JP2006227301A - Fluorescence observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To more suppress damage of a sample A, compared with a case where it is irradiated with continuous light, and to obtain a bright fluorescent image. <P>SOLUTION: The fluorescence observation device 1 is provided, which includes a pulse light source 2 for generating excitation light E in a pulse train to irradiate the sample A and an imaging device 5 for imaging fluorescent light F generated in the sample A. The pulse light source 2 irradiates each position of the sample A corresponding to each pixel of the imaging device 5 with the excitation light E in a plurality of pulses, within the imaging time of one frame of the imaging device 5. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fluorescence observation apparatus.

  Conventionally, a pulse laser fluorescence microscope has been used as an apparatus for capturing an instant of a high-speed phenomenon that changes instantaneously as an image by irradiating a sample with excitation light for a short time using a pulse laser light source and detecting fluorescence generated in the sample. Is known (for example, Non-Patent Document 1). According to this pulse laser fluorescence microscope, a sufficient amount of fluorescence can be generated by irradiating a selected pulse light of high energy instantaneously, and a bright fluorescence image can be obtained.

Also, conventionally, a multiphoton absorption phenomenon is observed in a very limited region of the sample by irradiating the sample with an ultrashort pulse laser beam having a pulse width in femtosecond units, and the obtained photon is observed. An excitation type fluorescence microscope is known (for example, refer to Patent Document 1).
According to this multiphoton excitation type fluorescence microscope, the multiphoton absorption phenomenon is caused only at the imaging position of the laser beam in the sample, so that high spatial resolution can be achieved. In addition, fluorescence observation can be performed by allowing the laser beam to reach a deep portion in the sample.
Junichi Takaya, one other person, “Biomicroscope beyond the limits-seeing invisible things, the Spectroscopical Society of Japan Measurement Series,” Japan Society for the Science of Publishing, June 30, 1991, Volume 21, p. 105-119 Japanese Patent No. 2848952

  However, there is a problem that the instantaneous image obtained by the conventional pulse laser fluorescence microscope is not suitable for accurately capturing subtle changes in the sample because it has a lot of noise and low spatial resolution. In addition, in order to obtain a sufficient amount of fluorescence, high-energy pulsed light is irradiated instantaneously. For example, there is a problem in that a sample such as a cell is damaged and fading occurs. Temporarily, when irradiating the pulse light which reduced energy to the extent that the sample is not damaged, there is a problem that the fluorescent image becomes dark.

On the other hand, the multiphoton excitation type fluorescence microscope has no such inconvenience. That is, since fluorescence is generated by a plurality of photons, laser light having a long wavelength with low energy of each photon can be used, so that the sample is less damaged. Therefore, fading of the sample in a portion other than the focal plane that is not observed is prevented, and observation over a long time is also possible.
However, since the multiphoton excitation type fluorescence microscope uses an ultrashort pulse laser beam, a dispersion compensation device is indispensable to compensate for dispersion generated when passing through an optical element, and the device is complicated and large. There is an inconvenience of becoming. There is also a problem that the ultrashort pulse laser itself is large and expensive.

  The present invention has been made in view of the above-described circumstances, and provides a fluorescence observation apparatus capable of suppressing damage to a sample and obtaining a bright fluorescent image as compared with the case of irradiating continuous light. It is aimed.

In order to achieve the above object, the present invention provides the following means.
The present invention includes a pulse light source device that generates pulse train-shaped excitation light and irradiates a sample, and an image sensor that images fluorescence generated in the sample, and the pulse light source device captures one frame of the image sensor. Provided is a fluorescence observation apparatus that irradiates each position of a sample corresponding to each pixel of the imaging element with a plurality of pulsed excitation light within a time period.

  According to the present invention, the sample is irradiated with excitation light in the form of a pulse train emitted from the pulse light source device. At each position of the sample, every time the pulsed excitation light is irradiated, the fluorescent material is excited to emit fluorescence, and the generated fluorescence is imaged by the imaging element.

As a result of the study, the inventors of the present invention have found that the fluorescence generated when the pulsed excitation light is irradiated a plurality of times is brighter than the fluorescence generated when the continuous excitation light is continuously irradiated. It was found that an image could be obtained and that the sample was less damaged.
According to the present invention, pulsed excitation light is irradiated to each position on the sample a plurality of times within the acquisition time of a single-frame fluorescence image by the imaging device. Thereby, a brighter fluorescent image can be obtained than when scanning with continuous light, and damage to the sample can be suppressed. In addition, since it is not necessary to use an ultrashort pulse laser that generates a multiphoton absorption phenomenon, dispersion compensation is unnecessary, and the size of the apparatus can be reduced by simplifying the configuration of the apparatus.

Further, since pulsed fluorescence from the sample is incident and accumulated several times within the imaging time of one frame by the image sensor, it is sent to the signal processing system, so that signal processing is performed for each incident without accumulating fluorescence. Compared to the case, cumulative noise can be reduced. As a result, the image quality can be improved.
In the present invention, the phrase “pulse shape” or “pulse train shape” includes a case where there is a fluctuation in light intensity such as sinusoidal modulation or the case where the light intensity between pulses does not become zero. It is.

In the above invention, it is preferable that the pulse light source device includes a light source and a modulation device that modulates the light emitted from the light source into a plurality of pulse trains.
With this configuration, it is possible to change the continuous light emitted from the light source into a pulse train light by the operation of the modulation device, or arbitrarily adjust the frequency of the pulse train light emitted from the light source. Therefore, it is possible to generate stable pulse train-like excitation light regardless of the performance of the light source.

In the above invention, the modulation device has a plurality of slits or pinholes and is arranged so as to block the optical axis from the light source toward the objective lens, and the disk is rotated about an axis substantially parallel to the optical axis. It is good also as providing the rotation means to rotate it.
With this configuration, when the disk is rotated by operating the rotating means, the slit or pinhole provided in the disk moves in a direction intersecting the optical axis. As a result, the position of the light spot on the sample also changes from moment to moment. A plurality of light spots formed by passing through a plurality of slits or pinholes provided on the disc move by the rotation of the disc and pass sequentially. Light that fluctuates in a pulse shape is irradiated multiple times. As a result, a brighter fluorescent image can be obtained than when scanning with continuous light, and damage to the sample can be suppressed. In this case, the light source may be a continuous light source or a pulsed light source.

Moreover, in the said invention, it is preferable that the said excitation light source consists of a continuous light source, and the said modulation | alteration apparatus modulates the continuous light from the said continuous light source, and forms a pulse-train-shaped light.
The strobe light source generates pulsed light by instantaneously releasing stored energy, and thus has a problem that it is inferior in high speed and stability. In the present invention, by adopting a continuous light source, the peak power is sufficiently smaller than the pulsed light generated by the strobe light source, and the stability of the light source itself can be improved. By applying high-speed modulation to be formed, a pulse light source device having high stability and high speed can be obtained. Thereby, it is possible to irradiate the pulse train-like excitation light uniformly over the entire observation range and obtain a highly accurate fluorescent image.

Further, in the above invention, the excitation light source includes a plurality of strobe light sources that generate pulse train-like excitation light having different phases in the same period, and the modulation device includes the pulse train-like excitation light from the plurality of strobe light sources. It is good also as comprising the switching device which synthesize | combines.
As described above, the strobe light source has a problem that it cannot be increased in speed to obtain stability, but has an advantage that it is less expensive than a pulse laser light source or the like. In the present invention, pulse train light from a plurality of strobe light sources can be combined by a switching device, and high-frequency pulse train light that is several times the number of strobe light sources can be stably obtained.

In the above invention, it is preferable that the pulse light source device is provided with a laser speckle removing device that emits laser light in a pulse train and removes speckle noise from the laser light emitted from the pulse light source device. .
Although laser light has a relatively large peak power and can be supplied as high-speed and stable excitation light, illumination unevenness due to speckle noise may occur due to the coherence of the laser. According to the present invention, speckle noise can be removed by the operation of the laser speckle removing device. Therefore, uniform illumination can be obtained and a clear fluorescent image can be obtained.

  According to the present invention, fluorescence is generated by each pulsed excitation light that is intermittently applied to each position of the sample, so that the damage to the sample is kept low compared to the case where continuous excitation light is applied. However, bright fluorescence can be obtained. Therefore, by obtaining bright fluorescence while suppressing damage to the sample over the entire observation range, there is an effect that a bright fluorescence image can be acquired.

Hereinafter, a fluorescence observation apparatus 1 according to an embodiment of the present invention will be described below with reference to FIGS. 1 and 2.
As shown in FIG. 1, the fluorescence observation apparatus 1 according to this embodiment includes a pulse light source device 2 that generates a pulse train-like excitation light E, and a pulse train-like excitation light E emitted from the pulse light source device 2. An objective lens 3 that irradiates the sample A, a dichroic mirror 4 disposed between the objective lens 3 and the pulse light source device 2, and an imaging device 5 that captures the fluorescence F separated by the dichroic mirror 4. I have.

  In the figure, reference numeral 6 denotes a barrier filter that removes the excitation light E component contained in the fluorescence F branched by the dichroic mirror 4, and reference numeral 7 connects the fluorescence F that has passed through the barrier filter 6 to the imaging surface of the image sensor 5. A condensing lens for imaging, reference numeral 8 is a signal processing device that processes electrical signals generated in the image sensor 5 to form image information, and reference numeral 9 is a monitor that displays image information formed by the signal processing device 8. .

  As shown in FIG. 1, the pulse light source device 2 includes a continuous light source 10 such as a xenon lamp, a collimator lens 11 that converts light from the continuous light source 10 into substantially parallel light, and the collimator lens 11. The modulation device 12 that converts the parallel light converted by the above into a pulse train light, the relay lens 13 that guides the pulse train light to the objective lens 3, and the light relayed by the relay lens 13 is made incident to the excitation light E. And an excitation filter 14 that transmits only the light.

  For example, as shown in FIG. 2, the modulation device 12 includes a rotary disk 16 having a plurality of slits 15 and a motor 17 that rotationally drives the rotary disk 16. The rotating disk 16 is arranged substantially orthogonal to the optical axis so as to block the parallel light that has passed through the collimating lens 11, and the light of the slit 15 portion disposed within the incident range of the parallel light incident on the rotating disk 16. Is allowed to pass only. The rotating disk 16 is arranged at a position conjugate with the focal position of the objective lens 3.

  It is preferable that the slit 15 provided in the rotating disk 16 is sufficiently smaller than the interval of the slits in the confocal disk. Further, the pattern of the slits 15 is not limited to the form shown in FIG. 2, and the slits 15 may be formed radially.

The wavelength characteristics of the dichroic mirror 4 are set so as to reflect the excitation light E and transmit the fluorescence F. The dichroic mirror 4 is disposed with an inclination of 45 ° with respect to the incident optical axis of the excitation light E. Thereby, the dichroic mirror 4 reflects the excitation light E, thereby deflecting it by 90 ° and directing it toward the objective lens 3.
The image sensor 5 is, for example, a cooled CCD (Cooled Charge Coupled Device) or a CMOS (Complementary Metal).
Oxide Semiconductor) imaging device.

The operation of the fluorescence observation apparatus 1 according to this embodiment configured as described above will be described below.
In order to perform fluorescence observation of the sample A using the fluorescence observation apparatus 1 according to the present embodiment, the continuous light source 10 constituting the pulse light source apparatus 2 is activated and the modulation apparatus 12 is activated. When the modulation device 12 is operated, the motor 17 rotates the rotary disk 16.

  The continuous light emitted from the continuous light source 10 is irradiated onto the rotating disk 16, so that only the light of the slit 15 portion provided in the rotating disk 16 is allowed to pass through the rotating disk 16 and is relayed by the relay lens 13. By being relayed and passing through the excitation filter 14, the excitation light E is incident on the dichroic mirror 4. The excitation light E incident on the dichroic mirror 4 is reflected and deflected by 90 ° and imaged on the sample A by the objective lens 3.

  At each moment, an image of a plurality of slits 15 provided on the rotating disk 16 is formed on the sample A. In the present embodiment, when the rotary disk 16 is rotated, the slits 15 through which light from the continuous light source 10 passes are sequentially moved. As a result, the image of the slit 15 on the sample A also moves sequentially.

Therefore, when the rotary disk 16 is rotated by a predetermined angle, the excitation light E in the form of slits 15 is irradiated over the entire observation range on the sample A.
In this case, when looking at each position on the sample A, the excitation light E is irradiated when the image of the slit 15 is formed, and the excitation light E is irradiated when it is arranged between the images of the slit 15. Stopped. As a result, when viewed over a predetermined time, at all positions on the sample A, the time during which the excitation light E is irradiated and the time during which the excitation light E is stopped alternately occur a plurality of times. That is, at each position on the sample A, the intensity of the excitation light E varies in a pulse train shape.

  Further, in the fluorescence observation apparatus 1 according to the present embodiment, since the modulation device 2 is configured by the rotating disk 16 and the motor 17, the pulse train shape can be easily selected by appropriately selecting the rotation speed of the rotating disk 16. The frequency of the excitation light E can be adjusted as appropriate.

When the sample A is irradiated with the excitation light E, the fluorescent molecules contained in the sample A are excited and the fluorescence F is emitted.
Here, the excitation of the fluorescent molecules and the fluorescence emission mechanism will be described with reference to FIG.
In general, a large number of fluorescent molecules exist in a stable ground level level (E ₀ ). When a molecule of this state absorbs light (E ₁ −E ₀ = hν ₁ ) corresponding to the difference from the upper energy state (E ₁ ), it is excited with a certain probability (F ₀₁ ). Occurs and transitions to the upper energy state.
Here, h is Planck's constant, and ν ₁ is a frequency corresponding to an energy difference of E ₁ −E ₀ .

As a light excitation method, laser light or lamp light is mainly used, and light having energy suitable for hν ₁ is selected.
The upper-level energy state has a relatively short lifetime and is unstable, and shifts to another stable energy state. For example, transition to the lower level energy state (E ₂ ) with a certain probability γ ₁ , light (hν ₂ ) is emitted from the level, and transition to the ground state (E ₀ ). The light generated at this time is fluorescence.

In addition, it is conceivable that the composition changes due to transition to another energy level, ionization, a different molecular state, or molecular dissociation without emitting hν ₂ light. Such a phenomenon occurs at a certain probability γ ₂ and does not emit light. Therefore, when observation is performed for a long time, the amount of fluorescence may decrease with time. This is because the number of molecules that return to the ground state decreases, resulting in a decrease in the number of molecules that fluoresce and is generally referred to as fading.
The model used in this description is a three-level system model, but the basic mechanism does not change even if it is two or four levels.

Note that the amount of fluorescence depends on the energy per unit time of excitation light given to the fluorescent molecule and its change over time, the excitation probability of the fluorescent molecule, the lifetime of the upper level energy, and the like. For example, even if high energy excitation light is continuously applied, strong fluorescence is not always obtained, molecules at the upper level are saturated, and many fading phenomena occur, It is not an efficient fluorescence generation process.
For long-term observation and avoidance of cell damage, it is very important to efficiently excite molecules with as little excitation energy as possible and to reduce the fading phenomenon as much as possible to obtain efficient fluorescence.

  The fluorescence F generated in the sample A is collected by the objective lens 3, is branched from the excitation light E by passing through the dichroic mirror 4, passes through the barrier filter 6, and forms an image on the image sensor 5 by the condenser lens 7. Be made. The imaging device 5 continues to image the fluorescence F from the sample A for a predetermined time until obtaining sufficient information to form a single-frame fluorescence image, and then according to the intensity of the fluorescence F acquired in each pixel. An electrical signal for each pixel is sent to the signal processing device 8. In the signal processing device 8, a fluorescent image is formed based on the electrical signal for each pixel sent from the image sensor 5, and the formed fluorescent image is displayed on the monitor 9.

In the present embodiment, a plurality of pulse trains of excitation light E are applied to each position on the sample A during a predetermined time until the image pickup device 5 obtains sufficient information to form a single-frame fluorescence image. Is done.
Compared with a conventional pulsed laser fluorescence microscope that obtains a sufficient amount of fluorescence with a single pulse, the peak power of each pulsed excitation light E for obtaining the same amount of fluorescence can be sufficiently reduced. Further, compared with the case where continuous excitation light E is continuously irradiated to each position, even if the peak power of each pulse train-like excitation light E is increased, by providing a pause period arranged between the pulses, Average power can be reduced.

  As a result, the peak power can be reduced as compared with the case of a single pulse to prevent instantaneous damage to the sample A, and the average power can be reduced as compared with the case where the continuous excitation light E is irradiated. Therefore, it is possible to prevent damage to the sample A due to excessive total energy applied to the sample A. Further, according to the inventor's knowledge, bright fluorescence F can be obtained even with a sufficiently low average power as compared with the case where the excitation light E is continuously irradiated. Therefore, there is an advantage that a bright fluorescent image can be acquired while reducing the power of the excitation light E.

  That is, by using the pulse light source device 2, the average power applied to the sample A can be reduced and a bright fluorescent image can be obtained. Therefore, it is possible to observe for a long period of time by reducing damage to the sample A. Further, unlike a multiphoton excitation type fluorescence observation apparatus using an ultrashort pulse laser beam, it is not necessary to consider the dispersion of the excitation light E, so that the configuration of the apparatus can be simplified and the size can be reduced. .

  Further, the pulsed fluorescence F generated from each position on the sample A during the predetermined time until the image pickup device 5 obtains sufficient information to form a single-frame fluorescent image shows the image pickup surface of the image pickup device 5. And is accumulated in the image sensor 5. The imaging element 5 converts the accumulated fluorescence into an electrical signal and sends it to the signal processing device 8. Therefore, every time one pulsed fluorescence F is incident, it is converted into an electrical signal and integrated by the signal processing device 8. Compared to the case, there is an advantage that accumulated noise can be reduced.

  In the fluorescence observation apparatus 1 according to the present embodiment, the rotating disk 16 having the plurality of slits 15 has been described as an example of the modulation apparatus 12, but instead, the rotating disk 16 having a plurality of pinholes is employed. May be. Further, although the continuous light source 10 is employed as the light source, a pulse light source may be employed instead.

Next, the fluorescence observation apparatus 20 according to the second embodiment of the present invention will be described below with reference to FIGS.
In the description of the present embodiment, portions having the same configuration as those of the fluorescence observation apparatus 20 according to the first embodiment described above are denoted by the same reference numerals and description thereof is omitted.

As shown in FIG. 4, the fluorescence observation apparatus 20 according to this embodiment includes a pulse light source device 21, a dichroic mirror 4, an objective lens 3, a barrier filter 6, a condensing lens 7, an image sensor 5, a signal processing device 8, and a signal processing device 8. The basic configuration having the monitor 9 is common to the fluorescence observation apparatus 1 according to the first embodiment.
The fluorescence observation apparatus 20 according to the present embodiment is different from the fluorescence observation apparatus 1 according to the first embodiment in a pulse light source device 21.

  In the present embodiment, the pulse light source device 21 includes two strobe light sources 22A and 22B, such as a xenon flash lamp, and a collimator lens 11 that converts light from the strobe light sources 22A and 22B into substantially parallel light. And a switching device (modulation device) 23 for combining the two lights converted into light. The switching device 23 includes a rotating disk 24 and a motor 25 that rotates the rotating disk 24. Reference numeral 26 denotes a belt for transmitting the power of the motor 25 to the rotary disk 24.

As shown in FIG. 5, the rotary disk 24 is configured by alternately arranging mirror portions 24 a that reflect approximately 100% of light and transmissive portions 24 b that transmit approximately 100% of light in the circumferential direction.
As shown in FIG. 4, the two strobe light sources 22A and 22B are disposed at an angle of 90 ° to each other, and the rotating disk 24 is disposed at the intersection of the optical axes. As a result, the light emitted from the first strobe light source 22A is transmitted through the rotating disk 24 and incident on the relay lens 13 when the transparent portion 24b of the rotating disk 24 is disposed on the optical axis. It is like that. Further, the light emitted from the second strobe light source 22B is reflected by the rotating disk 24 when the mirror portion 24a of the rotating disk 24 is disposed on the optical axis, and is deflected by 90 ° to be the first strobe light. The light is incident on the same optical axis as the light from the light source 22A.

  As shown in FIGS. 6A and 6B, each of the strobe light sources 22A and 22B emits a pulse train having the same frequency. Further, the first strobe light source 22A and the second strobe light source 22B have a period shifted by 90 °. As a result, after being synthesized by the switching device 23, as shown in FIG. 6C, light in a pulse train having a frequency twice that of the strobe light sources 22A and 22B is emitted.

  According to the fluorescence observation apparatus 20 according to the present embodiment, the switching device 23 can cause the excitation light E having a pulse train having a frequency higher than that of each of the strobe light sources 22A and 22B to be incident on the sample A. Therefore, even when using the relatively low-speed strobe light sources 22A and 22B, it is possible to make the sample A enter the pulsed excitation light E having a high frequency and obtain a bright fluorescent image. Since the frequency of the pulse train light emitted from the strobe light sources 22A and 22B may remain low, the strobe light sources 22A and 22B can be used under high stability conditions, and stable fluorescence observation can be performed.

  In the fluorescence observation apparatus 20 according to the present embodiment, the two strobe light sources 22A and 22B are used. However, the present invention is not limited to this. Three or more strobe light sources are used, and two or more switching light sources are used. The apparatus 23 may be connected in cascade to irradiate the sample A with pulsed excitation light E having a higher frequency.

Next, a fluorescence observation apparatus 30 according to the third embodiment of the present invention will be described below with reference to FIG.
Also in the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the fluorescence observation apparatus 30 according to the first embodiment, and the description thereof is omitted.

The fluorescence observation apparatus 30 according to this embodiment is also different from the fluorescence observation apparatus 1 according to the first embodiment in the pulse light source device 31.
In the present embodiment, the pulse light source device 31 includes a continuous light emission laser light source 32 that generates continuous light emission laser light L, and a high-speed light modulation element (modulation device) that shapes the generated laser light L intermittently into a pulse shape. 33 may be provided. As the high-speed light modulation element 33, an acousto-optic element or an electro-optic element can be employed. By so doing, it is possible to generate a pulsed laser beam L ₁ having a more stable peak power and pulse width and pulse interval. Accordingly, each position of the specimen A is irradiated with uniform pulse laser light L _1, it is possible to obtain a high fluorescence image accuracy. Further, pulsed light having a high repetition frequency can be irradiated with an inexpensive continuous light emitting laser 32.

  Further, as shown in FIG. 8, a pulse light source device 31 composed of a combination of continuous-wave laser light sources 32A and 32B having a plurality of wavelengths and high-speed light modulation elements 33A and 33B may be used. In this case, the pulsed laser light emitted from the continuous light emission laser light sources 32A and 32B can be synthesized by the mirror 34 and the dichroic mirror 35, and the sample A can be irradiated simultaneously. Further, as shown in FIG. 9, the high-speed light modulation element 33 may be shared by a plurality of continuous emission laser light sources 32A and 32B.

  Further, as shown in FIG. 10, when two continuous light emitting laser light sources 32A and 32B are connected by a dichroic mirror 35, only the laser light L ′ emitted from one continuous light emitting laser light source 32B is ½ wavelength. By passing the light through the plate 36, the polarization plane of the laser light L ′ when entering the high-speed light modulation element 33 may be made different from the laser light L emitted from the other continuous light emission laser light source 32A. . By doing so, the single high-speed light modulation element 33 can modulate the two types of laser beams L and L ′ emitted from the two continuous emission laser light sources 32A and 32B alternately with different intensity. it can.

Further, as shown in FIG. 11, the pulsed laser light L ₁ modulated by the high-speed light modulation element 33 may be passed through the laser speckle removing means 37. As the laser speckle removing means 37, for example, a multi-length fiber in which a plurality of optical fibers each having a different length is bundled can be cited. When the laser beam L is incident on the multi-length fiber, the coherence of the laser beam L can be significantly reduced. Normally, when the surface is irradiated with the laser beam L, the laser irradiation surface becomes a bright and dark pattern due to the interference of the laser beam L, and significant illumination unevenness (speckle noise) occurs. However, speckle noise is caused by passing through a multi-length fiber. Can be almost eliminated, and uniform illumination can be obtained.

  Further, as the laser speckle removing means 37, instead of the multi-length fiber, a diffusion plate is rotated and the obtained images are integrated. The bright and dark images of grains by random speckle pattern illumination become a uniform image by integration.

  In addition, the total reflection illumination method is known as a method for illuminating only a limited area of the surface of the sample. In the present invention, any one of the pulse light source devices described above is employed as a light source for the total reflection illumination method. You may decide to do it.

  In each of the above-described embodiments, the epi-illumination type fluorescence observation apparatus that irradiates the excitation light from the objective lens 3 side has been described, but instead, it is mounted on the transparent member 40 as shown in FIG. Even if the sample A is irradiated with excitation light E from the side opposite to the objective lens 3 and the present invention is applied to a transmission fluorescence system in which the fluorescence F emitted from the sample A in the direction opposite to the excitation light E is observed. Good. In FIG. 12, reference numeral 41 denotes a pulse light source, reference numeral 42 denotes a mirror, and reference numeral 43 denotes a condenser lens that guides the excitation light E to the sample A.

1 is a schematic diagram schematically showing an overall configuration of a fluorescence observation apparatus according to a first embodiment of the present invention. It is a front view which shows an example of the rotating disk used for the modulation apparatus of the fluorescence observation apparatus of FIG. It is a figure which shows the 3 level system model of the fluorescent molecule for demonstrating excitation of a fluorescent molecule, and a fluorescence light emission mechanism. It is a schematic diagram which shows schematically the whole structure of the fluorescence observation apparatus which concerns on the 2nd Embodiment of this invention. It is a front view which shows an example of the switching apparatus of the fluorescence observation apparatus of FIG. It is a graph which shows the relationship between the continuous emission light source of the fluorescence observation apparatus of FIG. It is a schematic diagram which shows schematically the whole structure of the fluorescence observation apparatus which concerns on the 3rd Embodiment of this invention. It is a schematic diagram which shows the 1st modification of the fluorescence observation apparatus of FIG. It is a schematic diagram which shows the 2nd modification of the fluorescence observation apparatus of FIG. It is a schematic diagram which shows the 3rd modification of the fluorescence observation apparatus of FIG. It is a schematic diagram which shows the 4th modification of the fluorescence observation apparatus of FIG. It is a schematic diagram which shows the other modification of the fluorescence observation apparatus of this invention.

Explanation of symbols

A Sample E Excitation light F Fluorescence L, L1 Laser light (Excitation light)
DESCRIPTION OF SYMBOLS 1 Fluorescence observation apparatus 2,31 Pulse light source device 3 Objective lens 4 Dichroic mirror 5 Imaging element 10 Light source 12, 33 Modulator 15 Slit 16 Disc (rotary disc)
17 Motor 22A, 22B Strobe light source 23 Switching device 32, 32A, 32B Continuous light source 33A, 33B High-speed modulation element (modulation device)

Claims (6)

A pulse light source device for generating pulsed excitation light and irradiating the sample; and
An imaging device for imaging fluorescence generated in the sample,
A fluorescence observation apparatus in which the pulsed light source device irradiates each position of a sample corresponding to each pixel of the image sensor a plurality of times of pulsed excitation light within an imaging time of one frame of the image sensor.   The fluorescence observation apparatus according to claim 1, wherein the pulse light source device includes a light source and a modulation device that modulates light emitted from the light source into a plurality of pulse trains.   The modulation device has a plurality of slits or pinholes and is arranged so as to block the optical axis from the light source toward the objective lens, and a rotating means for rotating the disk around an axis substantially parallel to the optical axis The fluorescence observation apparatus according to claim 2, comprising: The light source comprises a continuous light source;
The fluorescence observation apparatus according to claim 2, wherein the modulation device modulates continuous light from the continuous light emission light source to form pulse train light. The light source comprises a plurality of strobe light sources that generate light in a pulse train having different phases at the same period,
The fluorescence observation apparatus according to claim 2, wherein the modulation device includes a switching device that synthesizes pulse train light from the plurality of strobe light sources. The pulse light source device emits a pulse train of laser light,
The fluorescence observation device according to any one of claims 1 to 4, further comprising a laser speckle removal device that removes speckle noise from the laser light emitted from the pulse light source device.

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