a pulse width of 0.1 to 10 nanoseconds;
An optical element that divides a light pulse emitted from the light source into a first light pulse and a second light pulse;
A photonic crystal fiber that receives the first light pulse and generates a supercontinuum light by broadening the first light pulse;
Means for condensing the supercontinuum light into a wide-band Stokes light, the second light pulse as a narrow-band pump light, and superimposing them on an analysis target without using an optical delay path ;
A detection unit for detecting coherent anti-Stokes Raman scattered light emitted from the analysis target;
A nonlinear spectroscopic measurement system. A first laser light source emitting a first light pulse having a pulse width of 0.1 to 10 nanoseconds;
A second laser light source emitting a second light pulse having a pulse width of 0.1 to 100 nanoseconds;
A photonic crystal fiber that receives the first light pulse and generates a supercontinuum light by broadening the first light pulse;
Means for condensing the supercontinuum light into a wide-band Stokes light, the second light pulse as a narrow-band pump light, and superimposing them on an analysis target without using an optical delay path ;
A detection unit for detecting coherent anti-Stokes Raman scattered light emitted from the analysis target;
A nonlinear spectroscopic measurement system. The nonlinear spectroscopic measurement system according to claim 1 or 2, wherein the pulse width of 0.1 to 10 nanoseconds is 1 nanosecond or less. Spectral width of the pump light is ^{1 cm -1} or less, the nonlinear spectroscopy system according to any one of claims 1-3. The supercontinuum light, used as a light source of a two-photon excitation, it is possible fluorescence measurement caused by two-photon excitation, nonlinear spectroscopy system according to any claims 1 to 4. Dividing an optical pulse emitted from a laser light source emitting an optical pulse having a pulse width of 0.1 to 10 nanoseconds into a first optical pulse and a second optical pulse;
Generating a supercontinuum light the first light pulse incident on the photonic crystal fiber, and broadband the first light pulse,
The supercontinuum light is used as broadband Stokes light, the second optical pulse is used as narrowband pump light, and these are superposed and condensed on the analysis target without using an optical delay path ;
Detecting coherent anti-Stokes Raman scattering light emitted from the analysis object;
A nonlinear spectroscopic measurement method comprising: A first light pulse is emitted from a first laser light source emitting a light pulse having a pulse width of 0.1 to 10 nanoseconds, and a second light is emitted from a second laser light source emitting a light pulse having a pulse width of 0.1 to 100 nanoseconds. Emitting each light pulse;
Generating a supercontinuum light the first light pulse incident on the photonic crystal fiber, and broadband the first light pulse,
The supercontinuum light is used as broadband Stokes light, the second optical pulse is used as narrowband pump light, and these are superposed and condensed on the analysis target without using an optical delay path ;
Detecting coherent anti-Stokes Raman scattering light emitted from the analysis object;
A nonlinear spectroscopic measurement method comprising: