JP4914542B2 - Scanning microscope equipment - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a scanning laser microscope apparatus that irradiates a specimen to be observed with a laser beam (laser beam), and more particularly to a multiphoton excitation scanning laser microscope apparatus for detecting chemical reaction and fluorescence due to multiphoton absorption of a specimen. .
[0002]
[Prior art]
In a multi-photon excitation scanning laser microscope, excitation that is normally performed with one photon (single photon) is performed with multi-photons. For example, in a two-photon excitation scanning laser microscope, fluorescence excitation performed at a wavelength of 400 nm (single photon) is performed at 800 nm, which is a double wavelength. That is, at this wavelength of 800 nm, fluorescence excitation is performed using two photons.
[0003]
Normally, mercury lamps used in fluorescent microscopes and continuous-wave laser light have a low photon density per unit time, so enormous light intensity is required to generate the multi-photon excitation phenomenon. Since there is a problem such as damage to the system and specimen, it is not suitable for practical use as a light source for generating a multiphoton excitation phenomenon.
[0004]
For this reason, a light source for a two-photon excitation scanning laser microscope that oscillates a sub-picosecond pulse laser beam is used, for example. This is because, by using this sub-picosecond pulse laser beam, the two-photon excitation phenomenon occurs with a probability almost proportional to the square of the photon density per unit area and unit time. This is because the probability of doing so increases.
[0005]
For example, Japanese Laid-Open Patent Publication No. 5-503149 discloses a laser light source that emits a sub-picosecond pulse laser beam and scanning optics for scanning a sample surface (focal plane) with the pulse laser beam emitted from the laser light source. A two-photon excitation scanning laser microscope combined with a unit is described.
[0006]
[Problems to be solved by the invention]
In general, such a two-photon excitation scanning laser microscope is expensive because of its complicated structure, and is very difficult for a user to handle. In particular, such a two-photon excitation scanning laser microscope is configured so that the oscillation wavelength of the laser beam can be adjusted, so that observation at various excitation wavelengths is possible, but the oscillation wavelength of the laser beam is not limited. There is a problem that the adjustment work (change work) becomes very complicated. The excitation wavelength used for two-photon excitation is considered to be twice as long as the wavelength used for normal one-photon excitation. However, in practice, the wavelength is shorter than twice the wavelength due to the phenomenon of blue shift. It had to be excited. Therefore, it is difficult to determine what wavelength is the appropriate excitation wavelength (oscillation wavelength), and trial and error is required to select the oscillation wavelength for each observation, and the setting of the oscillation wavelength becomes complicated. It was. In particular, this is a significant problem for users who have no knowledge of the relationship between the excitation wavelength for the fluorescent reagent and the adjustment of the oscillation wavelength of the laser beam.
[0007]
In view of the above circumstances, an object of the present invention is to provide a scanning microscope apparatus that facilitates setting of an oscillation wavelength of laser light.
[0008]
[Means for Solving the Problems]
The invention according to claim 1 is a scanning microscope apparatus that observes a sample by scanning a laser beam on a sample placed on a stage, and a laser light source capable of continuously changing the wavelength region of the laser light; Storage means for storing excitation wavelength information by multiphotons corresponding to the fluorescent reagent, which is set from the wavelength range, and the wavelength range of the laser beam become the excitation wavelength corresponding to the designated fluorescent reagent in the control means for controlling the laser light source, it has a, the laser light source oscillates the ultrashort pulsed laser light to allow observation of the fluorescence by multiphoton excitation, scanning microscope apparatus characterized by such is there.
[0009]
According to the above configuration, when a fluorescent reagent is designated, an optimum laser corresponding to the designated fluorescent reagent is stored based on the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent stored in the storage unit. The excitation wavelength of light is automatically set as the oscillation wavelength of the laser light source. Accordingly, the oscillation wavelength can be easily set.
[0010]
According to a second aspect of the present invention, in the first aspect of the present invention, the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent stored in the storage means is information that can be arbitrarily set. According to this configuration, it is possible to arbitrarily set (change or edit) information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent.
[0011]
According to a third aspect of the invention, in the first aspect of the invention, the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent stored in the storage means is information set based on past observation results. It is a certain configuration. According to this configuration, the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent can be set based on past observation results.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a diagram showing a system configuration of a scanning microscope apparatus according to an embodiment of the present invention.
[0014]
The scanning microscope apparatus shown in the figure is a multiphoton excitation scanning laser microscope apparatus for detecting a chemical reaction and fluorescence due to multiphoton absorption of a specimen, and includes a scanning laser microscope 1, an operation panel 2, a controller 3, A storage means (memory) 4 and an image monitor 5 are included.
[0015]
The scanning laser microscope 1 further includes a laser light source 6, a mirror 7, a dichroic mirror 8, a scanning unit 9, a revolver 10, an objective lens 11, a stage 12, a sample 13, a lens 14, a pinhole plate 15, and a photodetector 16. Have.
Here, the laser light source 6 is for generating (oscillating) laser light as spot light that scans the surface of the sample 13 serving as a specimen. For example, an extremely short pulse that enables observation of fluorescence by multiphoton excitation. Oscillates laser light. The laser light source 6 is configured so that the wavelength range of the laser light can be continuously varied, and generates laser light corresponding to the oscillation wavelength (excitation wavelength) set by the controller 3. The laser light from the laser light source 6 is reflected by the mirror 7 and guided to the scanning unit 9 through the dichroic mirror 8.
[0016]
The scanning unit 9 is for two-dimensionally scanning the laser beam from the laser light source 6 provided from the mirror 7 on the sample 13 based on a control command from the controller 3. It has a mirror and a galvanometer mirror for scanning in the Y-axis direction, and is configured to swing the optical path of the spot light with respect to the objective lens 11 in the XY direction by swinging these galvanometer mirrors in the X-axis direction and the Y-axis direction. Yes.
[0017]
The revolver 10 holds a plurality of objective lenses 11 having different magnifications. By switching the revolver 10, a plurality of objective lenses 11 having a desired magnification are selectively inserted into the observation optical path of the microscope. The spot light that is two-dimensionally scanned by the scanning unit 9 through the objective lens 11 that has been selectively inserted is irradiated onto the sample 13 on the stage 12.
[0018]
Image information of reflected light from the sample 13 or image information of fluorescence generated from the sample 13 is returned to the scanning unit 9 through the objective lens 11 and returned to the dichroic mirror 8 through the scanning unit 9. The dichroic mirror 8 is a semi-transparent mirror provided on the emission light path of the laser light source 6 with respect to the scanning unit 9 and guides image information from the sample 13 given through the scanning unit 9 to the light detection system. It is.
[0019]
Image information from the scanning unit 9 obtained through the dichroic mirror 8 is condensed by the lens 14 and given to a pinhole plate 15 forming a confocal optical system. The pinhole plate 15 is formed with a pinhole having a predetermined diameter, and is arranged so that the pinhole is positioned at the imaging position on the front surface of the light receiving surface of the photodetector 16. The light detector 16 is composed of a light detecting element that converts light obtained through the pinhole of the pinhole plate 15 into an electric signal corresponding to the light amount.
[0020]
In addition to the keyboard, the operation panel 2 includes a pointing device such as a trackball, a joystick, or a mouse. Various instructions such as a scanning instruction and an image input instruction are given to the controller 3 according to instructions from the user. Is output.
[0021]
The controller 3 outputs control commands to various places as necessary to control the entire scanning microscope apparatus. For example, by outputting a predetermined control command to the laser light source 6, the oscillation wavelength of the predetermined laser light is set in the laser light source 6. Further, in response to a scanning instruction received via the operation panel 2, a scanning command is output to the scanning unit 9, the image information of the sample 13 detected by the photodetector 16 is captured, and the image information is transferred to the storage unit 4. At the same time, an image based on the image information is displayed on the image monitor 5.
[0022]
The storage means 4 stores excitation wavelength information corresponding to various fluorescent reagents. Further, the storage means 4 stores image information of the sample 13 from the photodetector 16 transferred via the controller 3 described above. In the present embodiment, the storage unit 4 includes a memory unit that stores information on excitation wavelengths corresponding to various fluorescent reagents, a memory unit that stores image information of the sample 13, and the like.
[0023]
The image monitor 5 displays an image based on the image information of the sample 13 stored in the storage unit 4, a list of fluorescent reagents based on information on excitation wavelengths corresponding to various fluorescent reagents, and the like.
The information on the excitation wavelength corresponding to the various fluorescent reagents stored in the storage means 4 is the optimum excitation wavelength corresponding to the designated fluorescent reagent when a predetermined fluorescent reagent is designated. Is information that can be determined. However, the excitation wavelength at this time is assumed to be a wavelength within a wavelength range that can be varied by the laser light source 6. Information on the excitation wavelengths corresponding to the various fluorescent reagents is stored in the storage unit 4 as, for example, a correspondence table between the fluorescent reagents and the excitation wavelengths.
[0024]
FIG. 2 is a diagram showing an example of a correspondence table between the fluorescent reagent and the excitation wavelength.
As shown in the figure, the correspondence table shows the optimum excitation wavelength (WaveLength) for each fluorescent reagent name (Name). Therefore, by designating a predetermined fluorescent reagent by this table, it becomes possible to uniquely determine the optimum excitation wavelength corresponding to the designated fluorescent reagent. In this correspondence table, the excitation wavelength corresponding to the fluorescent reagent name is described in the correspondence table. However, the correspondence source is not limited to the fluorescent reagent name, and instead of the fluorescent reagent name, for example, a user ( It may be the name of an observer or the like) or an experiment number.
[0025]
Next, an example of the operation of the scanning microscope apparatus configured as described above will be described. In this example, it is assumed that the correspondence table shown in FIG. 2 is stored in the storage unit 4 as information on excitation wavelengths corresponding to various fluorescent reagents.
First, in order to set the oscillation wavelength of laser light from the laser light source 6, the controller 3 displays a list of fluorescent reagents on the image monitor 5 based on a correspondence table of fluorescent reagents and excitation wavelengths stored in the storage unit 4. Then, the user is prompted to select and instruct the fluorescent reagent used for the sample 13 to be observed from the list of fluorescent reagents displayed on the display screen.
[0026]
At this time, the user can instruct selection of the fluorescent reagent used for the sample 13 from the list of fluorescent reagents displayed on the image monitor 5. The selection instruction by the user is made via the operation panel 2.
When the controller 3 receives an instruction for selecting a predetermined fluorescent reagent from the user via the operation panel 2, the controller 3 refers to the correspondence table stored in the storage unit 4 and performs excitation corresponding to the selected fluorescent reagent. Read the wavelength. For example, when the user instructs to select “TRITC” as the fluorescent reagent, “1000” is read as the corresponding excitation wavelength from the correspondence table shown in FIG.
[0027]
Then, the read excitation wavelength is set as the oscillation wavelength of the laser light source 6. The oscillation wavelength is set by the controller 3 outputting a predetermined control command to the laser light source 6.
Subsequently, the controller 3 receives a scanning start instruction from the user via the operation panel 2, and when this is received, outputs a scanning command to the scanning unit 9, and actually scans the sample 13, Image information of the sample 13 is detected by the photodetector 16, and an image based on the image information is displayed on the image monitor 5.
[0028]
As described above, according to the operation described above, the user simply selects and instructs the fluorescent reagent used for the sample 13 as the specimen from the list of various fluorescent reagents displayed on the image monitor 5. It becomes possible to set the optimum excitation wavelength for the fluorescent reagent as the oscillation wavelength of the laser light. Therefore, the user can easily set the oscillation wavelength of the laser beam without knowledge of the relationship between the excitation wavelength for the fluorescent reagent and the adjustment of the oscillation wavelength of the laser beam. Further, since the excitation wavelength corresponding to the fluorescent reagent is uniquely determined, observation under the same conditions is always possible.
[0029]
In this embodiment, the contents of the excitation wavelength information corresponding to the various fluorescent reagents stored in the storage unit 4 can be freely edited by the user, and the user can arbitrarily enter the excitation wavelength information corresponding to the fluorescent reagent. It may be configured to be settable. Thereby, the user can freely set the excitation wavelength corresponding to the fluorescent reagent, and can be applied to experiments using various fluorescent reagents.
[0030]
Further, the content of the excitation wavelength information corresponding to various fluorescent reagents stored in the storage unit 4 may be set based on past observation results (experimental results). For example, it can be set as follows. The sample 13 is irradiated with laser light while continuously changing the oscillation wavelength in a predetermined wavelength range, and the fluorescence intensity is measured for each oscillation wavelength during that time. Set as the optimal excitation wavelength for the reagent. For example, when the fluorescence intensity for each oscillation wavelength of the laser light as shown in FIG. 3 is measured, the oscillation wavelength (W) at which the fluorescence intensity is maximum (Imax) corresponds to the fluorescence reagent. What is necessary is just to set as an optimal excitation wavelength.
[0031]
Further, the information on the excitation wavelength corresponding to the various fluorescent reagents is not limited to the storage means 4 and is stored, for example, a computer hard disk, a portable storage medium (floppy (registered trademark) disk, CD-R, etc. ) Or the like, and may be read from the storage medium and used. In this case, if the information is stored as a file of a general-purpose file format, the information can be changed more easily by editing the file. Alternatively, the information may be stored in a storage medium of an external device, transferred by communication, read, and used.
[0032]
【Effect of the invention】
As described above in detail, according to the present invention, it is possible to automatically set the optimum excitation wavelength corresponding to the designated fluorescent reagent as the oscillation wavelength of the laser beam, so that the user can Even without knowledge of the relationship between the excitation wavelength for the reagent and the adjustment of the oscillation wavelength of the laser beam, the oscillation wavelength of the laser beam can be easily set. In addition, since the excitation wavelength corresponding to the fluorescent reagent is uniquely determined as the oscillation wavelength of the laser light, observation under the same conditions is always possible.
[Brief description of the drawings]
FIG. 1 is a diagram showing a system configuration of a scanning microscope apparatus according to an embodiment of the present invention.
FIG. 2 is a diagram showing an example of a correspondence table of excitation wavelengths corresponding to fluorescent reagents.
FIG. 3 is an example of a graph showing measurement results of fluorescence intensity at each laser oscillation wavelength.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Scanning microscope apparatus 2 Operation panel 3 Controller 4 Memory | storage means (memory)
5 Image Monitor 6 Laser Light Source 7 Mirror 8 Dichroic Mirror 9 Scanning Unit 10 Revolver 11 Objective Lens 12 Stage 13 Sample 14 Lens 15 Pinhole Plate 16 Photodetector

Claims (3)

In a scanning microscope apparatus that observes a sample by scanning a laser beam on the sample placed on the stage,
A laser light source capable of continuously changing the wavelength range of the laser light;
Storage means for storing information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent set from the wavelength range;
Control means for controlling the laser light source so that the wavelength range of the laser light becomes an excitation wavelength corresponding to a designated fluorescent reagent;
I have a,
The laser light source oscillates an extremely short pulse laser beam that enables fluorescence to be observed by multiphoton excitation.
Scanning microscope device comprising a call. The scanning microscope apparatus according to claim 1, wherein the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent stored in the storage means is information that can be arbitrarily set. The scanning microscope apparatus according to claim 1, wherein the information on the excitation wavelength by multiphotons corresponding to the fluorescent reagent stored in the storage means is information set based on past observation results.

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