JP2006226848A - Fluorescence imaging device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To easily image only a necessary fluorescence from a fluorescent pigment. <P>SOLUTION: This fluorescence imaging device 1 comprises a xenon flash lamp 2 as a pulse light source, an optical fiber 3, an irradiation means 6 for irradiating a sample 5, and an imaging unit 9 for imaging fluorescence. The imaging unit 9 has an amplifying function for converting light into an electron and for amplifying the electron, and an image intensifier 31 having an electric shutter function, and passes the fluorescence in an optional time. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fluorescence imaging apparatus for observing fluorescence from a fluorescent dye of a sample.

  Conventionally, when observing fluorescence of a plurality of fluorescent dyes, each of the fluorescence from the plurality of fluorescent dyes is simultaneously imaged with a camera or the like.

  In such a case, each fluorescent dye has its own absorption wavelength (excitation wavelength) and fluorescence wavelength, and the difference in wavelength characteristics is used to separate and observe the desired fluorescence wavelength with an optical filter. ing. However, when the wavelengths of fluorescence to be observed overlap, there is a problem that wavelength separation cannot be performed with an optical filter.

  On the other hand, there is a technique for imaging the fluorescence of a desired fluorescent dye after the lifetime of the fluorescence from one of the fluorescent dyes is exhausted, paying attention to the time domain due to the difference in fluorescence lifetime of each fluorescent dye.

For example, Non-Patent Document 1 describes that a disc having a plurality of fan-shaped slits called an optical chopper is rotated to discontinuously receive light to the detector. This is because the fluorescence is blocked by an optical chopper until the fluorescence lifetime of one of the fluorescent dyes is exhausted, so that the fluorescence does not reach the detector. In this case, only the fluorescence of a fluorescent dye having a long fluorescence lifetime is imaged.
J.AM.CHEM.SOC.2004,126,4888-4896

  However, when such an optical chopper is used, the size of the slit and the time during which the slit shields light must be controlled in accordance with the end of the life of the fluorescent dye having the shorter fluorescence lifetime.

  In particular, when switching between light shielding and transmission for a short time, there is also an observation in a state where a slit passes in the light beam, so that control with good response cannot be performed, and the amount of observation light is lost. Also, since the rotation of the optical chopper has speed irregularities and errors, it is difficult to control in a short time of the order of μsec. For example, due to the difference in the light shielding time, there may be a case where fluorescent light that should have been shielded for each observation may be mixed, and reliable separation cannot be performed. On the other hand, if the light shielding time is delayed for the sake of certainty, the amount of fluorescence emitted in the second half is acquired even though the amount of fluorescence emitted immediately after excitation is the largest, so the total amount of light is acquired. It is not an efficient acquisition method because the amount is reduced. In addition, the S / N becomes worse.

  In addition, when changing the setting of light shielding or transmission time, or when it is necessary to change the fluorescent dye, it is necessary to prepare a plurality of slits according to it and adjust the light shielding timing every time it is replaced. There was a drawback.

  The present invention has been made in view of such circumstances, and an object of the present invention is to provide a fluorescence imaging apparatus capable of easily imaging only the fluorescence of a necessary fluorescent dye from a plurality of fluorescent dyes having overlapping wavelength widths. To do.

  In the fluorescent imaging device according to claim 1 of the present invention, fluorescence from a sample in which at least the first fluorescent dye and the second fluorescent dye having a fluorescent lifetime longer than that of the first fluorescent dye are present is observed. In the fluorescence observation apparatus, the pulse light source, the irradiation means for irradiating the sample with light from the pulse light source, the imaging means for imaging the fluorescence from the second fluorescent dye, and the second fluorescent dye And adjusting means for arbitrarily adjusting an imaging start time for starting imaging of fluorescence from the image pickup means by the imaging means.

  In the fluorescence imaging apparatus according to claim 2, the adjusting means may further arbitrarily adjust an imaging end time at which imaging of the fluorescence of the second fluorescent dye ends.

  In the fluorescence imaging apparatus according to a third aspect, the adjusting means is an image intensifier.

  In the present invention, only the fluorescence from the necessary fluorescent dye can be easily imaged.

Embodiments will be described below with reference to the drawings.

  Embodiment 1 of the present invention will be described below with reference to FIGS.

  The configuration of the first embodiment will be described with reference to FIG.

  FIG. 1 is a diagram illustrating the entire fluorescence imaging apparatus.

  As shown in FIG. 1, a fluorescence imaging apparatus 1 according to the first embodiment is disposed on the emission side of a xenon flash lamp 2 as a pulse light source for fluorescence excitation and transmits light. An optical fiber 3, an irradiation means 6 for irradiating the sample 5 on the cover glass 4 with the light from the optical fiber 3, a mirror 7 for reflecting the fluorescence from the pre-stained fluorescent dye existing in the sample 5, The barrier filter 8 is disposed on the exit side of the mirror 7 and allows only necessary fluorescence to pass therethrough, and the imaging unit 9 captures the fluorescence that has passed through the barrier filter 8.

  The irradiating means 6 is disposed on the emission side of the excitation filter 21 through which the light from the optical fiber 3 passes and excites the light of the xenon flash lamp 2, and the excitation light excited by the excitation filter 21. Is reflected to the sample 5 side, and the fluorescence from the sample 5 is transmitted to the imaging unit 9 side. The dichroic mirror 22 is disposed on the emission side of the dichroic mirror 22 and the objective lens 23 collects the light on the sample 5. ing.

  The imaging unit 9 receives light that has passed through the barrier filter 8 and converts the light into electrons, and also amplifies the electrons, an image intensifier 31 having an electrical shutter function, and the image intensifier. The relay lens 32 condenses the light that has passed through 31, and a camera 34 having a solid-state image sensor 33 such as a CCD disposed at the condensing position of the relay lens 32.

  Further, as described above, the image intensifier 31 has an amplification function and a shutter function, can easily change the amplification amount, and is used as optical shutter control for transmitting and blocking observation light. Be able to.

  Further, the fluorescence imaging apparatus 1 controls the filter controller 42 for inserting and removing the excitation filter 21 and the barrier filter 8 from the optical axis 41 as necessary, and the amplification function and shutter function of the image intensifier 31 II. The controller 43, the timing controller 44 for controlling the lighting timing of the xenon flash lamp 2 and the II controller 43, and the filter controller 42, the II controller 43 and the computer 45 for controlling the timing controller 44 are connected. .

  The operation of the first embodiment will be described with reference to FIGS.

  2 is a diagram showing the relationship between fluorescence lifetime and fluorescence to be imaged, FIG. 3 is a diagram showing fluorescence characteristics (during normal fluorescence imaging and time-resolved fluorescence imaging), and FIG. 4 is a microscopic observation image (DIC of the same part of the sample) It is a figure which shows a microscope observation image, a normal fluorescence observation image, and a time-resolved fluorescence observation image.

  A signal Expose signal of the imaging period of the imaging unit 9 is sent, and this signal becomes a signal output start signal of the timing controller 44. The timing controller 44 sends a signal Trigger signal 1 for causing the xenon flash lamp 2 to emit light and a signal Trigger signal 2 to the II controller 43 adjusted in time with the signal Trigger signal 1.

  The light from the xenon flash lamp 2 emitted at the timing of the signal Trigger signal 1 passes through the optical fiber 3, passes through the wavelength-selected light as excitation light by the excitation filter 21, and is reflected by the dichroic mirror 22 to be objective. The sample 5 is irradiated after passing through the lens 23.

  On the other hand, the sample 5 has two types of fluorescent dyes having different fluorescence lifetimes as shown in FIG. One of these two types of fluorescent dyes has a lifetime of fluorescence at T1, as indicated by A in FIG. On the other hand, as shown by B, the fluorescence lifetime is exhausted at T2.

  When the excitation light is irradiated, the fluorescence from the two types of fluorescent dyes A and B passes through the objective lens 23, reflects off the mirror 7, passes through the barrier filter 8, and then enters the image intensifier 31. To do. The II controller 43 receives the signal Trigger signal 2, converts it into a signal Gate Signal for driving the image intensifier 31, and operates it. The signal Trigger signal2 is a start signal for starting imaging at a time T1 when the fluorescence lifetime of A is exhausted as shown in FIG. 2, and an end signal for ending the imaging at a time T2 when the fluorescence lifetime of B is exhausted. The fluorescence imaging period for pulse irradiation is a period from T1 to T2.

  The image intensifier 31 is controlled by the computer 45 and the II controller 43 so that the shutter function is closed until T1 and the shutter function is opened for the period from T1 to T2. That is, only the B fluorescence in the period from T1 to T2 passes through the image intensifier 31.

  The B fluorescence from T1 to T2 that has passed through the image intensifier 31 passes through the relay lens 32, and only the B fluorescence in the period from T1 to T2 is imaged by the solid-state imaging device 33.

  Here, the fluorescence image will be described.

  As an example, a description will be given using a sample in which HeLa cells are injected with a compound having a relatively long fluorescence lifetime as Rhodamin 6G and B as A in FIG.

  FIG. 3 (1) shows the fluorescence characteristics when A and B are imaged for a period of 800 μsec (corresponding to T2 in FIG. 2) after irradiating the excitation light pulse with the fluorescence microscope.

  On the other hand, the fluorescence characteristic when imaged for a period of about 50 μsec (corresponding to T1 in FIG. 2) to 800 μsec after irradiation with excitation light is as shown in FIG. 3 (2).

Rhodamin 6G (A) is excited by ultraviolet excitation light of 300 to 400 nm, has a fluorescence wavelength of about 500 to 650 nm, and a fluorescence lifetime of several nsec (T1). This is indicated by (ii) in FIG. Compound (B) is also excited by ultraviolet excitation light of 300 to 400 nm, the fluorescence wavelength is about 550 to 650 nm, and the fluorescence lifetime is several msec (T2). In FIG. 3 (1) and (2), it is indicated by (iii).

  Thus, in FIG. 3 (1), since the fluorescence wavelengths of A and B overlap, it cannot be distinguished whether the fluorescence is due to A or B, but in FIG. The wavelength does not appear in FIG. 3 (2), and only the fluorescence wavelength B in (iii) appears.

  That is, although the fluorescence wavelengths of A and B overlap, there is a difference in fluorescence lifetime between several nsec (T1) and several msec (T2). Therefore, the fluorescence characteristics of FIG. Can be obtained.

  Here, the scale of the vertical axis in FIGS. 3 (1) and (2) is different.

  Next, fluorescent images actually captured are shown.

  FIG. 4 (1) is an image obtained by imaging the sample by the DIC microscope observation method. In this image, first, it is possible to capture the entire shape of the cell. In addition, the outline of a plurality of cells can be clearly captured.

  FIG. 4 (2) corresponds to FIG. 3 (1). The same part as (1) is excited with 360 ± 40 nm ultraviolet light and then has a wavelength region of 617 ± 37 nm from 800 μsec. Is captured by the camera 34. In this image, since the fluorescence of A and B is imaged simultaneously, the fluorescence of the entire cell is imaged, and A and B cannot be distinguished.

  FIG. 4 (3) corresponds to FIG. 3 (2), and is an image captured by the camera 34 until 800 μsec after the same portion is irradiated with the same excitation light and about 50 μsec elapses. In this image, only the fluorescence of the portion where only B is injected is imaged, and the portion where A is injected is not imaged. In other words, by not irradiating excitation light and imaging for a period of up to about 50 μsec after light emission, and by imaging the period up to 800 μsec (time-resolved fluorescence imaging), unnecessary fluorescence and stray light are not imaged. Thus, it is possible to obtain an image of only the target B fluorescence, and it is possible to perform image observation and subsequent reaction analysis without being disturbed by the fluorescence of Rhodamin 6G (A).

  In addition to artificially stained fluorescence such as Rhodamin 6G, in general, substances that cells naturally shine with ultraviolet rays, stray light emitted from optical elements, etc. are present in the time domain of several nsec. These can also be removed.

  Next, the effect of the first embodiment will be described.

  In Embodiment 1 of the present invention, by controlling the image intensifier 31, only the B fluorescence can be imaged by the solid-state imaging device 34 without imaging the A fluorescence. Is possible.

  In the first embodiment, since the image intensifier 31 is used, the shutter function can be easily changed from the closed state to the opened state by the control of the computer 45 as compared with the conventional optical chopper. Furthermore, the time of the closed state and the time of the open state can be arbitrarily changed, and if necessary, the imaging start time and the imaging end time for imaging with the solid-state imaging device can be arbitrarily changed and are easy to use. .

  Furthermore, the rate at which electrons are easily amplified by the image intensifier 31 can be changed as necessary.

  Hereinafter, Example 2 of the present invention will be described with reference to FIG.

  FIG. 5 is a diagram showing the relationship between the fluorescence lifetime and the fluorescence to be imaged.

  The configuration of the present invention is the same as that of the first embodiment, and images fluorescence during the periods X, Y, and Z in FIG.

  In the second embodiment, the computer 45 controls the B fluorescence in the periods X, Y, and Z in FIG. 5 so as to pass through the image intensifier 31.

  The fluorescence of each period of X, Y, and Z in FIG. 5 captured by the solid-state imaging device 33 of the camera 34 is controlled by the computer 45 so as to be discriminated and stored for each period.

  In the second embodiment, the periods passing through the image intensifier 31 are set as X, Y, and Z, and the respective periods are stored. Therefore, it is possible to capture more detailed changes in B fluorescence over time, and the fluorescence time lapses. Etc. can also be tracked. These periods can be arbitrarily changed a plurality of times, such as a time width and an interval.

  In the above embodiment, a xenon flash lamp is used. However, a lamp light source capable of pulse oscillation or a pulse laser light source may be used. Further, for example, even if the light source body is continuous light, it may be introduced as pulsed light via an optical element (AOTF, AO, EO, etc.) that can control transmission / cutoff of light in the illumination optical path.

  In the embodiment, the excitation light is introduced into the microscope through the optical fiber, but it may be introduced directly without the optical fiber, or a relay system using a normal lens may be used.

  Further, if a plurality of types of excitation filters and barrier filters of the embodiment are prepared and controlled so as to be switched by a known switching unit as necessary, the types of fluorescent dyes that can be used in the fluorescence imaging apparatus can be further increased.

It is a figure explaining the whole fluorescence imaging device. It is a figure which shows the relationship between the fluorescence lifetime and the fluorescence imaged. It is a figure which shows the fluorescence characteristic of a fluorescent pigment | dye. It is a figure which shows a microscope observation image. It is a figure which shows the relationship between the fluorescence lifetime and the fluorescence imaged.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescence imaging device 2 Xenon pulse lamp 3 Optical fiber 31 Image intensifier 33 Solid-state image sensor 43 II controller

Claims (3)

In a fluorescence observation apparatus for observing fluorescence from a sample in which at least a first fluorescent dye and a second fluorescent dye having a fluorescence lifetime longer than that of the first fluorescent dye are present, a pulse light source, and the pulse An irradiating means for irradiating the sample with light from a light source, an imaging means for imaging the fluorescence from the second fluorescent dye, and an imaging start time for starting imaging of the fluorescence from the second fluorescent dye by the imaging means And a fluorescent imaging apparatus characterized by comprising: adjusting means for arbitrarily adjusting the angle. 2. The fluorescence imaging apparatus according to claim 1, wherein the adjusting unit is further capable of arbitrarily adjusting an imaging end time at which imaging of fluorescence of the second fluorescent dye is ended. The fluorescence imaging apparatus according to claim 1, wherein the adjustment unit is an image intensifier.