WO2012086195A1

WO2012086195A1 - Time-resolved fluorescence measurement device and method

Info

Publication number: WO2012086195A1
Application number: PCT/JP2011/007143
Authority: WO
Inventors: 真也荻窪; 貴志足立
Original assignee: 富士フイルム株式会社
Priority date: 2010-12-21
Filing date: 2011-12-20
Publication date: 2012-06-28
Also published as: JP2012132742A

Abstract

[Problem] To improve the efficiency of detection of fluorescence emitted from an object to be measured in a time-resolved fluorescence measurement device. [Solution] An object to be measured (30) is irradiated with excitation light. A fluorescence detection means (24) detects fluorescence emitted from the object to be measured (30). The fluorescence detection means (24) has a detector group in which a plurality of detectors are arranged in a line in a predetermined direction. An optical element (23) is disposed in a light path to the position of incidence of the fluorescence on the fluorescence detection means (24). The optical element (23) changes the direction of travel of the fluorescence such that the fluorescence is detected by different detectors among the plurality of detectors of the detector group as time passes after the object to be measured (30) is irradiated with the excitation light. A PC (26) calculates the life of the fluorescence on the basis of the result of the detection of the fluorescence by the detector group.

Description

Time-resolved fluorescence measuring apparatus and method

The present invention relates to a time-resolved fluorescence measurement apparatus and method, and more particularly, to a time-resolved fluorescence measurement apparatus and method for measuring fluorescence using a time domain measurement method, particularly a time gate method.

It is known that fluorescence is emitted when a substance emitting fluorescence is irradiated with visible light or ultraviolet light. When a substance that emits fluorescence is irradiated with light having a short pulse width, fluorescence is observed from the picosecond to nanosecond order after the irradiation. In general, the emission intensity of this fluorescence becomes maximum immediately after light irradiation, and thereafter decays exponentially. The decay constant in this exponential function is called the fluorescence lifetime. A technique for observing the fluorescence lifetime under a microscope and performing two-dimensional mapping is called FLIM (FluorescenceFluorLifetime Imaging Microscopy).

Here, the characteristic of the fluorescence lifetime is that it can be based on an index that is excellent in time and quantitative. Fluorescence characteristics can also be quantified by fluorescence emission intensity, but fluorescence emission intensity is determined by concentration of fluorescent substance, fading, wavelength of excitation light, intensity, light collection efficiency, detector sensitivity, observation It is affected by various factors such as light attenuation in the sample and transmittance of the optical system. On the other hand, the fluorescence lifetime is different from the fluorescence emission intensity that can change depending on the measurement conditions, and is a value inherent to the substance that emits fluorescence, so that quantitative discussion is possible. Fluorescence lifetime provides important information about the structure and electronic state of the molecule, as well as information about the surrounding environment (ion concentration, pH, oxygen concentration, refractive index, viscosity, temperature, etc.).

There are two main methods for observing the fluorescence lifetime. One is a time-domain measurement method using pulsed light as excitation light, and the other is a frequency-domain measurement method using frequency-modulated light in the form of a sine wave as excitation light. domain measurement). In the time domain measurement method, a short pulse laser is incident on a sample, and the time response is observed with a high-speed detector. Typical observation methods in the time domain measurement method include time-correlated single photon counting (TCSPC), time gate method using CCD with image intensifier, and streak camera. A method of observing a spectrum and a time waveform at one time by spatial transformation is mentioned. By combining these methods with an array-type high-sensitivity detector or scanner mirror, fluorescence lifetime mapping, that is, imaging can be performed.

In the time gating method, the fluorescence decay curve is divided into several windows in the time axis direction, and after irradiating the sample with pulsed light, the detector is momentarily moved with a delay time corresponding to the window (time domain) to be measured. Just turn it on. Fluorescence is detected in a plurality of windows, the value of the integrated emission intensity in each window is plotted against time, and the fluorescence lifetime is obtained on the assumption that the integrated emission intensity decays in a single exponential function. The time gate method can shorten the measurement time compared to the time correlated single photon counting method. However, for example, assuming that the time domain is divided into four, in order to obtain the integrated emission intensity in each of the four windows, it is necessary to perform measurement at least four times at the same measurement location. For this reason, it is difficult to measure the fluorescence lifetime at the same time for samples such as cells that change every moment. Further, there are problems that the fluorescent material is discolored by irradiating pulsed light many times, and that if the measurement site is a cell, the cell is damaged.

In response to the above problem, Patent Document 1 discloses a method of measuring the integrated emission intensity in two windows of a single fluorescence decay curve with a single excitation light. The time-resolved imaging apparatus described in the cited document 1 includes a separation optical system, a delay optical system, and a synthesis optical system. The separation optical system separates and outputs the fluorescence generated in the measurement target into first light and second light. The first light is output to the combining optical system via the delay optical system, and the second light is output to the combining optical system as it is. The delay optical system sets a longer optical path length for the first light than the optical path length of the second light. The combining optical system combines the image of the measurement object formed by each of the second light output from the separation optical system and the first light that has passed through the delay optical system into a non-overlapping region on the imaging surface, The synthesized image is output to the imaging unit. Since the first light is delayed by the amount of delay given by the delay optical system, the integrated light emission intensity in the two time regions can be obtained by imaging the first light and the second light at the same time in the imaging unit. Can be measured.

JP 9-43146 A

Here, in the time gate method, the detector is turned on only for a time corresponding to the window width, and the fluorescence that is incident when the detector is turned on is detected by the detector, but the fluorescence in other time regions is detected. Is not detected by the detector. This is a factor that lowers the detection efficiency of fluorescence with respect to excitation light. Also in Patent Document 1, the separated first light and second light are imaged by the imaging unit only for the window width, and fluorescence in the time region outside the window is not captured by the imaging unit. Therefore, it is not possible to improve the fluorescence detection efficiency with respect to the excitation light.

In view of the above, an object of the present invention is to provide a time-resolved fluorescence measuring apparatus and method capable of improving the detection efficiency of fluorescence emitted from a measurement object.

In order to achieve the above object, the present invention is a light irradiation means for irradiating a measurement object with light having a predetermined wavelength as excitation light, and a fluorescence detection means for detecting fluorescence emitted from the measurement object. A plurality of detectors arranged in a line along a predetermined direction, a fluorescence detection means, and arranged in an optical path between the fluorescence entering the fluorescence detection means, the excitation light An optical element that changes the traveling direction of the fluorescence so that the fluorescence is detected by different detectors of the plurality of detectors of the detector group over time after the measurement object is irradiated. There is provided a time-resolved fluorescence measuring apparatus comprising fluorescence lifetime calculation means for calculating a fluorescence lifetime based on a fluorescence detection result in the detector group.

In the time-resolved fluorescence measuring apparatus of the present invention, the fluorescence lifetime calculating means obtains the fluorescence emission intensity in each of a plurality of time regions based on the fluorescence detection results by the plurality of detectors, and the obtained fluorescence emission A configuration for calculating the fluorescence lifetime based on the intensity can be employed.

The fluorescence lifetime calculating means calculates the fluorescence emission intensity in each time region based on the fluorescence detection result of one or more detectors corresponding to the time region of the fluorescence emission intensity to be obtained among the plurality of detectors. You may ask for it.

The optical element may continuously change the traveling direction of the fluorescence from one end side to the other end side of the plurality of detectors arranged in a row over the fluorescence measurement period.

The optical element is supported so as to be elastically displaceable, and is movable in a first direction and a second direction opposite to the first direction, and a physical acting force in the first direction on the movable part. It is possible to use an electric device element including a first driving source for applying a second driving source for applying a physical acting force in the second direction to the movable portion.

The excitation light can be a point light source. In this case, the fluorescence detector means can detect fluorescence emitted from the portion of the measurement object irradiated with the excitation light with a plurality of detectors of the detector group.

Instead of the above, a line light source may be used for the excitation light. In that case, the fluorescence detection means includes a plurality of detector groups corresponding to an area of 1 row × m columns (m is an integer of 2 or more) irradiated with the excitation light of the measurement object, and the measurement object Each of the fluorescence emitted from an area of m columns in one row irradiated with the excitation light of the object can be detected by a corresponding detector group of the plurality of detector groups.

Further, instead of the above, a surface light source may be used for the excitation light. In that case, each of the fluorescence detection means corresponds to an area of n rows × m columns (n and m are integers of 2 or more) irradiated with the excitation light of the measurement object, each of which is 1 row × m. N sets of a plurality of detector groups corresponding to the column region, and the optical element emits fluorescence emitted from the m column region of each row irradiated with the excitation light of the measurement target region. The fluorescence detection means emits each of the fluorescence emitted from the m-column region of each row irradiated with the excitation light of the measurement object. What is necessary is just to detect with the corresponding detector group among the several detector groups of a set to perform.

The fluorescence detection means may include an image intensifier having a gate function and a charge imaging device.

The time-resolved fluorescence measuring apparatus of the present invention includes a scanning unit that scans the excitation light on the surface of the measurement object, the emission intensity of the fluorescence corresponding to each position of the scanned excitation light, and the calculation. And a fluorescence image generating means for generating a fluorescence image representing at least one distribution of the fluorescence lifetime.

The scanning unit scans the condensing position of the excitation light on the measurement object in the depth direction in addition to the surface of the measurement object, and the fluorescence image generation unit 3 scans the fluorescence image. It can be generated as dimensional information.

The present invention provides a microscope system comprising the time-resolved fluorescence measuring device of the present invention.

The present invention also provides an endoscope fiber probe system comprising the time-resolved fluorescence measuring device of the present invention.

The present invention further includes a step of irradiating the measurement object with light having a predetermined wavelength as excitation light, and fluorescence emitted from the measurement object after the excitation light is irradiated on the measurement object. The step of changing the traveling direction of the fluorescence so as to be detected by different detectors out of the plurality of detectors arranged in a line along a predetermined direction as time passes, and detection of fluorescence in the plurality of detectors And a step of calculating a fluorescence lifetime based on the result.

In the time-resolved fluorescence measuring apparatus and method of the present invention, when detecting fluorescence emitted from an object to be measured, different detectors out of a plurality of detectors in which the fluorescence is arranged in a line along a predetermined direction over time Change the direction of travel of the fluorescence as detected by. In this way, for example, the fluorescence can be sequentially detected by different detectors over the measurement period, and the detection efficiency of the fluorescence with respect to the excitation light can be increased.

1 is a block diagram showing a time-resolved fluorescence measuring device according to an embodiment of the present invention. The flowchart which shows the operation | movement procedure of fluorescence lifetime measurement. The figure which shows a MEMS mirror and CCD typically. The figure which shows a MEMS mirror and CCD typically. The perspective view which shows the basic structure of DMD. Sectional drawing which shows the basic operation principle of DMD. Sectional drawing which shows the basic operation principle of DMD. The graph which shows the relationship between time when a drive signal is supplied, and a rotation angle. The perspective view which shows typically an optical element and fluorescence detection means when the excitation light of a line light source is irradiated. The perspective view which shows typically an optical element and fluorescence detection means when the excitation light of a surface light source is irradiated. The block diagram which shows a microscope system. The block diagram which shows an endoscope probe system. The perspective view which shows the cross section of an endoscope probe.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. FIG. 1 shows a time-resolved fluorescence measuring apparatus according to an embodiment of the present invention. The time-resolved fluorescence measurement apparatus 10 includes a laser light source 11, a pulse picker 12, mirrors 13 and 21, a beam splitter 14, an excitation light detection means 15, a nonlinear optical crystal 16, a dichroic mirror 17, an objective lens 18, an excitation light removal filter 19, A spectroscope 20, a condensing lens 22, an optical element 23, a fluorescence detection means 24, an optical element control means 25, and a computer (PC: Personal Computer) 26 are provided.

The time-resolved fluorescence measuring apparatus 10 obtains the fluorescence lifetime using a time gate method. That is, the fluorescence emission decay curve is divided into several divided windows (time domain), the sample is irradiated with pulsed light as excitation light, the fluorescence emission intensity is detected in multiple windows, and the fluorescence emission intensity in each window is detected. Is plotted against time, and the fluorescence lifetime is obtained assuming that the integrated emission intensity decays exponentially. Note that the light to be measured is not limited to the fluorescence emitted from the measurement object, and may be light having characteristics similar to that, for example, phosphorescence. That is, the lifetime of phosphorescence may be obtained using the time-resolved fluorescence measuring device 10.

The laser light source 11 generates excitation light having a predetermined wavelength that is irradiated onto the measurement object 30 that is a sample. As the laser light source 11, for example, a TiSa laser or the like can be used. The laser light source 11 generates pulsed laser light. The laser light source 11 generates, for example, pulsed light having a pulse time from picoseconds to femtoseconds as pulse excitation light. For example, the pulse picker 12 thins out the pulse excitation light periodically generated by the laser light source 11 at a predetermined rate. When the pulse picker 12 thins out the pulse excitation light, the measurement target 30 can be irradiated with the pulse excitation light at a desired cycle. When the cycle in which the laser light source 11 generates the pulse excitation light is a desired cycle, the pulse picker 12 may be omitted.

The beam splitter 14 reflects a part of the pulse excitation light and transmits the rest. The excitation light detector 15 detects the light reflected by the beam splitter 14. A photodiode or the like can be used for the excitation light detection means 15. The nonlinear optical crystal 16 receives the light transmitted through the beam splitter 14 and generates a second harmonic or a third harmonic of the wavelength of the incident light. The dichroic mirror 17 transmits light in the excitation light wavelength band and reflects light in the fluorescence wavelength band emitted from the measurement object 30.

The objective lens 18 condenses the excitation light transmitted through the dichroic mirror 17 at a desired location on the measurement object 30. Moreover, the objective lens 18 enters the fluorescence generated at the portion irradiated with the excitation light of the measurement object 30 in the opposite direction to the excitation light, and converts it into parallel light and emits it. The excitation light removal filter 19 and the spectroscope 20 remove light in the wavelength band of excitation light from incident light. The condensing lens 23 condenses incident light on the optical element 23.

The fluorescence detection means 24 detects fluorescence emitted from the measurement object 30. The fluorescence detection means 24 has a detector group in which a plurality of detectors are arranged in a line along a predetermined direction. The optical element 23 changes the traveling direction of the fluorescence so that the fluorescence is detected by different detectors among the plurality of detectors of the detector group as time elapses after the measurement object 30 is irradiated with the excitation light. Let The optical element control means 25 drives and controls the optical element 23. The PC 26 performs driving of the fluorescence detection unit 24 and a control instruction to the optical element control unit 25. Further, the PC 26 calculates the fluorescence lifetime based on the detection result of the fluorescence in the detector group of the fluorescence detection means 24.

A series of operations from excitation light irradiation to fluorescence lifetime calculation will be described. The pulse excitation light emitted from the laser light source 11 is reflected by the mirror 13 to change the traveling direction, is incident on the beam splitter 14, is partially reflected, and the rest is transmitted through the beam splitter 14. The light transmitted through the beam splitter 14 becomes excitation light that is irradiated onto the measurement object 30. On the other hand, the light reflected by the beam splitter 14 is used to detect that the excitation light has been irradiated. The excitation light detection means 15 detects the light reflected by the beam splitter 14 and transmits a signal indicating that the excitation light has been detected to the PC 26. When the PC 26 receives a signal indicating that the excitation light has been detected, the PC 26 starts detecting fluorescence in the fluorescence detection means 24.

The excitation light that has passed through the beam splitter 14 enters the nonlinear optical crystal 16. The nonlinear optical crystal 16 generates a second harmonic or a third harmonic of the wavelength of incident light. The excitation light emitted from the nonlinear optical crystal 16 enters the dichroic mirror 17. The dichroic mirror 17 transmits light having a wavelength (wavelength band) of excitation light to the objective lens 18 side. The excitation light that has passed through the dichroic mirror 17 is condensed at a desired location on the measurement object 30 by the objective lens 18. In addition, each part arrange | positioned in the optical path from the laser light source 11 to the objective lens 18 is corresponded to the excitation light irradiation means which irradiates the measurement object 30 with excitation light.

Fluorescence is generated at the location where the excitation light of the measurement object 30 is irradiated. The fluorescence emitted from the measurement object 30 passes through the objective lens 18 in the direction opposite to the excitation light and enters the dichroic mirror 17 as parallel light. The dichroic mirror 17 reflects the incident fluorescence, and the reflected light enters the excitation light removing filter 19 and the spectroscope 20, and only a necessary wavelength is taken out. Instead of the spectroscope 20, a low pass filter or a band pass filter may be used. The fluorescence from which the necessary wavelength component has been extracted is reflected by the mirror 21 to change its direction, and is condensed on the optical element 23 by the condenser lens 22. Instead of using the condenser lens 22, the optical element 23 may be irradiated with fluorescence as parallel light.

The optical element 23 is configured as a micromirror, for example. As the micromirror, a MENS (Micro Electro Mechanical Systems) mirror created by a semiconductor process can be suitably used. For example, the optical element 23 is supported so as to be elastically displaceable, and is movable in a predetermined direction and in a direction opposite to the predetermined direction, a first drive source that applies a physical acting force in a predetermined direction to the movable part, An electric device element including a second driving source that applies a physical acting force in a direction opposite to the predetermined direction can be used. A reflection mirror is formed on the upper surface of the movable part. An electrostatic force or an electromagnetic force can be used as the physical acting force for driving the movable part. The optical element control means 25 controls driving of the movable part (reflection mirror) in the optical element 23.

Fluorescence reflected from the optical element 23 enters the fluorescence detection means 24 that detects fluorescence. The fluorescence detection means 24 includes a detector group in which a plurality of detectors are arranged in a line along a predetermined direction. As the fluorescence detection means 24, a charge imaging device (CCD image sensor: ChargeCharCoupled Device Image Sensor) can be used. In this case, each pixel constituting the CCD corresponds to each detector. The fluorescence detection means 24 may have an image intensifier with a gate function in addition to the CCD. The fluorescence detection unit 24 may detect the fluorescence emitted from the measurement object 30 as fluorescence of one or a plurality of wavelengths. A microlens or a microlens array may be provided in front of the fluorescence detection unit 24, and fluorescence emitted from the measurement object 30 may be incident on each detector as parallel light or convergent light.

When the PC 26 receives a signal indicating that the excitation light has been detected from the excitation light detection means 15, the PC 26 rotates and displaces the reflection mirror of the optical element 23 via the optical element control means 25. The optical element 23 changes the traveling direction (reflection direction) of the fluorescence reflected by the reflection mirror surface and directed to the fluorescence detection means 24 by the rotational displacement of the reflection mirror. The optical element 23 allows the fluorescence emitted from the measurement object 30 to be detected by different detectors of the plurality of detectors constituting the detector group of the fluorescence detection means 24 with time. Change the direction of travel. In other words, the fluorescence detection means 24 detects fluorescence with the different detectors sequentially as time advances in the detector group. For example, the optical element 23 continuously changes the traveling direction of the fluorescence from one end side to the other end side of the plurality of detectors arranged in a line over the fluorescence measurement period.

Each detector of the fluorescence detection means 24 outputs an electrical signal corresponding to the detected fluorescence emission intensity. In the fluorescence detection means 24, since the fluorescence emitted from the measurement object 30 is sequentially detected by different detectors when the optical element 23 is rotationally displaced, the emission intensity of the fluorescence detected by each detector is the fluorescence. Represents the integrated value of the fluorescence emission intensity in the time width across one detector. The PC 26 also functions as a fluorescence lifetime calculation unit, and calculates the fluorescence lifetime based on the detection result of the fluorescence detected by the detector group of the fluorescence detection unit 24. More specifically, the PC 26 obtains an integrated value of the fluorescence emission intensity in each of a plurality of time regions based on the fluorescence detection results of the plurality of detectors constituting the detector group, and based on these, the fluorescence decay Determine curve and fluorescence lifetime.

FIG. 2 shows the operation procedure of fluorescence lifetime measurement. The laser light source 11 emits pulse excitation light (step S1). The pulse excitation light is thinned out by the pulse picker 12 as necessary, then reflected by the mirror 13 and incident on the beam splitter 14. The beam splitter 14 reflects a part of the pulsed excitation light and transmits the rest in the direction of the measurement object 30. The excitation light that has passed through the beam splitter 14 passes through the nonlinear optical crystal 16 and the dichroic mirror 17, and is collected at a desired location on the measurement object 30 by the objective lens 18. At this time, the excitation light applied to the measurement object 30 is assumed to be a point light source.

Fluorescence is generated at the location irradiated with the excitation light of the measurement object 30 (step S2). The fluorescence passes through the objective lens 18 in the reverse direction, is reflected by the dichroic mirror 17, and the excitation light component is removed through the excitation light removal filter 19 and the spectroscope 20. Fluorescence emitted from the measurement object 30 is reflected by the mirror 21 after the undesired components are removed, and the direction of the fluorescence is changed. Then, the light is condensed on the reflection mirror surface of the optical element 23 by the condenser lens 22.

On the other hand, a part of the excitation pulse light reflected by the beam splitter 14 enters the excitation light detection means 15, and the excitation light detection means 15 detects that the excitation light has been irradiated (step S3). The excitation light detection unit 15 notifies the PC 26 that the excitation light irradiation has been detected, and the PC 26 instructs the optical element control unit 25 to rotate the reflection mirror in the optical element 23. The optical element control means 25 gives a signal to the movable part of the optical element 23 and rotationally displaces the movable part. The optical element 23 changes the reflection direction of the fluorescence emitted from the measurement object 30 with the lapse of time as the movable part is rotationally displaced (step S4). As the direction of fluorescence changes with time, the fluorescence emitted from the measurement object 30 moves on a plurality of detectors arranged in a one-dimensional array with respect to the time axis. The instrument detects fluorescence in different time domains.

The PC 26 calculates the fluorescence detection result in the fluorescence detection means 24 and calculates the fluorescence decay curve and the fluorescence lifetime value (step S5). In step S5, the PC 26 obtains an integrated value of the fluorescence emission intensity in each of the plurality of time regions based on the fluorescence detection results of the plurality of detectors constituting the detector group. At that time, for example, the PC 26 obtains the fluorescence emission intensity in each time region based on the detection result of the fluorescence in one or more detectors corresponding to the time region of the fluorescence emission intensity to be obtained among a plurality of detectors. be able to. The PC 26 plots the integrated emission intensity in each window with respect to the time axis, and obtains the fluorescence lifetime assuming that the integrated emission intensity decays exponentially.

For example, when the number of detectors constituting the detector group is 100 and the number of windows (time domain) used for obtaining the fluorescence attenuation characteristics is five, the PC 26 adjoins a total of 100 detectors. These 20 detectors may be grouped, and the sum of the fluorescence emission intensities detected by the 20 detectors in each group may be used as the integrated value of the fluorescence emission intensities in each window. As the number of detectors constituting the detector group increases, the number of window divisions for obtaining the fluorescence attenuation characteristic can be increased. Multi-component analysis is also possible by increasing the number of window divisions.

Subsequently, the MENS mirror used as the optical element 23 and its periphery will be described. 3A and 3B schematically show a MEMS mirror 51 that is an optical element and a CCD 52 that is fluorescence detection means. The electrostatically driven MEMS mirror 51 is rotationally displaced in accordance with an electrostatic force acting between a movable electrode provided on the back of the mirror and a fixed electrode located below the movable electrode. FIG. 3A shows a state in which no electrostatic force is supplied. At this time, it is assumed that the movable mirror is in a flat state. When the movable mirror is driven in synchronization with the excitation light, the movable mirror is tilted in a predetermined direction with respect to the initial state as shown in FIG. 3B.

In the CCD 52, pixels (detectors) are arranged in a line along an arc centered on the MEMS mirror. By rotating and moving the movable mirror over the fluorescence measurement period, the fluorescence emitted from the measurement object 30 is detected by separate pixels of the CCD 52 in which the pixels are one-dimensionally arranged corresponding to the time axis. It will be. Instead of tilting the movable mirror from a flat state in a predetermined direction, the movable mirror may be tilted in a direction opposite to the predetermined direction in the initial state, and tilted from that state in the predetermined direction.

The rotation angle of the movable mirror in the MEMS mirror 51 is 10 °, and the time until the movable mirror comes into contact with the lower part from the flat state is 2 μsec. In other words, the movable mirror changes the rotation angle by 10 ° during 2 μsec. Assuming that the time axis for measuring fluorescence is 200 nsec, the angle at which the movable mirror is rotationally displaced during 200 nsec is 10 ° × (200 nsec / 2 μsec) = 1 °. If the number of pixels of the one-dimensional array CCD 52 is 2000 pixels and the size of one pixel is 10 μm, the length of the fluorescence detection surface of the CCD 52 is 2000 × 10 μm = 20 mm. A suitable distance r between the MEMS mirror 51 and the CCD 52 is found to be approximately 1.1 m from the arc relationship (2πr × (1/360) = 20 mm). In other words, by arranging the CCD 52 at a position approximately 1.1 m away from the MEMS mirror 51, the fluorescence whose traveling direction changes at a rotation angle of 1 ° during the measurement period of 200 nsec can be detected by the 20 mm CCD.

When 10 pixels of the CCD 52 correspond to 1 window of the time gate method, the number of windows is 2000 pixels / 10 pixels = 200 windows. The one window time width is 200 nsec / 200 windows = 1 nsec because the total measurement time width is 200 nsec. That is, the time resolution of the time gate method is 1 nsec. When 100 pixels of the CCD correspond to one window of the time gate method, the time resolution is 10 nsec and the number of windows is 20. Depending on how many pixels of the CCD 52 correspond to one window of the time gate method, the number of windows and the time resolution can be arbitrarily set. In the case where the pixels are not arranged along a circular arc centered on the movable mirror in the CCD, for example, when the CCD is a flat surface, the detection pixel may be corrected according to the distance difference from the surface of the MES mirror.

Next, the configuration of the MEMS mirror will be described. The MEMS mirror is used as a general term for a micro-sized mirror manufactured using a semiconductor process or the like. In the present embodiment, a DMD element (Digital Micro-mirror Device) that can be suitably used as a MEMS mirror will be described. DMD is a mirror array device for two-dimensional light modulation, which was invented in 1987 by TI Corporation (Texas Instrument) and mass-produced in 1996. The basic structure and operating principle of the DMD will be described below.

FIG. 4 shows the basic structure of DMD. The DMD 60 has a structure in which mirrors 62 each having a side of 10 μm to 13 μm are two-dimensionally arranged on a CMOS (Complementary Metal Metal Oxide Semiconductor) driving circuit formed on the Si substrate 61. One of the two-dimensionally arranged mirrors is used as a micromirror that changes the traveling direction of fluorescence emitted from one point of the measurement object 30 (FIG. 1). The mirror 62 is a rotatable structure formed by thin film surface MEMS technology, and can be tilted and displaced by ± 10 deg to 12 deg by electrostatic driving.

On the Si substrate 61, a 1-bit SRAM (Static Random Access Memory) circuit is formed for each pixel by a CMOS process. In this SRAM, data for determining the direction of displacement of the mirror 62 is written. A first electrode and a second electrode are formed on the top of the SRAM circuit via an insulating layer, and each is connected to the output of the SRAM circuit. Address voltages Va1 and Va2 corresponding to the data written in the SRAM circuit and its complementary data are supplied to the first electrode and the second electrode, respectively.

Moreover, a landing pad is formed on the Si substrate 61. A beam called a hinge 63 having both ends supported through a gap is formed on the upper part thereof. A rigid film called a yoke 64 is formed on the hinge 63, and a rigid mirror 62 is formed on the yoke 64 via a support column. The mirror 62 can be elastically twisted integrally with the yoke 64 by a hinge 63. The first electrode, the second electrode, the yoke 64, and the mirror 62 are arranged symmetrically with respect to the hinge axis. Therefore, the yoke 64 and the mirror 62 can be rotated (tilted) left and right by the torsional elasticity of the hinge shaft. Become.

When the mirror 62 and the yoke 64 are tilted, the end of the yoke 64 comes into contact with the landing pad, and the mirror 62 and the yoke 64 are not tilted any further. The landing pad functions as a stopper. The hinge 63, the yoke 64, the mirror 62, and the landing pad are made of Al or an Al alloy and are electrically at the same potential. A bias voltage Vb is supplied to the hinge 63, the yoke 64, the mirror 62, and the landing pad.

5A and 5B show the basic operating principle of DMD. A bias voltage Vb is supplied to the yoke 64 / mirror 62 constituting the movable portion 67. On the other hand, when a drive signal, that is, data corresponding to the rotation direction of the movable portion 67 (mirror 62) is written to the SRAM circuit, the address voltages Va1 and Va2 are supplied to the first electrode 65 and the second electrode 66. . The address voltages Va1 and Va2 are digital voltages and are complementary to each other. Here, if ΔV1 = | Vb−Va1 | and ΔV2 = | Vb−Va2 |, the pixel electrode (the first electrode 65 and the second electrode 66) and the movable portion 67 facing each other are set according to the values of ΔV1 and ΔV2. Electrostatic force (electrostatic torque for rotation) is generated during When ΔV1> ΔV2, the movable portion 67 is inclined toward the first electrode 65 as shown in FIG. 5A. Conversely, when ΔV1 <ΔV2, the movable portion 67 is inclined toward the second electrode 66 as shown in FIG. 5B.

In an actual DMD, the movable part 67 is tilted to one side when the power is on, and the movable part 67 is tilted to the opposite side of the initial state or the same side as the initial state according to the relationship between ΔV1 and ΔV2. Lean on. The operation tilted to the opposite side is called CROSS operation, and the operation tilted to the same side is called STAY operation. FIG. 6 shows the relationship between the time when the drive signal is supplied and the rotation angle. In FIG. 6, the relationship between the time and the rotation angle in the CROSS operation is indicated by a solid line, and the relationship between the time and the rotation angle in the STAY operation is indicated by a dotted line. When the electrostatic torque due to ΔV1 or ΔV2 is sufficiently larger than the hinge elastic torque (more than the pull-in torque), the movable portion 67 stops in contact with the landing pad, and the rotation angle is determined.

In the present embodiment, the optical element 23 is disposed in the optical path until the fluorescence emitted from the measurement object 30 enters the fluorescence detection unit 24, and the fluorescence detection unit 24 is used with time using the optical element 23. The direction of travel of fluorescence toward is changed. The fluorescence detection means 24 sequentially detects the fluorescence emitted from the measurement object 30 with different detectors among a plurality of detectors constituting the detector group over time. In this embodiment, in a detector group in which a plurality of detectors are arranged one-dimensionally with respect to the time axis, fluorescence is sequentially detected by different detectors over a measurement period. For this reason, in the fluorescence detection means 24, the fluorescence emitted from the measuring object 30 can be detected without waste, and the detection efficiency of the fluorescence with respect to the excitation light can be increased.

In the normal time gate method, for example, in order to obtain the integrated fluorescence emission intensity in three windows, it was necessary to irradiate the excitation light pulse at least three times. Although the technique described in Patent Document 1 can reduce the number of times of excitation light pulse irradiation by half, it still requires irradiation of at least two excitation light pulses. In the present embodiment, when the detector group has three or more detectors, the integrated fluorescence emission intensity can be obtained in three or more windows with at least one excitation light pulse irradiation, and a plurality of excitation light pulses can be obtained. The measurement time can be shortened as compared with the prior art that required irradiation. In this embodiment, since the measurement can be completed with at least one excitation light pulse, it is possible to measure the fluorescence lifetime of a sample such as a cell that changes every moment at the same time. In addition, since measurement can be performed with one pulse or a small number of pulses, the influence of photobleaching can be suppressed.

In the present embodiment, the integrated fluorescence emission intensity can be obtained with a window corresponding to the number of detectors constituting the detector group in the fluorescence detection means 24 at the maximum. In that case, the fluorescence detection result of one detector corresponds to the integrated fluorescence emission intensity in one window. Several detectors may be combined to correspond to one window, and the fluorescence detection results of one or more adjacent detectors may be added together. As the number of detectors constituting the detector group increases, the number of windows in the time gate method can be increased, and the number of points for fitting the fluorescence decay curve can be increased to improve the accuracy of analysis. Also, multi-component analysis can be performed by increasing the number of fitting of the fluorescence decay curves.

The time-resolved fluorescence measuring apparatus 10 may have a scanning unit that scans the irradiation position of the excitation light on the measurement object 30 in addition to the configuration shown in FIG. The scanning unit scans, for example, the irradiation position of excitation light, which is a point light source, on the surface of the measurement object 30 in two directions, an x direction and a y direction, which are orthogonal to each other. As the scanning means, for example, a galvanometer mirror, a polygon mirror, a resonant mirror, a piezo stage, or the like can be used. The PC 26 (fluorescence lifetime calculation means) obtains the fluorescence emission intensity (attenuation characteristic) and the fluorescence lifetime at each scanned position.

In addition to the function as the fluorescence lifetime calculation means, the PC 26 may function as a fluorescence image generation means for generating a fluorescence image representing the fluorescence emission intensity distribution corresponding to each position of the scanned excitation light. Instead of the fluorescence emission intensity, the PC 26 may generate an image representing a fluorescence lifetime distribution calculated corresponding to each scanned position as a fluorescence image. Alternatively, the PC 26 may generate an image representing the distribution of both the emission intensity and the fluorescence lifetime as a fluorescence image. The PC 26 displays the generated fluorescent image on a display device such as a display.

The above scanning means may scan not only the surface of the measurement object 30 but also the condensing position of the excitation light with respect to the measurement object 30 in the depth direction. A piezo stage or a condensing lens (objective lens 18) can be used for scanning in the depth direction. The fluorescence lifetime measuring means only needs to obtain the fluorescence emission intensity (attenuation characteristic) and the fluorescence lifetime not only for each position on the surface of the measurement object but also for each position scanned in the depth direction. Further, the fluorescence image generating means may generate a fluorescence image as three-dimensional information by adding a depth direction to two directions on the surface of the measurement object 30.

In the above description of the embodiment, an example in which an electrostatically driven MEMS element is used as the optical element 23 has been described. However, a MEMS element having an electromagnetic force, such as an electromagnetic scanner, may be used as the optical element 23. Electromagnetic drive is commonly used in scanner elements. In the case of electromagnetic driving, when a current is passed through the movable part, a Lorentz force is generated by the direction of the current and the direction of the magnetic force of the permanent magnet, thereby driving the movable part. By alternately changing the direction in which the current flows, the movable portion can be oscillated.

In the description of the above embodiment, a point light source is assumed as the excitation light, the excitation light is irradiated to one point (one minute region) of the measurement object 30, and the fluorescence emitted from the minute region is converted into the optical element 23. In the above description, the fluorescence detection means 24 detects the signal. Instead of this, it is also possible to irradiate the excitation light to a plurality of locations of the measurement object 30 at a time using a line light source. Also in this case, the mechanism in which the fluorescence emitted from each minute region (each point on one line) irradiated with the excitation light of the measurement object 30 is detected by the fluorescence detection unit 24 is described in the above embodiment. It is the same.

In the case of using a line light source, the optical element 23 applies fluorescence emitted from a portion irradiated with one line of excitation light of the measurement object 30 to the fluorescence detection unit 24 while changing the traveling direction of the fluorescence with time. What is necessary is just to radiate | emit toward. Moreover, the fluorescence detection means 24 should just have the number of detector groups corresponding to each point of 1 line to which excitation light is irradiated. For example, the fluorescence detection unit 24 may have m detector groups corresponding to a minute region of 1 row × m columns irradiated with the excitation light (line light) of the measurement target 30. In each detector group, a plurality of detectors are arranged in a line, and a plurality of detector groups are arranged side by side, so that the plurality of detectors are arranged in a two-dimensional array in the fluorescence detection means 24. .

FIG. 7 shows the optical element 23 and the fluorescence detection means 24 when the excitation light of the line light source is irradiated. The optical element 23 is configured as a MEMS mirror array 53, for example. When the DMD 60 (FIG. 4) is used for the MEMS mirror array 53, the DMD 60 irradiates the line light of the measurement object 30 using, for example, one row of the two-dimensionally arranged mirrors (each pixel constituting one row). It is only necessary to reflect the fluorescence emitted from the formed portion (a minute region of 1 row × m columns). The same drive signal (data to be stored in the SRAM) may be supplied to the pixels constituting one row of the DMD at the same timing. Instead of the MEMS mirror array 53 that can be driven independently for each pixel, a single MEMS mirror having a length corresponding to one line of excitation light, for example, the length of five pixels in FIG. 7 may be used. Good.

The fluorescence detection means 24 is configured as a CCD array 54 in which pixels are two-dimensionally arranged, for example. Among the two-dimensionally arrayed pixels of the CCD array 54, a plurality of pixels arrayed along a direction corresponding to the rotational displacement direction of the mirror in the MEMS mirror array 53 is one point of the measurement object 30 (one minute region). ) Is configured. In the CCD array 54, the detector groups are arranged in a direction corresponding to the pixel arrangement direction of the MEMS mirror array 53. The CCD array 54 detects each of the fluorescence emitted from the minute region of 1 row × m columns irradiated with the excitation light of the measurement object 30 by a corresponding detector group among the m detector groups.

By irradiating the measurement object 30 with excitation light as line light, one line of the measurement object can be excited at a time, and fluorescence emitted from one line can enter the MEMS mirror array 53 at a time. Can do. The MEMS mirror array 53 emits (reflects) the fluorescence emitted from one line toward the CCD array 54 while changing the traveling direction of the fluorescence with time. If a two-dimensional array CCD array 54 is used as the fluorescence detection means 24, it is possible to detect fluorescence from one line by time resolution. When line light is used, imaging can be performed by scanning line excitation light in one direction on the sample surface.

The excitation light applied to the measurement object 30 may be a surface light source. When irradiating a surface light source as excitation light, the fluorescence detection means 24 corresponds to a micro area of n rows × m columns irradiated with the excitation light of the measurement object 30, and each is in an area of 1 row × m columns. It is only necessary to have n sets of a plurality of corresponding detector groups. Further, the optical element 23 reflects the fluorescence emitted from the minute column of m columns in each row irradiated with the excitation light of the measurement object 30 toward a plurality of detector groups in a corresponding set of the fluorescence detection unit 24. (Exit) may be performed. The fluorescence detection means 24 detects each of the fluorescence emitted from the area of m columns of each row irradiated with the excitation light of the measurement object 30 by the corresponding detector group among the plurality of detector groups of the corresponding set. To do.

FIG. 8 shows the optical element 23 and the fluorescence detection means 24 when the excitation light of the surface light source is irradiated. For example, the optical element 23 is configured as a MEMS mirror array 55 in which MEMS mirrors are two-dimensionally arranged. DMD 60 (FIG. 4) can be used for the MEMS mirror array 55. The fluorescence detection means 24 is configured as a CCD array 56 in which pixels are two-dimensionally arranged, for example. The CCD array 56 may be formed by connecting n (n sets) CCD arrays 54 shown in FIG. The CCD array 54 shown in FIG. 7 includes a plurality of detector groups corresponding to one row irradiated with the excitation light of the measurement object 30, and the CCD array 56 includes a plurality of detectors corresponding to the one row. Has n sets of groups.

The same drive signal (data to be stored in the SRAM) may be supplied to each pixel constituting one row of the MEMS mirror array 55 (DMD) at the same timing. A drive signal is supplied to each row of the DMD at a different timing. The timing for supplying the drive signal to each row is such that the fluorescence emitted from each row irradiated with the excitation light of the measurement object 30 is emitted toward a plurality of detector groups in the corresponding set of the CCD array 56. Adjust it. The CCD array 56 detects each of the fluorescence emitted from the minute region of m columns in each row irradiated with the excitation light of the measurement object 30 by the corresponding detector group among the plurality of detector groups in the corresponding set. To do. The measurement object 30 is excited at once using a surface light source, and the fluorescence emitted from the region irradiated with the excitation light of the measurement object 30 is detected by the fluorescence detection means 24, whereby the excitation light is detected on the measurement object 30. Fluorescent images can be acquired at once without scanning.

For example, in FIG. 8, the pixels constituting the first set (first light receiving portion) of the CCD array 56 correspond to the region of the first row of the measurement object 30 and constitute the second set (second light receiving portion). The pixels correspond to the second row area of the measurement object 30, and the pixels constituting the third set (third light receiving unit) correspond to the third row area of the measurement object 30. The first light receiving unit has five detector groups corresponding to the five micro regions in the first row, and the second light receiving unit has five detector groups corresponding to the five micro regions in the second row. The third light receiving unit has five detector groups corresponding to the five minute regions in the third row.

The drive signals are supplied from the first row to the third row of the MEMS mirror array 55 at different timings, so that the fluorescence emitted from each region of the first row of the measurement object 30 is transmitted from the first light receiving unit. Fluorescence emitted from each region of the second row of the measurement object 30 is detected by the second light receiving unit, and fluorescence emitted from each region of the third row of the measurement object 30 is detected by the third light receiving unit. Detected. In each light receiving unit, each of the fluorescence emitted from the minute regions of the five columns in each corresponding row of the measurement object 30 is detected by the corresponding detector group among the five detector groups constituting each light receiving unit. For example, the fluorescence emitted from the minute regions in the first column of the first row to the third row respectively corresponds to the detector group corresponding to the minute region in the first column of the first light receiving unit and the first column of the second light receiving unit. And a detector group corresponding to the first row of the third light receiving unit.

Next, a microscope system incorporating the above time-resolved fluorescence measuring device will be described. FIG. 9 shows a microscope system. The configuration of the microscope system 100 is basically the same as that of the time-resolved fluorescence measurement device 10 except that a part of the means constituting the time-resolved fluorescence measurement device 10 shown in FIG. It is the same.

The pulse excitation light emitted from the laser light source 11 is thinned to a desired repetition frequency by the pulse picker 12 as necessary, enters the beam splitter 14 via the mirror 13, and is separated into two lights. One of the separated lights is reflected by the beam splitter 14 and travels toward the excitation light detection means 15. The other of the separated lights is incident on the nonlinear optical crystal 16, and the nonlinear optical crystal 16 generates the second harmonic or the third harmonic of the incident wavelength.

The excitation light in which the second harmonic or the third harmonic is generated by the nonlinear optical crystal 16 is introduced into the microscope 101. The introduced excitation light enters the dichroic mirror 17 through the excitation filter 102, passes through the dichroic mirror 17, and is irradiated to a desired portion of the measurement object 30 by the objective lens 18. The fluorescence emitted from the measurement object 30 is converted into parallel light by the objective lens 18 and passes through the dichroic mirror 17. The light transmitted through the dichroic mirror 17 passes through the excitation light removal filter 19, is reflected by the mirror 103, and is emitted to the outside of the microscope 101.

The light emitted from the microscope 101 is split by the spectroscope 20, reflected by the mirror 21, passes through the condenser lens 22, and enters the optical element 23 as convergent light. The optical element 23 allows the fluorescence emitted from the measurement object 30 to be detected by different detectors of the plurality of detectors constituting the detector group of the fluorescence detection means 24 with time. Change the direction of travel. In the detector group, the fluorescence detection means 24 detects fluorescence sequentially with different detectors as time advances. The PC 26 calculates the fluorescence lifetime based on the detection result of the fluorescence detected by the detector group of the fluorescence detection means 24.

When a point light source is used as the excitation light, the excitation light is two-dimensionally fluorescent if it is scanned two-dimensionally on the surface of the measurement object 30 using scanning means such as a galvanometer mirror, polygon mirror, resonant mirror, or piezo stage. An attenuation curve can be obtained, and two-dimensional image information of the fluorescence lifetime can be obtained. When a line light source is used for the excitation light, two-dimensional image information of the fluorescence lifetime can be obtained by scanning the excitation light in one direction. When a surface light source is used for the excitation light, a two-dimensional image of fluorescence lifetime can be obtained without using a scanning means. In addition to the two directions on the surface, three-dimensional image information is obtained by scanning also in the depth direction (Z-axis direction) with a condenser lens or a piezo stage that collects excitation light on the measurement object 30. Can do.

Next, an endoscopic probe system incorporating the above time-resolved fluorescence measuring device will be described. FIG. 10 shows an endoscopic probe system. The configuration of the endoscope probe system 200 is basically the same as that of the time-resolved fluorescence measuring apparatus 10 shown in FIG. 1 except that a part of the means is incorporated in the endoscope probe apparatus 201. The configuration is the same as that of the decomposition fluorescence measuring apparatus 10.

The excitation light in which the second harmonic or the third harmonic is generated by the nonlinear optical crystal 16 is introduced into the endoscope probe apparatus 201. The introduced excitation light enters the dichroic mirror 17 through the excitation filter 202 and passes through the dichroic mirror 17. The excitation light that has passed through the dichroic mirror 17 is guided to the endoscope probe 204 through the optical fiber 203, and is irradiated to a desired portion of the measurement object 30 from the tip of the endoscope probe 204. Fluorescence emitted from the measurement object 30 passes through the endoscope probe 204 in the reverse direction and enters the dichroic mirror 17 through the optical fiber 203.

FIG. 11 shows a cross section of the endoscope probe 204. The endoscope probe 204 reaches the affected part, which is the measurement object 30, through the forceps opening of the endoscope. The endoscope probe 204 includes an optical fiber 206 formed in the center and a plurality of optical fibers 207 formed in the periphery. The optical fiber 206 formed in the center functions as an incident light that guides the excitation light to the measurement object 30 (FIG. 10). The plurality of optical fibers 207 formed in the periphery function as a light receiving unit for receiving fluorescence emitted from the measurement object 30 and guiding it to the optical fiber 203 side.

10, the light incident on the dichroic mirror 17 from the optical fiber 203 side passes through the excitation light removal filter 19, is reflected by the mirror 205, and is emitted to the outside of the endoscope probe apparatus 201. The light emitted from the endoscope probe device 201 is split by the spectroscope 20, reflected by the mirror 21, passes through the condenser lens 22, and enters the optical element 23 as convergent light. The optical element 23 allows the fluorescence emitted from the measurement object 30 to be detected by different detectors of the plurality of detectors constituting the detector group of the fluorescence detection means 24 with time. Change the direction of travel. In the detector group, the fluorescence detection means 24 detects fluorescence sequentially with different detectors as time advances. The PC 26 calculates the fluorescence lifetime based on the detection result of the fluorescence detected by the detector group of the fluorescence detection means 24.

When a point light source is used as the excitation light, if the excitation light is scanned two-dimensionally on the surface of the measurement object 30 using a scanning means such as a MEMS scanner mirror provided at the distal end of the endoscope, the fluorescence is attenuated two-dimensionally. A curve can be obtained, and two-dimensional image information of fluorescence lifetime can be obtained. Further, a fluorescence decay curve may be obtained two-dimensionally by receiving a signal from the measurement object 30 two-dimensionally with a fiber handle or the like. When a line light source is used for the excitation light, two-dimensional image information of the fluorescence lifetime can be obtained by scanning the excitation light in one direction. When a surface light source is used for the excitation light, a two-dimensional image of fluorescence lifetime can be obtained without using a scanning means. In addition to the two directions on the surface, three-dimensional image information can be obtained by scanning in the depth direction (Z-axis direction) with a condensing lens that collects excitation light on the measurement object 30. .

Examples will be described below. Using DMSO (dimethyl sulfoxide) as a solvent, Pd meso-Tetra (4 carboxyphenyl) porphine was dissolved to prepare a 5.0 × 10 ⁻⁴ mol / L solution. An appropriate amount of this solution is put into a 10 mm square sealable quartz cell, dissolved oxygen is removed by nitrogen bubbling, the quartz cell is covered, the excitation wavelength is 405 nm, the fluorescence wavelength is 697 nm, and the time-resolved fluorescence measuring apparatus 10 ( The fluorescence lifetime was measured using FIG. The light emitted from the sample was phosphorescence, and its attenuation curve was measured in a time range of about 5 msec.

As the optical element 23, a scanner element having a rotation angle of ± 10 ° and a driving frequency of 100 Hz was used. The fluorescence detecting means 24 is a one-dimensional array CCD having 2000 pixels and a pixel size of 10 μm. The time for the scanner element to drive from −10 ° to + 10 ° is 5 msec, which is approximately equal to the measurement time of the fluorescence decay curve. When 100 pixels of the CCD correspond to one window of the time gate method, 5 msec × (100 pixels / 2000 pixels) = 250 μsec, and the time resolution of the time gate method is 250 μsec.

The phosphorescence lifetime of Pdmeso-Tetra (4-carboxyphenyl) -porphine was measured by the time gate method using the above apparatus. Since 100 pixels of the CCD correspond to one window, the number of windows is 20. Plots of 20 points were obtained with respect to the time axis, and by fitting these 20 points, the phosphorescence lifetime of Pd meso-Tetra (4 carboxyphenyl) porphine was determined to be about 690 μsec.

Although the present invention has been described based on the preferred embodiment, the time-resolved fluorescence measuring apparatus and method of the present invention are not limited to the above embodiment, and various modifications can be made from the configuration of the above embodiment. Further, modifications and changes are also included in the scope of the present invention.

Claims

A light irradiating means for irradiating the measurement object with light having a predetermined wavelength as excitation light;
Fluorescence detection means for detecting fluorescence emitted from the measurement object, the fluorescence detection means having a detector group in which a plurality of detectors are arranged in a line along a predetermined direction;
The fluorescence is arranged in an optical path until it enters the fluorescence detection means, and after the excitation light is irradiated onto the measurement object, the fluorescence is emitted from a plurality of detectors of the detector group over time. An optical element that changes the direction of travel of the fluorescence so that it can be detected by a different detector,
A time-resolved fluorescence measuring apparatus comprising: a fluorescence lifetime calculating means for calculating a fluorescence lifetime based on a fluorescence detection result in the detector group.
The fluorescence lifetime calculating means calculates fluorescence emission intensity in each of a plurality of time regions based on the fluorescence detection results of the plurality of detectors, and calculates the fluorescence lifetime based on the determined fluorescence emission intensity. The time-resolved fluorescence measuring apparatus according to claim 1, wherein
The fluorescence lifetime calculating means calculates the fluorescence emission intensity in each time region based on the fluorescence detection result of one or more detectors corresponding to the time region of the fluorescence emission intensity to be obtained among the plurality of detectors. The time-resolved fluorescence measuring device according to claim 2, wherein the time-resolved fluorescence measuring device is obtained.
The optical element is configured to continuously change the traveling direction of the fluorescence from one end side to the other end side of the plurality of detectors arranged in a row over the measurement period of the fluorescence. The time-resolved fluorescence measuring device according to any one of claims 1 to 3.
The optical element is supported so as to be elastically displaceable, and is movable in a first direction and a second direction opposite to the first direction, and a physical acting force in the first direction is applied to the movable part. 5. The electric device element according to claim 1, wherein the electric device element includes a first drive source to be applied and a second drive source to apply a physical acting force in the second direction to the movable portion. The time-resolved fluorescence measuring apparatus described.
The time-resolved fluorescence measuring device according to any one of claims 1 to 5, wherein the excitation light is a point light source.
The said fluorescence detector means detects the fluorescence emitted from the location where the said excitation light of the said measuring object was irradiated with the several detector of the said detector group, It is characterized by the above-mentioned. The time-resolved fluorescence measuring apparatus described.
The time-resolved fluorescence measuring device according to any one of claims 1 to 5, wherein the excitation light is a line light source.
The fluorescence detection means includes a plurality of detector groups corresponding to an area of 1 row × m columns (m is an integer of 2 or more) irradiated with the excitation light of the measurement object, and the measurement object of the measurement object 9. The method according to claim 8, wherein each of the fluorescence emitted from the region of m columns in one row irradiated with the excitation light is detected by a corresponding detector group of the plurality of detector groups. The time-resolved fluorescence measuring apparatus described.
The time-resolved fluorescence measuring apparatus according to any one of claims 1 to 5, wherein the excitation light is a surface light source.
The fluorescence detection means corresponds to an area of n rows × m columns (n and m are each an integer of 2 or more) irradiated with the excitation light of the measurement object, each of which is an area of 1 row × m columns. N sets of a plurality of detector groups corresponding to
The optical element emits fluorescence emitted from the m-column region of each row irradiated with the excitation light of the measurement target region toward a plurality of detector groups in a corresponding set of the fluorescence detection unit,
The fluorescence detection means detects each of the fluorescence emitted from the m-column area of each row irradiated with the excitation light of the measurement object, and the corresponding detector group of the plurality of detector groups of the corresponding set. The time-resolved fluorescence measuring device according to claim 10, wherein the time-resolved fluorescence measuring device is detected by the method.
12. The time-resolved fluorescence measuring apparatus according to claim 1, wherein the fluorescence detecting means includes an image intensifier having a gate function and a charge image pickup device.
Scanning means for scanning the excitation light on the surface of the measurement object;
Fluorescence image generation means for generating a fluorescence image representing at least one distribution of the fluorescence emission intensity corresponding to each position of the scanned excitation light and the calculated fluorescence lifetime, The time-resolved fluorescence measuring device according to any one of claims 1 to 12.
The scanning means scans the condensing position of the excitation light on the measurement object in the depth direction in addition to the surface of the measurement object, and the fluorescence image generation means 3 scans the fluorescence image. The time-resolved fluorescence measuring device according to claim 13, wherein the time-resolved fluorescence measuring device is generated as dimensional information.
A microscope system comprising the time-resolved fluorescence measuring device according to any one of claims 1 to 14.
An endoscope fiber probe system comprising the time-resolved fluorescence measuring device according to any one of claims 1 to 14.
Irradiating the object to be measured with light having a predetermined wavelength as excitation light;
After the excitation light is irradiated onto the measurement object, fluorescence emitted from the measurement object is detected by different detectors out of a plurality of detectors arranged in a line along a predetermined direction over time. Changing the direction of travel of the fluorescence,
A time-resolved fluorescence measurement method comprising: calculating a fluorescence lifetime based on fluorescence detection results of the plurality of detectors.