JP2013003204A - Laser microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microscope device, despite a single device, suppressing deterioration in generation efficiency of CARS light and in spatial resolution and suited to a simultaneous observation of a CARS light observation and a differential interference observation.SOLUTION: The laser microscope device includes: a light source section 100 for generating first and second luminous fluxes L1 and L2 having different wavelengths; a laser combiner 105 for multiplexing the two luminous fluxes L1' and L2'; a polarizer 104 provided at least one of two optical paths and creating a luminous flux of linearly polarized light; a two-dimensional scanning part 201; a birefringence prism 202 for dividing a luminous flux into two; an objective lens 203 for converging the multiplexed luminous flux; a birefringence prism 209 for synthesizing two luminous fluxes from a sample; an analyzer 210; a luminous flux separation section 207; a first detecting section 301 and a second detection section 302 for detecting a CARS signal and a DIC signal; and an image processing section 401 for forming an image on an observation surface in the sample on the basis of information by the first and second detection sections and position information of light spot by the two-dimensional scanning section 201.

Description

  The present invention relates to a laser microscope apparatus.

  A CARS microscope is known that uses a specific vibration of a molecule in a specimen to generate coherent anti-Stokes Raman scattering (CARS) light (CARS signal) from the molecule and detects the CARS light to detect the specimen. (See Patent Document 1). Since this CARS microscope uses a specific vibration of a sample molecule, it is not necessary to label an observation target with a fluorescent probe in advance, unlike a fluorescence microscope. Moreover, the molecule | numerator to observe can be changed by changing the vibration to utilize.

 Conventionally, a pulse laser having two different frequencies is used as a light source of the CARS microscope. According to such a microscope, the two pulsed laser beams (pump light and Stokes light) are focused on the sample surface in a state where the frequency difference is adjusted to coincide with a specific vibration frequency of the sample molecule. At this time, in an extremely narrow space where the photon density spreading near the focal plane is high, the frequency difference between the pump light and the Stokes light resonates with a specific vibration frequency of the molecule, and strong CARS light is generated. This CARS light has a frequency higher than the frequency of the irradiated pump light and Stokes light (that is, has a short wavelength). Therefore, the molecules of the specimen can be observed by spectrally selecting and detecting only the CARS light.

  Currently, using femtosecond pulse laser light or picosecond pulse laser light as a light source, switching between CARS light observation, multiphoton excitation (MPE) fluorescence observation, and second harmonic generation (SHG) light observation is performed. There is known a laser microscope capable of performing (see Patent Document 2).

  On the other hand, the sample is irradiated by irradiating the sample with two light beams separated into several tens of nm to several μm, and after passing through the sample, the sample is observed by detecting a light beam (DIC signal) obtained by synthetic interference of the two light beams. Differential interference (DIC) microscopes are known. This DIC microscope is an observation method that visualizes the fine structure of a transparent specimen as light and dark, like a phase contrast microscope.

  Usually, a DIC microscope makes polarized light incident on a birefringent prism (Nomarski prism), illuminates the sample with two light beams that are separated slightly together with the illumination condenser lens, and places it in the objective lens and the observation optical path. The fine structure of the specimen is visualized by recombining the light beam with the birefringent prism, and finally allowing the light beam to pass through the polarizer to cause interference. In this method, the phase difference (for example, step difference or refractive index difference) of the sample in the optical path through which the two light beams pass can be observed as bright and dark, and a minute step of about 10 nm in the optical axis direction can be detected as in the case of the phase contrast microscope. . Further, unlike the phase contrast microscope, there is no restriction on the illumination NA, so that the resolving power in the direction perpendicular to the optical axis can be exhibited to the limit of the microscope objective lens. Furthermore, the contour does not shine compared to the phase difference method, and an image with high resolution can be obtained.

  In a DIC microscope, a linearly polarized light beam is divided into two light beams that are spatially separated according to the polarization component, irradiated onto the specimen, and the light spot formed on the specimen is scanned two-dimensionally and reflected by the specimen. Combining the two luminous fluxes, selectively extracting only the luminous flux from the vicinity of the observation plane out of the synthesized luminous flux, detecting a specific linearly polarized luminous flux from the luminous flux near the observation plane, and detecting the detected information and An image of the observation surface of the specimen can also be formed based on the position information of the light spot. This makes it possible to observe a step of several nm in a transparent specimen in real time (see Patent Document 3).

Japanese translation of PCT publication No. 2002-520612 JP 2010-96667 A JP 2005-309415 A

  One of the features of the CARS microscope is that there is no need to label the observation target with a fluorescent probe in advance because it uses a specific vibration of a sample molecule. On the other hand, a DIC microscope is suitable for observing the fine structure of a transparent specimen (for example, a cell) that is not labeled with a fluorescent probe. However, simply incorporating a birefringent prism, a polarizer, and an analyzer into the CARS microscope causes the following problems when performing CARS light observation and DIC observation simultaneously.

  The first defect is a decrease in CARS light intensity generated from the specimen. In CARS light observation, the polarized light of pump light and Stokes light is usually linearly polarized light, adjusted so that the polarization directions of pump light and Stokes light are parallel, and condensed on the specimen. In this way, CARS light can be generated most efficiently from specific molecules in the specimen. On the other hand, in DIC observation, a linearly polarized light beam is divided into two light beams whose polarization directions are orthogonal to each other, and then the two light beams are collected on a sample. For this purpose, the direction of the birefringent prism and / or the direction of polarization of the linearly polarized light beam to be incident so that the linearly polarized light beam is incident on the birefringent prism and divided into two light beams whose polarization directions are orthogonal to each other. Adjust. In other words, when a birefringent prism is combined with a CARS microscope in order to perform CARS light observation and DIC observation at the same time, both the pump light and Stokes light whose polarization directions are parallel to each other are orthogonal to each other by the birefringence prism. Will be divided into two luminous fluxes. In this case, as described below, the CARS light intensity generated from the specimen decreases.

Using pump light and stokes light of linearly polarized light, the intensity I _CARS of CARS light generated when focusing on the specimen to the polarization direction of the pump light and the Stokes light by adjusting in parallel, the intensity of the pump light I _P If the intensity of Stokes light is I _S , the following is obtained.

  FIG. 2A is a schematic diagram showing a state in which a linearly polarized light beam incident on the birefringent prism is divided into two light beams by the birefringent prism. The polarization directions of the two light beams divided by the birefringent prism are orthogonal to each other. Here, a surface including both two light beams divided by the birefringent prism is defined as an R surface. The axis parallel to the optical axis of the light beam incident on the birefringent prism is the z axis, the axis parallel to the R plane and perpendicular to the z axis is the x axis, and the axis perpendicular to the R plane and perpendicular to the z axis is the y axis. Define.

As shown in FIG. 2B, for example, when the polarization directions of the pump light and the Stokes light incident on the birefringent prism are inclined by 45 degrees from the x axis, respectively, the birefringent prism as shown in FIG. Divided into two luminous fluxes. That is, the pump light incident on the birefringent prism is divided into a component in the polarization direction parallel to the x axis and a component in the polarization direction parallel to the y axis. Similarly, the Stokes light incident on the birefringent prism is also divided into a component having a polarization direction parallel to the x axis and a component having a polarization direction parallel to the y axis. Here, each I _P the intensity of the pump light and the Stokes light incident on birefringent prism, I _S, one of the two light beams separated by the birefringent prism, polarization direction pump light and stokes light parallel to the x-axis strength of each I _'Px, I' _Sx, respectively polarization direction the intensity of parallel pump light and stokes light on the y-axis I _'Py, I' when defined as _Sy, the following relationship holds.

I ′ _Px = I _P / 2 (Formula 2)

I ′ _Sx = I _S / 2 (Formula 3)

I ′ _Py = I _P / 2 (Formula 4)

I ′ _Sy = I _S / 2 (Formula 5)

Therefore, the intensity I ′ _CARSx of the component in the polarization direction parallel to the x axis and the polarization direction in the direction parallel to the y axis of the CARS light generated when the pump light and Stokes light that have passed through the birefringent prism are collected on the sample. The intensity I ′ _{CARSy of the} component is as follows from the formulas 1 to 5.

Therefore, as shown in FIG. 2B, the intensity I of CARS light generated when the pump light and the Stokes light inclined by 45 degrees from the x-axis are divided into two light beams by the birefringent prism and condensed on the sample, respectively. ' _CARS is as follows from Equation 7 and Equation 8.

Based on Equations 1 and 9, using linearly polarized pump light and Stokes light, the polarization direction of pump light and Stokes light is adjusted in parallel, and compared with the intensity I _{CARS of} CARS light generated when the sample is condensed on the sample. Then, as shown in FIG. 2B, the pump light and the Stokes light whose polarization directions are inclined by 45 degrees from the x axis are divided into two light beams by the birefringent prism and are collected on the sample. It can be seen that the intensity I ' _CARS of the CARS light decreases to a quarter.

  The second problem is a reduction in the spatial resolution of CARS light observation. In order to perform CARS light observation and DIC observation at the same time, when a birefringent prism is combined with a CARS microscope, both the pump light and Stokes light are split into two linearly polarized light beams orthogonal to each other as described above. When these two linearly polarized lights are collected on the sample, the respective light spots are formed at slightly shifted positions on the sample, and the CARS light generated from the two light spots is detected simultaneously. The two light spot deviation amounts (shear amounts) vary depending on the type of the objective lens, and the spatial resolution of CARS light observation decreases as the shear amount increases. Thereby, when performing both CARS light observation and DIC observation, it has the technical subject that it is very difficult to detect the same place simultaneously.

  The present invention has been made in view of the above-described circumstances, and is used to simultaneously perform DIC observation and CARS light observation using the same apparatus having one light source while suppressing a decrease in the generation efficiency of CARS light. The purpose is to acquire a suitable new technique and simultaneously acquire molecular information by CARS light observation and fine structure information by DIC observation.

In order to achieve the above object, the present invention employs the following means.
The present invention is a laser microscope apparatus that simultaneously detects a DIC signal and a CARS signal, and a light source unit (for example, one light source such as a pulse light source) that generates a first light beam and a second light beam having different wavelengths. And means for branching the light beam generated from the light source into two, and a frequency conversion means for converting the frequency of at least one of the two light beams), and a combination of the two light beams. A two-dimensional scanning unit that scans a light spot formed on a sample, and a linear polarization adjustment unit that is provided in at least one of two optical paths that guide the two light beams to the multiplexing unit. A scanning unit, a light beam dividing unit that divides a light beam into two light beams according to a polarization component, a light collecting unit that collects the combined light beam on a sample, and a light beam obtained from the sample by the light beam dividing unit. Two luminous flux A light beam combining unit that combines the divided light beams, a linearly polarized light extraction unit that extracts a light beam of specific linear polarization from the combined light beam, and a light beam separation unit that separates at least one of the first light beam and the second light beam. , A first detection unit for detecting a CARS signal, a second detection unit for detecting a DIC signal, information detected by the first detection unit and the second detection unit, and the two-dimensional Based on the position information of the light spot obtained from the scanning unit, a laser microscope apparatus including an image processing unit that forms an image of the observation surface in the specimen and a display unit that displays the formed image is employed.

  According to the laser microscope apparatus of the present invention, a light source unit (for example, one light source such as a pulse light source, means for branching a light beam generated from the light source into two, and at least one of the two light beams 2) light beams (pump light and Stokes light) having different wavelengths from each other are combined by a combining unit. By converging the combined light flux on the sample and scanning the light spot formed on the sample by the two-dimensional scanning unit, it is possible to observe the CARS light generated from the sample. In addition, the direction of the light beam dividing unit is adjusted so that at least one of the pump light and the Stokes light is not divided into two light beams by the light beam dividing unit. Next, the linearly polarized light adjusting unit is adjusted so that only one of the pump light and the Stokes light is divided into two light beams by the light beam dividing unit. The pump light or Stokes light divided into two light beams by the light beam dividing unit is condensed on the sample, and the light spot formed on the sample is scanned by the two-dimensional scanning unit. The DIC image of the sample can be observed by combining the light beam obtained from the sample by the light beam combining unit and detecting the light beam of a specific linear polarization from the combined light beam.

  By doing so, one of the pump light and Stokes light is divided into two linearly polarized light beams orthogonal to each other for DIC observation, and the other is not divided. The decrease can be suppressed. In addition, one of the pump light and Stokes light forms two light spots on the specimen, and the other forms one light spot. Since the CARS light generated from the sample has the relationship of (Equation 1), the CARS light is generated only from the portion where the light spots of the pump light and the Stokes light overlap. That is, it is possible to avoid a reduction in the spatial resolution of CARS light observation due to the above-described splitting of the pump light and the Stokes light into two linearly polarized lights orthogonal to each other.

  Here, at the time of CARS light observation, the frequency conversion unit converts the frequency of at least one of the light beams so that the frequency difference between the two light beams generated from the light source unit is approximately equal to the specific vibration frequency of the molecules in the sample. . As a result, the CARS light can be generated by using the light flux having the higher frequency as the pump light and the light flux having the lower frequency as the Stokes light.

In the above laser microscope apparatus, instead of the light beam splitting unit and the light beam combining unit, a light beam splitting and combining unit that splits the light beam into two light beams according to the polarization component and combines the light beams returned from the sample, and the return from the sample A light beam separating unit that separates the light beam synthesized by the light beam splitting and synthesizing unit, and the second detection unit may be arranged in an epi-illumination type.
By adopting such a configuration, it is possible to acquire a DIC image of a specimen in an epi-illumination type. In the case of the epi-illumination type, the optical path length in the specimen is twice that of the transmission type. For this reason, the phase change of the observation surface can be detected with twice the sensitivity as compared with the transmission type.

In the laser microscope apparatus, the light beam separation unit may be a light beam separation unit whose separation ratio does not depend on polarization of incident light.
By using such a light beam separation unit, even if the light beam incident on the light beam separation unit is a light beam other than P-polarized light or S-polarized light with respect to the incident surface of the light beam separation unit, the light beam separation unit has a constant separation ratio regardless of the polarization. , And can be separated into a transmitted light beam and a reflected light beam. Therefore, the light beam can be guided to the light beam separation unit while maintaining the polarization direction of the linearly polarized light generated by the linear polarization adjustment unit. Furthermore, the light beam can be guided to the linearly polarized light extraction unit while maintaining the polarization direction of the light beam returned from the sample and synthesized by the light beam splitting and synthesizing unit. Thereby, a DIC image can be observed using the phase difference produced by the fine structure of the specimen.

  ADVANTAGE OF THE INVENTION According to this invention, the new technique suitable for performing CARS light observation and DIC observation simultaneously using the same apparatus is offered. Thereby, the reduction in the generation efficiency of CARS light in CARS observation can be suppressed, and the molecular information obtained by CARS light observation and the fine structure information obtained by DIC observation can be obtained simultaneously without reducing the spatial resolution.

1 is a block diagram illustrating an overall configuration of a laser microscope apparatus according to a first embodiment of the present invention. (A) It is the schematic diagram showing a mode that the linearly polarized light beam which injected into the birefringent prism was divided | segmented into two light beams by the birefringent prism. (B) It is the schematic diagram showing the pump light and Stokes light which incline 45 degree | times from x-axis and inject into a birefringent prism. It is a block diagram which shows the whole structure of the laser microscope apparatus which concerns on the 2nd Embodiment of this invention.

[First Embodiment]
A laser microscope apparatus 1 according to a first embodiment of the present invention will be described below with reference to FIG.

  The laser microscope apparatus 1 includes a light source unit 100 that generates a first light beam and a second light beam having different wavelengths. The light source unit 100 includes one pulsed light source 101 that can emit a light beam L1 having a wavelength of 900 nm to 1100 nm and a light beam L2 of the second harmonic, and two optical paths 001 and 001 that pass the light beams L1 and L2, respectively. 002 and a laser combiner (multiplexing unit) 105 that combines two light beams L1 ′ and L2 ′ that have passed through the two optical paths 001 and 002.

  The first optical path 001 is provided with a delay optical path 102 for adjusting the pulse timing of the light beam L1, and a polarizer (linear polarization adjusting unit) 104 for generating a linearly polarized light beam L1 ′ from the light beam L1. .

  The delay optical path 102 is configured by a mirror (reflector), for example (not shown). The optical path length of the light beam L1 can be changed by turning back the optical path of the light beam L1 using at least two sets of reflectors and adjusting the interval between at least two sets of reflectors. Thereby, the temporal timing of the pulse of the light beam L1 can be adjusted.

  The polarizer 104 is a polarizing plate, and selectively transmits only a light beam having a specific polarization component among incident light beams, and can arbitrarily change the direction of the linearly polarized light. Therefore, the polarizer 104 can generate a desired linearly polarized light beam L1 'from the light beam L1. Since the polarizer 104 is an element necessary only when observing a DIC image, the polarizer 104 may be incorporated into the optical path 001 only when observing a DIC image.

  In the second optical path 002, an optical parametric oscillator (OPO) 103 that allows the light beam L2 to pass therethrough is provided as a frequency conversion means.

  The OPO 103 generates a high-power and narrow-line-width light beam L2 ′ from visible to near-infrared using the light beam L2 to be passed as an excitation light source, and in the sample 205 into the light beams L1 ′ and L2 ′ guided through the optical paths 001 and 002. A frequency difference substantially equal to the specific frequency of the numerator molecule can be given.

  The laser microscope apparatus 1 also converts the light beam L3 combined by the laser combiner 105 into a two-dimensional scanning unit 201 that scans the light beam L3 two-dimensionally and the light beam L3 that has passed through the two-dimensional scanning unit 201 into two light beams according to polarization components. A birefringent prism (light beam splitting unit) 202 for separation and an objective lens (light collecting unit) 203 for condensing the light beam that has passed through the birefringent prism 202 on the surface of the specimen 205 are provided. In the figure, reference numeral 204 denotes a stage.

The birefringent prism 202 splits only one of the light beam L1 ′ and the light beam L2 ′ out of the light beam L3 that has passed through the two-dimensional scanning unit 201 into two light beams. The two divided light beams are spatially separated, and their polarization components are orthogonal to each other.
The direction of the birefringent prism 202 is adjusted so that at least one of the light beam L1 ′ and the light beam L2 ′ is not divided into two light beams. The polarizer 104 is adjusted so that only one of the light beam L1 ′ and the light beam L2 ′ is divided into two light beams by the birefringent prism 202. The light beams that have passed through the birefringent prism 202 pass through the objective lens (condenser) 203 and are condensed on the specimen 205 to form a light spot. Since the birefringent prism 202 is an element necessary only when the DIC image is observed, like the polarizer 104, the birefringent prism 202 may be incorporated into the optical path only when the DIC image is observed.

  The two-dimensional scanning unit 201 scans a light spot formed on the specimen 205 two-dimensionally. The two-dimensional scanning unit 201 may use a galvanometer mirror, for example. The two-dimensional scanning unit 201 may also use an acousto-optic element.

  The laser microscope apparatus 1 also includes a first detection unit 301 for detecting CARS light generated from the specimen 205 and collected by the condenser lens 206. In the figure, reference numeral 207 denotes a dichroic mirror (light beam separator), and reference numeral 208 denotes a bandpass filter for selectively allowing CARS light generated from the specimen 205 to pass therethrough.

  In addition, the laser microscope apparatus 1 is divided into two light beams by a birefringent prism 202, condensed on a specimen 205 through an objective lens 203, and a birefringent prism for synthesizing the two light beams obtained from the specimen 205. (Flux synthesizer) 209 and an analyzer (linearly polarized light extractor) 210 for extracting a linearly polarized light beam out of the light beams synthesized by the birefringent prism 209, and a light beam that has passed through the analyzer 210 is detected. 2nd detection part 302 is provided. In the figure, reference numeral 211 denotes a band-pass filter that can selectively pass the wavelength of the light beam divided into two linearly polarized lights orthogonal to each other by the birefringent prism 202.

  The analyzer 210 is a polarizing plate like the polarizer 104, and selectively transmits only a light beam having a specific polarization component among incident light beams. The analyzer 210 is arranged in a crossed Nicols relationship with respect to the polarizer 104. Therefore, the analyzer 210 selectively transmits a linearly polarized light beam orthogonal to the linearly polarized light transmitted through the polarizer 104.

  The 1st detection part 301 and the 2nd detection part 302 are comprised with a photomultiplier tube, for example.

  Further, the laser microscope apparatus 1 is based on the information detected by the first detection unit 301 and the second detection unit 302 and the position information (scanning information) of the light spot obtained from the two-dimensional scanning unit 201. An image processing unit 401 that forms an image of the observation surface of the specimen 205 and a display unit 402 that displays the formed image are provided.

The operation of the laser microscope apparatus 1 configured as described above will be described below.
First, the case where the sample 205 is observed with CARS light using the laser microscope apparatus 1 according to the present embodiment will be described below.

  The light beam L1 generated from one pulse light source 101 passes through the delay optical path 102 provided on the first optical path 001, and the temporal timing of the pulse is adjusted. On the other hand, the light beam L2, which is the second harmonic of the light beam L1 generated from one pulse light source 101, is converted to a high frequency side by the OPO 103 provided on the second optical path 002, and the light beam L2 ′ Become. As a result, the two light beams L1 and L2 'guided along the optical paths 001 and 002 can be given a frequency difference substantially equal to the specific frequency of the molecules in the sample 205.

  Thereafter, the two light beams L1 and L2 'are combined by the laser combiner 105 to become a light beam L3.

  The pulse laser beam L3 combined in this manner is scanned by the two-dimensional scanning unit 201 and then condensed on the surface of the specimen 205 via the objective lens 203. Thereby, CARS light can be generated from the specific vibration frequency of the molecules in the sample 205 at each position where the light beam L3 is collected.

  The CARS light generated in the specimen 205 is collected by a condenser lens 206 disposed on the opposite side of the objective lens 203 with the specimen 205 interposed therebetween, and is detected by the first detection unit 301. Then, based on the information detected by the first detection unit 301 and the position information (scanning information) of the light spot obtained from the two-dimensional scanning unit 203, an image of the observation surface of the specimen 205 is formed by the image processing unit 401. Then, an image formed by the display unit 402 can be displayed.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, using the pulse light source 101, the light beam L2 ′ whose frequency is converted to the high frequency side by the OPO 103 is pump light, and the light beam whose frequency is not converted. CARS light can be generated using L1 as Stokes light.

  Next, the observation of the DIC image in the case where the observation of the CARS light generated from the specimen 205 and the observation of the DIC image of the specimen 205 are simultaneously performed using the laser microscope apparatus 1 according to the present embodiment will be described below. The observation of the CARS light is as described above.

  When the DIC image of the specimen 205 is observed simultaneously with the observation of the CARS light generated from the specimen 205, the polarizer 104, the birefringent prism (light beam splitter) 202, and the birefringent prism (light flux combiner) are added to the laser microscope apparatus 1. ) 209 and the analyzer 210 are used.

  The direction of the birefringent prism 202 is adjusted so that at least one of the light beam L1 'and the light beam L2' out of the light beam L3 that has passed through the two-dimensional scanning unit 201 is not divided into two light beams. The polarizer 104 is adjusted so that only one of the light beam L 1 ′ and the light beam L 2 ′ is divided into two light beams by the birefringent prism 202. The two divided light beams are spatially separated, and their polarization components are orthogonal to each other. The two divided light beams are converged by the objective lens 203 and condensed on the surface of the sample 205, and scanned by the two-dimensional scanning unit 201.

  The two light fluxes that have passed through the specimen 205 are partially reflected by the dichroic mirror 207 through the condenser lens 206, enter the birefringent prism 209, and are combined into one light flux by the birefringent prism 209. The light beam combined by the birefringent prism 209 is incident on the analyzer 210. The analyzer 210 selectively transmits linearly polarized light orthogonal to the linearly polarized light transmitted through the polarizer 104. The light beam that has passed through the analyzer 210 is detected by the second detection unit 302. Then, based on the information detected by the second detection unit 302 and the light spot position information (scanning information) obtained from the two-dimensional scanning unit 201, an image of the observation surface of the specimen 205 is formed by the image processing unit 401. Then, an image formed by the display unit 402 can be displayed. The image processing unit 401 repeatedly obtains two-dimensional image information from the same observation area by continuous driving of the two-dimensional scanning unit 201, forms time-series moving image information, and displays (screens) it on the display unit 402. You can also.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, the DIC image of the specimen 205 can be observed using the two linearly polarized light beams that are divided by the birefringent prism 202 and orthogonal to each other. .

  Since the light beam divided into two by the birefringent prism 202 is spatially separated, it is irradiated to two different points in the sample. A phase difference (retardation) corresponding to the difference between the optical path lengths of the portions where the two light beams pass through the sample is generated between the two light beams. That is, a phase difference occurs between the two light beams due to the difference in the optical thickness of the portion through which the two light beams pass. The phase difference between the two light beams generates a linearly polarized component that is orthogonal to the linearly polarized light that is transmitted through the polarizer 104. Therefore, the phase difference between the two light beams is detected as the intensity of the light beam that has passed through the analyzer 210.

 That is, when the optical thickness of the part in the sample 205 through which the two light beams pass is equal, no signal is output from the second detection unit 302, but the optical part of the part in the sample 205 through which the two light beams pass. When the typical thickness is different, a signal corresponding to the difference in optical thickness is output. Thereby, even if the observation object in the specimen 205 is optically transparent, information on the level difference and inclination of the surface can be acquired.

Here, regarding the CARS light generated from the specimen, the reduction in CARS light intensity that can occur when CARS light observation and DIC observation are performed at the same time, and the effect of preventing the reduction in CARS light intensity in the present embodiment will be described.

As described above, if the birefringent prism is combined with the CARS microscope, both the pump light and the Stokes light are split into two linearly polarized light beams orthogonal to each other. As a result, the CARS light generated from the sample is obtained. The strength of is reduced. For example, as shown in FIG. 2B, the intensity I of CARS light generated when pump light and Stokes light inclined 45 degrees from the x-axis are divided into two light beams by a birefringent prism and condensed on a sample. ' _CARS is reduced by a _{factor of} four compared to the CARS light intensity I _CARS generated when the polarization directions of the pump light and Stokes light are adjusted in parallel to be condensed on the specimen.

On the other hand, according to the laser microscope apparatus 1 according to the present embodiment, it is possible to adjust so that only Stokes light is divided into two linearly polarized light beams orthogonal to each other. When the intensity of the CARS light generated when Stokes light inclined 45 degrees from the x-axis as shown in FIG. 2B is divided into two light beams by the birefringent prism and condensed on the sample is calculated, I ′ ′ Twice the _CARS . That is, the decrease in CARS light intensity when compared with I _CARS can be suppressed to a half.

  Next, a description will be given of a reduction in the spatial resolution of CARS light observation that can occur when CARS light observation and DIC observation are performed at the same time, and the effect of preventing the reduction in spatial resolution in the present embodiment.

  As described above, when the birefringent prism is combined with the CARS microscope, both the pump light and the Stokes light are split into two linearly polarized light beams orthogonal to each other. As a result, CARS light generated from two light spots formed at slightly shifted positions on the specimen is detected at the same time, and the spatial resolution of CARS light observation is reduced.

  On the other hand, according to the laser microscope apparatus 1 according to the present embodiment, it is possible to adjust so that only Stokes light is divided into two linearly polarized light beams orthogonal to each other. Therefore, only Stokes light forms two light spots on the specimen, and pump light forms only one light spot. Thereby, it is possible to avoid a reduction in the spatial resolution of CARS light observation due to the splitting of the pump light and the Stokes light into two linearly polarized lights orthogonal to each other. Thereby, when performing both CARS light observation and DIC observation, there exists an advantage that it becomes possible to detect the same place simultaneously. The ability to detect the same location at the same time means that a CARS light observation image can be obtained even when imaging a sample that exhibits various movements, such as a living body, or when the scanning speed changes during the optical scanning process. Since the DIC observation image and the DIC observation image completely coincide with each other in time and space, there is a remarkable effect that a clear overlapping display is possible for the entire observation region.

  As described above, according to the laser microscope apparatus 1 according to the present embodiment, the CARS light observation and the DIC observation can be simultaneously performed on the same place without suppressing the decrease in the generation efficiency of the CARS light and without reducing the spatial resolution. it can.

[Second Embodiment]
A laser microscope apparatus 2 according to a second embodiment of the present invention will be described below with reference to FIG.
The laser microscope apparatus 2 according to the present embodiment is different from the first embodiment in that the DIC observation is performed by an epi-illumination type. Hereinafter, with respect to the laser microscope apparatus 2 of the present embodiment, description of points that are common to the first embodiment will be omitted, and different points will be described.

  The laser microscope apparatus 2 two-dimensionally scans the light beam separating unit 501 that directs the light beam L3 combined by the laser combiner (combining unit) 105 to the sample 205 and the light beam L3 that has passed through the light beam separating unit 501. A two-dimensional scanning unit 201, a birefringent prism (light beam splitting / combining unit) 503 that separates the light beam L3 that has passed through the two-dimensional scanning unit 201 into two light beams according to the polarization component, and a light beam that has passed through the birefringent prism 503 is a sample 205 An objective lens (light condensing unit) 203 that condenses the light is provided. In the figure, reference numeral 502 denotes a deflection mirror, and reference numeral 204 denotes a stage.

  The light beam separation unit 501 directs the light beam L3 combined by the laser combiner 105 to the objective lens 203, and separates the light beam returning from the sample 205 through the objective lens 203 from the light beam directed to the objective lens 203. . The light beam separation unit 501 is configured by, for example, a mirror that partially reflects and partially transmits light. The light beam separation unit 501 is constituted by, for example, a non-polarizing beam splitter whose split ratio of reflection and transmission of incident light does not depend on the polarization of incident light.

The birefringent prism 503 splits only one of the light beam L1 ′ and the light beam L2 ′ out of the light beam L3 that has passed through the two-dimensional scanning unit 201 into two light beams according to the polarization component. The two divided light beams are spatially separated, and their polarization components are orthogonal to each other.
The direction of the birefringent prism 503 is adjusted so that at least one of the light beam L1 ′ and the light beam L2 ′ is not divided into two light beams. The polarizer (linear polarization adjusting unit) 104 is adjusted so that only one of the light beam L1 ′ and the light beam L2 ′ is split into two light beams by the birefringent prism 503. The light beams that have passed through the birefringent prism 503 pass through the objective lens (condenser) 203 and are condensed on the specimen 205 to form a light spot. Further, the birefringent prism 503 combines the two light beams split by the birefringent prism 503 that return from the sample 205 through the objective lens 203. Since the birefringent prism 503 is an element necessary only when the DIC image is observed, similarly to the polarizer 104, it may be incorporated into the optical path only when the DIC image is observed.

  In addition, the laser microscope apparatus 2 includes a first detection unit 301 for detecting CARS light generated from the specimen 205 and collected by the condenser lens 206. In the figure, reference numeral 504 is a short-pass filter for selectively passing the CARS light generated from the sample 205 from the light beam transmitted through the sample 205, and reference numeral 505 is a band-pass filter.

  In addition, the laser microscope apparatus 2 includes an analyzer (linear polarization extraction unit) 210 for extracting a light beam having a specific linear polarization from the light beam returned from the sample 205 separated by the light beam separation unit 501, and a light beam that has passed through the analyzer 210. The band-pass filter 506 for selectively extracting the wavelength of the light beam divided into two linearly polarized light orthogonal to each other by the birefringent prism 503, and the second light beam for detecting the light beam that has passed through the band-pass filter 506. And a detection unit 302.

  Further, the laser microscope apparatus 2 may include a confocal pinhole for excluding unwanted light from other than the observation surface from the light beam that has passed through the analyzer 210. By using a confocal optical system, undesired light from other than the observation surface, such as reflected light and scattered light from parts outside the vicinity of the observation surface, or reflected light from optical elements in the optical system, is confocal. Blocked by a pinhole. This makes it possible to observe the fine structure of the specimen while suppressing noise.

  Further, the laser microscope apparatus 2 is based on the information detected by the first detection unit 301 and the second detection unit 302 and the position information (scanning information) of the light spot obtained from the two-dimensional scanning unit 201. An image processing unit 401 that forms an image of the observation surface of the specimen 205 and a display unit 402 that displays the formed image are provided.

The operation of the laser microscope apparatus 2 configured as described above will be described below.
First, the case of observing the specimen 205 with CARS light using the laser microscope apparatus 2 according to the present embodiment is as described in the first embodiment.

  The observation of the DIC image when the observation of the CARS light generated from the specimen 205 and the observation of the DIC image of the specimen 205 are simultaneously performed using the laser microscope apparatus 2 according to the present embodiment will be described below. The observation of the CARS light is as described above.

  When the DIC image of the specimen 205 is observed simultaneously with the observation of the CARS light generated from the specimen 205, the laser microscope apparatus 2 is used in a state where the polarizer 104 and the birefringent prism 503 are arranged.

  The direction of the birefringent prism 503 is adjusted so that at least one of the light beam L1 'and the light beam L2' out of the light beam L3 that has passed through the two-dimensional scanning unit 201 is not divided into two light beams. The polarizer 104 is adjusted so that only one of the light beam L 1 ′ and the light beam L 2 ′ is divided into two light beams by the birefringent prism 503. The two divided light beams are spatially separated, and their polarization components are orthogonal to each other. The two divided light beams are converged by the objective lens 203 and condensed on the surface of the sample 205, and scanned by the two-dimensional scanning unit 201.

  The two light beams reflected by the specimen 205 return to the birefringent prism 503 through the objective lens 203 and are combined into one light beam by the birefringent prism 503. The light beam from the birefringent prism 503 is reflected by the deflection mirror 502, passes through the two-dimensional scanning unit 201, and partially passes through the light beam separation unit 501. The light beam that has passed through the light beam separation unit 501 enters the analyzer 210. The analyzer 210 selectively transmits linearly polarized light orthogonal to the linearly polarized light transmitted through the polarizer 104. The light beam that has passed through the analyzer 210 is detected by the second detection unit 302. Then, based on the information detected by the second detection unit 302 and the light spot position information (scanning information) obtained from the two-dimensional scanning unit 201, an image of the observation surface of the specimen 205 is formed by the image processing unit 401. Then, an image formed by the display unit 402 can be displayed. The image processing unit 401 repeatedly obtains two-dimensional image information from the same observation area by continuous driving of the two-dimensional scanning unit 201, forms time-series moving image information, and displays (screens) it on the display unit 402. You can also.

  As described above, according to the laser microscope apparatus 2 according to the present embodiment, it is possible to observe the DIC image of the specimen 205 using the two linearly polarized light beams which are divided by the birefringent prism 503 and orthogonal to each other. .

  In addition, when a confocal pinhole for removing unwanted light from other than the observation surface from the light beam that has passed through the analyzer 210 is provided, the confocal pinhole is disposed at a position conjugate with the sample surface. Since it is a confocal optical system, undesired light from other than the observation surface, such as reflected light and scattered light from portions outside the vicinity of the observation surface, and reflected light from optical elements in the optical system, is blocked. This makes it possible to acquire information on the fine structure of the specimen that has been buried in noise in normal microscope observation such as bright-field observation. Further, laser light as a point light source is irradiated onto the aperture sample by the objective lens, and the generated light is imaged by the same objective lens, and only the light from the focal plane is detected through the confocal pinhole. Accordingly, since the image is obtained by converging the light beam twice, the plane spatial resolution is about 1.4 times better than the normal microscope observation.

Here, regarding the CARS light generated from the specimen, the reduction in CARS light intensity that can occur when CARS light observation and DIC observation are performed at the same time, and the effect of preventing the reduction in CARS light intensity in the present embodiment will be described.

As with the laser microscope apparatus 1 according to the first embodiment, according to the laser microscope apparatus 2 according to this embodiment, adjustment is performed so that only Stokes light is divided into two linearly polarized light beams orthogonal to each other. Is possible. When the intensity of the CARS light generated when Stokes light inclined 45 degrees from the x-axis as shown in FIG. 2B is divided into two light beams by the birefringent prism and condensed on the sample is calculated, I ′ ′ Twice the _CARS . That is, the decrease in CARS light intensity when compared with I _CARS can be suppressed to a half.

  Next, a description will be given of a reduction in the spatial resolution of CARS light observation that occurs when CARS light observation and DIC observation are performed simultaneously.

  As with the laser microscope apparatus 1 according to the first embodiment, according to the laser microscope apparatus 2 according to this embodiment, adjustment is performed so that only Stokes light is divided into two linearly polarized light beams orthogonal to each other. Is possible. Therefore, only Stokes light forms two light spots on the specimen, and pump light forms only one light spot. Thereby, it is possible to avoid a reduction in the spatial resolution of CARS light observation due to the splitting of the pump light and the Stokes light into two linearly polarized lights orthogonal to each other. Thereby, when performing both CARS light observation and DIC observation, there exists an advantage that it becomes possible to detect the same place simultaneously. The ability to detect the same location at the same time means that a CARS light observation image can be obtained even when imaging a sample that exhibits various movements, such as a living body, or when the scanning speed changes during the optical scanning process. Since the DIC observation image and the DIC observation image completely coincide with each other in time and space, there is a remarkable effect that a clear overlapping display is possible for the entire observation region.

  As described above, according to the laser microscope apparatus 2 according to the present embodiment, the CARS light observation and the DIC observation can be simultaneously performed on the same place without reducing the generation efficiency of the CARS light and reducing the spatial resolution. it can. Furthermore, the spatial resolution of the DIC observation can be increased by performing the DIC observation by the epi-illumination type.

  As mentioned above, although each embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design changes and the like without departing from the gist of the present invention. .

  For example, as disclosed in Japanese Patent Application Laid-Open No. 2010-96667, a femtosecond pulse laser light source is used as the pulse light source 101, and the frequency dispersion amount of the light beam emitted from the femtosecond pulse laser light source is adjusted in the first optical path 001. A frequency dispersion adjusting unit may be provided, and a frequency converting unit and a delay optical path may be provided in the second optical path 002. Here, it is preferable that the frequency dispersion adjusting unit includes, for example, a pair of prisms that can adjust the distance between each other, and a mirror, and the light beam that has passed through the pair of prisms is returned again after being folded by the mirror. It is preferable to pass through the prism pair and return to the same optical path. In this case, the amount of frequency dispersion given to the light beam passing through the frequency dispersion adjusting means can be adjusted by adjusting the interval between the prisms. A pair of diffraction gratings may be used instead of the pair of prisms.

  Further, instead of adjusting the amount of frequency dispersion by the frequency dispersion adjusting means, a band of a light beam emitted from the femtosecond pulse laser light source may be limited using a bandpass filter. In this case, the frequency conversion means is composed of, for example, a photonic crystal fiber. The photonic crystal fiber changes or expands the frequency band of the light beam to be passed, and causes the pulsed laser light L1, L2 ′ guided through the optical path 001, 002 to have a frequency difference substantially equal to the specific vibration frequency of the molecules in the sample. It is preferable to give.

  In addition, the two-dimensional scanning unit may have a configuration in which the focal point of the irradiation light is moved to a different position in the depth direction of the sample by adding another scanning unit as its own configuration. It is also possible to obtain a three-dimensional image.

L1, L2, L3, L4, L1 ′, L2 ′, L4 ′ Light flux 100 Light source unit 101 Pulse light source 102 Delayed optical path 103 Optical parametric oscillator (OPO)
104 Polarizer (Linear Polarization Adjuster)
105 Laser combiner
201 Two-dimensional scanning unit 202 Birefringent prism (beam splitting unit)
203 Objective lens (condenser)
204 Stage 205 Specimen 206 Condensing lens 207 Dichroic mirror (light beam separation unit)
208, 211, 505, 506 Band pass filter 209 Birefringent prism (light beam combining unit)
210 Analyzer (Linear polarization extractor)
301 First detection unit 302 Second detection unit 401 Image processing unit 402 Display unit 501 Light beam separation unit 502 Deflection mirror 503 Birefringence prism (light beam splitting and combining unit)
504 Short pass filter

Claims (3)

A laser microscope apparatus for simultaneously detecting a CARS signal and a DIC signal,
A light source unit for generating a first light beam and a second light beam having different wavelengths from each other;
A multiplexing unit for combining the two luminous fluxes;
A linearly polarized light adjusting unit that is provided in at least one of the two optical paths that guide the two light beams to the multiplexing unit, and generates a linearly polarized light beam;
A two-dimensional scanning unit that scans a light spot formed on the specimen;
A light beam splitting unit that splits a light beam into two light beams according to a polarization component;
A condensing part for condensing the combined light flux on the sample;
Of the light flux obtained from the sample, a light beam combining unit that combines the light beam split into two light beams by the light beam splitting unit;
A linear polarization extraction unit that extracts a specific linearly polarized light beam from the combined light beam;
A light beam separation unit for separating at least one of the first light beam and the second light beam;
A first detector for detecting a CARS signal;
A second detector for detecting a DIC signal;
An image processing unit that forms an image of an observation surface in the specimen based on information detected by the first detection unit and the second detection unit and position information of a light spot obtained from the two-dimensional scanning unit; A laser microscope apparatus comprising:   2. A beam splitting / combining unit that splits a light beam into two light beams according to a polarization component and combines a light beam returning from the sample, instead of the light beam splitting unit and the light beam combining unit, and a return from the sample. A laser beam splitting unit that splits the beam synthesized by the beam splitting and synthesizing unit, and the second detection unit is arranged in an epi-illumination type. 3. The laser microscope apparatus according to claim 1, wherein the light beam separation unit is a light beam separation unit whose separation ratio does not depend on polarization of incident light.

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