Fluorescence resonance energy transfer analyzer

An analyzer for measuring an FRET (fluorescence resonance energy transfer) efficiency of a specimen containing a donor and an acceptor. The analyzer has an illuminator, an optical system, a detector and a calculator. The illuminator emits light for donor excitation and acceptor bleaching. The detector detects fluorescence from the specimen. The calculator calculates the FRET efficiency using the output of the detector. The detector independently detects the fluorescence in wavelength regions. One of the regions has a larger overlap with the fluorescence spectrum of the acceptor than with that of the donor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to analysis of fluorescence resonance energy transfer.

2. Related Background Art

Fluorescence resonance energy transfer (FRET) is a phenomenon in which excitation energy transfers from a fluorescent molecule to another molecule. The molecule that supplies energy to another molecule is called a donor, and the molecule that receives the energy is called an acceptor. When FRET occurs, the fluorescence of the donor weakens. When the acceptor is a fluorescent molecule, fluorescence is emitted from the acceptor.

When FRET in a cell is measured by microscopic observation, the following method may be used: measure the fluorescence intensities of the donor and the acceptor when the donor is excited, and calculate the ratio between the measured intensities, i.e., the acceptor's intensity/the donor's intensity (see Atsushi Miyawaki et al., “Fluorescent indicators for Ca2+based on green fluorescent proteins and calmodulin,”Nature, vol. 388, pp. 882-887, 28 Aug., 1997). The fluorescence intensities of the donor and acceptor can be measured in turn by switching the bandpass filters disposed in front of the detector. Moreover, the fluorescence from the donor and the acceptor can be simultaneously detected with two detectors by separating the fluorescence from them with a dichroic mirror and then filtering the fluorescence with the bandpass filter. As the detector, a camera such as a cooled CCD camera, or a photomultiplier is used, for example.

By this method, change in the fluorescence where the fluorescence of the donor weakens and that of the acceptor intensifies, which is characteristic of the FRET, can be observed. Calculating the fluorescence intensity ratio between the donor and the acceptor clearly shows the amount of the change in the fluorescence intensities. Moreover, this method is advantageous because it can cancel variation in the fluorescence intensities due to the thickness of the cell, the distribution of the dyes and the illumination unevenness of the light source. However, this method is unfit for quantitative measurement of the fluorescence intensity ratio though it can detect the changes in the FRET. When the ratio between the amounts of the donors and the acceptors in a cell is changed, when the wavelength region where fluorescence is detected is changed, when the kind of the fluorescent reagent in use is changed, or when the spectral sensitivity characteristic of the detector is changed, the value of the fluorescence intensity ratio changes accordingly. Consequently, no quantitative comparison can be made between the measurement values obtained before and after these experimental conditions are changed.

An FRET efficiency (Et) is an example of a value that can be quantitatively compared even when the experimental condition changes. Et=1−Fd′/Fd is known as an expression for obtaining this, where Fd is the fluorescence intensity of the donor when no FRET occurs, and Fd′ is the fluorescence intensity of the donor when FRET occurs. To determine Fd, light with the absorption wavelength of the acceptor is used to illuminate a specimen containing the donor and acceptor, all the acceptor molecules in measurement region are broken by photo-bleaching, and then the fluorescence intensity of the donor is measured. By Measuring Fd′ at time intervals before this bleaching experiment, variations of Et over time can be calculated.

When the bleaching experiment is performed after the measurement of Fd′, the wavelength of the illumination light and the dichroic mirror are switched from ones for Fd′ measurement to ones for bleaching. Intense light not including the absorption wavelength region of the donor and including the absorption wavelength region of the acceptor as widely as possible is used to illuminate the specimen. This is done in order to bleach the acceptor as quickly as possible without the fluorescence of the donor being affected by bleaching or the like. However, the wavelength region of the light for bleaching frequently overlaps with the fluorescence wavelength region of the acceptor. Accordingly, part of the light for bleaching leaks from the dichroic mirror and enters the detector for monitoring the fluorescence of the acceptor. Since the leakage light has a very high intensity, it may be impossible to monitor the bleaching process of the acceptor by the detector.

For example, a case is assumed where the fluorescent dye ECFP is used as the donor, the fluorescent dye EYFP is used as the acceptor and the fluorescence of the donor and acceptor are measured after the separation with a filter.FIG. 7shows the absorption spectrum51and the fluorescence spectrum52of ECFP and the absorption spectrum53and the fluorescence spectrum54of EYFP. Considering these spectra, the following bandpass filters are used: the filter having a transmission wavelength region “a” for exciting ECFP at 440 nm with the half width of 20 nm, the filter having a transmission wavelength region “c” for filtering the fluorescence of ECFP at 480 nm with the half width of 30 nm, and the filter having a transmission wavelength region “d” for filtering the fluorescence of EYFP at 535 nm with the half width of 25 nm. In this case, a dichroic mirror that allows light with wavelengths not less than 455 nm to pass therethrough is used. Thereafter, in bleaching EYFP, a bandpass filter that has a transmission wavelength region “b” at 525 nm with the half width of 45 nm is used. The filter is selected so as not to include the absorption wavelength region of ECFP and widely cover the absorption wavelength region of EYFP. In this case, a dichroic mirror of 560 nm is used (see Atsushi Miyawaki, “GFP wo mochiita bunshikan FRET (Intermolecular FRET Using GFP),”Seitai no Kagaku,Vol. 53, No. 1, pp. 75-81, Feb., 2002, and Asako Sawano et al., “Multicolor Imaging of Ca2+and Protein Kinase C Signals Using Novel Epifluorescence Microscopy,”Biophysical Journal,Vol. 82, pp. 1076-1085, Feb., 2002). When an ND filter or the like is placed on the optical path, it is removed from the path in order to apply light as intense as possible to the specimen. Accordingly, part of this intense illumination light enters the detector for measuring EYFP in the EYFP bleaching experiment.

Therefore, in order to determine when the bleaching is completed and the illumination is to be stopped, the specimen is illuminated by the light for bleaching over a predetermined time, then the settings of the dichroic mirror, the filter and the like of the microscope are returned to the ones for the fluorescence measurement, and then the fluorescence of the acceptor is measured. Thereafter, the settings are returned to the ones for the bleaching, and the light for bleaching is used to illuminate the specimen. This operation is repeated until the fluorescence of the acceptor no longer attenuates (see Atsushi Miyawaki and Roger Y. Tsien, “Monitoring Protein Conformations and Interactions by Fluorescence Resonance Energy Transfer between Mutants of Green Fluorescent Protein,”Methods in Enzymology,vol. 327, pp. 472-500, 2000).

Furthermore, there is a method for measuring the bleaching speed of the fluorescence of the donor to quantitatively measure the FRET efficiency. In this method, the FRET efficiency Et is expressed as Et=1−τb1/τ′b1, where τb1is the bleaching speed of the donor fluorescence when no FRET occurs, and τ′b1is the bleaching speed of the donor fluorescence when FRET occurs. The bleaching speed of the donor fluorescence can be obtained as the attenuation speed of the fluorescence intensity when the fluorescence intensity is measured a number of times at time intervals while the specimen is continuously illuminated by the light with the absorption wavelength of the donor. However, since the fluorescence of the donor is bleached in the measurement, variations over time of the FRET efficiency for the same specimen cannot be determined. In addition, it is necessary to set a region for determining τb1in the specimen and previously break the acceptor molecules in the region by photo bleaching (see Fred S. Wouters et al., “FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes,”The EMBO Journal,Vol. 17, No. 24, pp. 7179-7189, 1998, and Philippe I. H. Bastiaens et al., “Imaging the molecular state of proteins in cells by fluorescence resonance energy transfer (FRET) Sequential photobleaching of Forster donor-acceptor pairs,” Proceedings of the Second Hamamatsu International Symposium on Biomolecular Mechanisms and Photonics:Cell-Cell Communication,pp. 77-82, 1995).

SUMMARY OF THE INVENTION

It is an object of the present invention to quantitatively measure an efficiency of a fluorescence resonance energy transfer with simple operation.

In one aspect, this invention relates to an FRET (fluorescence resonance energy transfer) analyzer for measuring an FRET efficiency of a specimen containing a donor and an acceptor. The analyzer comprises an illuminator for selectively emitting light for donor excitation and light for acceptor bleaching, a detector for detecting fluorescence emitted from the specimen in response to illuminating the specimen with the light for donor excitation, and generating an output corresponding to an intensity of the fluorescence, and a calculator for calculating the FRET efficiency using the output of the detector. The detector independently detects light in first, second and third wavelength regions different from one another. The first wavelength region has a larger overlap with a fluorescence spectrum of the donor than with a fluorescence spectrum of the acceptor. The second wavelength region has a larger overlap with the fluorescence spectrum of the acceptor than with the fluorescence spectrum of the donor. The third wavelength region has a larger overlap with the fluorescence spectrum of the acceptor than with the fluorescence spectrum of the donor, and has no substantial overlap with a wavelength region of the light for acceptor bleaching.

The illuminator may have an optical attenuator adapted to place a light dimming filter on an optical path. When the illuminator emits the light for donor excitation, the optical attenuator places the light dimming filter on the optical path so that the light for donor excitation passes through the light dimming filter. When the illuminator emits the light for acceptor bleaching, the optical attenuator removes the light dimming filter from the optical path so that the light for acceptor bleaching does not pass through the light dimming filter.

The illuminator may have a light source, an optical attenuator, and a wavelength selector. The light source emits light in both a wavelength region of the light for donor excitation and a wavelength region of the light for acceptor bleaching. The optical attenuator receives the light from the light source, and is adapted to place a light dimming filter on an optical path. The wavelength selector receives the light from the optical attenuator to extract either component in the wavelength region of the light for donor excitation or the optical component in the wavelength region of the light for acceptor excitation. When the illuminator emits the light for donor excitation, the optical attenuator places the light dimming filter on the optical path so that the light from the light source passes through the light dimming filter before entering the wavelength selector. When the illuminator emits the light for acceptor bleaching, the optical attenuator removes the light dimming filter from the optical path so that the light from the light source does not pass through the light dimming filter.

The third wavelength region may have an overlap with the fluorescence spectrum of the acceptor so that fluorescence emitted from the acceptor when the specimen is illuminated by the light for acceptor bleaching is detected without saturation of the detector. The third wavelength may overlap with only a low-intensity region of the fluorescence spectrum of the acceptor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred embodiments of the present invention will be described below in greater detail with reference to the accompanying drawings. To facilitate understanding, identical reference numerals are used, where possible, to designate identical or equivalent elements that are common to the embodiments, and, in subsequent embodiments, these elements will not be further explained.

First Embodiment

FIG. 1is a block diagram showing the structure of an FRET (fluorescence resonance energy transfer) analyzer of this embodiment. The FRET analyzer100measures an FRET efficiency of a specimen5. The specimen5contains fluorescent dyes ECFP and EYFP. ECFP is a donor, and EYFP is an acceptor.FIG. 2shows the fluorescence spectrum and the absorption spectrum of each of ECFP and EYFP.FIG. 2also shows various wavelength regions used by the FRET analyzer100.

The FRET analyzer100calculates the FRET efficiency by the following expression:
Et=1−Fd′/Fd(1),
where Et is the FRET efficiency, Fd is the fluorescence intensity of the donor when no FRET occurs, and Fd′ is the fluorescence intensity of the donor when FRET occurs.

First, the analyzer100causes the FRET by illuminating the specimen5with light for donor excitation, and measures the fluorescence intensity of the donor as Fd′. Then, the analyzer100breaks the acceptor by illuminating the specimen5with light for acceptor bleaching and measures the fluorescence intensity of the donor as Fd. The analyzer100calculates Et using the obtained Fd and Fd′.

As shown inFIG. 1, the FRET analyzer100comprises an illuminator section1, an optical system2, a detector section3and a processing section4. The illuminator section1, the optical system2and the detector section3are optically coupled with each other. The illuminator section1, the optical system2and the detector section3are electrically connected to the processing section4.

The illuminator section1selectively emits light for donor excitation and light for acceptor bleaching, which are to be used to illuminate the specimen5. The illuminator section1has a light source10, a shutter12, an ND filter switcher13, and a wavelength switcher14that are optically coupled with each other. The shutter12, the ND filter switcher13and the wavelength switcher14are electrically connected to the processing section4through signal lines.

The light source10emits light in both a wavelength region for donor excitation and a wavelength region for acceptor bleaching. In the present embodiment, a white light source such as a xenon lamp is used as the light source10.

The shutter12is disposed on the optical path between the light source10and the ND filter switcher13. The shutter12controls the incidence, on the ND filter switcher13, of the light emitted from the light source10. The light emitted from the light source10enters the ND filter switcher13when the shutter12is opened, and does not enter the ND filter switcher13when the shutter12is closed.

The ND filter switcher13is an optical attenuator for roducing the intensity of the light emitted from the light source10. The ND filter switcher13receives the light from the shutter12and transmits it to the wavelength switcher14. The ND filter switcher13includes a plurality of ND filters as light dimming filters. These ND filters have transmittances different from one another (for example, 10%, 1%, and 0.1%).

When causing FRET, the ND filter switcher13places one of the ND filters on the optical path. The light for donor excitation passes through the placed ND filter and its intensity is reduced. By placing an appropriate ND filter on the optical path, a fluorescent image of the specimen5having intensity appropriate to the sensitivity of the detector section3can be transmitted to the detector section3.

Moreover, it is possible for the ND filter switcher13to place none of the ND filters on the optical path. In this case, the light transmittance of the ND filter switcher13is substantially 100%. As mentioned later, when the acceptor is bleached, the ND filter switcher13removes the ND filter from the optical path. Accordingly, the light for acceptor bleaching does not pass through the ND filter, so that its intensity is not lowered.

The light emitted from the light source10passes through the shutter12and the ND filter switcher13, and then enters the wavelength switcher14. The wavelength switcher14switchably extracts a specific wavelength component from the light emitted from the light source10. That is, the wavelength switcher14is a kind of filter device. The wavelength switcher14can be formed by use of a monochromater, a bandpass filter, or other wavelength selecting means.

When receiving the light from the light source10, the wavelength switcher14transmits a component of the light having either a wavelength region λex1or a wavelength region λex2. λex1is the wavelength region not less than 430 nm and not more than 450 nm with the center wavelength of 440 nm and the half width of 20 nm. λex2is the wavelength region not less than 502.5 nm and not more than 547.5 nm with the center wavelength of 525 nm and the half width of 45 nm. Light in the wavelength region λex1excites the donor ECFP in the specimen5so as to cause fluorescence. Light in the wavelength region λex2bleaches the acceptor EYFP in the specimen5. The wavelength regions λex1and λex2are determined in consideration of the absorption spectra of both the donor ECFP and the acceptor EYFP.

The absorption spectra of ECFP and EYFP are shown inFIG. 2. InFIG. 2, the broken line51shows the absorption spectrum of ECFP, and the broken line53shows the absorption spectrum of EYFP. The solid line “a” shows the wavelength region λex1, and the solid line “b” shows the wavelength region λex2. The wavelength region λex1overlaps with the absorption spectrum51of ECFP, and includes the peak wavelength of the absorption spectrum51. The wavelength region λex2overlaps with the absorption spectrum53of EYFP, and includes the peak wavelength of the absorption spectrum53. The wavelength region λex2does not overlap with the absorption spectrum51of ECFP.

The optical system2receives the light emitted from the illuminator section1, and sends it to the specimen5. Moreover, the optical system2receives fluorescence emitted from the specimen5and sends it to the detector section3. The optical system2prevents the light reflected at the specimen5from the illuminator section1from entering the detector section3. Regarding the FRET analyzer100as a microscope, the optical system2corresponds to an epi-illumination optical system.

The optical system2has a dichroic mirror switcher16, an objective lens system18and a total reflection mirror20that are optically coupled with each other. The dichroic mirror switcher16is also optically coupled with the wavelength switcher14of the illuminator section1. Moreover, the dichroic mirror switcher16is electrically connected to the processing section4through a signal line.

The light exiting from the wavelength switcher14enters the dichroic mirror switcher16. The dichroic mirror switcher16includes two dichroic mirrors DM1and DM2having different characteristics. The dichroic mirror switcher16selectively places one of these dichroic mirrors on the optical path. Accordingly, the light from the wavelength switcher14enters one of the dichroic mirrors. Which mirror is placed on the optical path is determined on an instruction from the processing section4. The dichroic mirror DM1transmits light with the wavelengths not less than 455 nm, and reflects light with the wavelengths less than 455 nm. The dichroic mirror DM2transmits light with the wavelengths not less than 560 nm, and reflects light with the wavelengths less than 560 nm. These dichroic mirrors are used for reflecting the light from the illuminator section1toward the specimen5, and for blocking the reflected light to prevent its incidence on the detector section3.

The objective lens system18is disposed between the dichroic mirror switcher16and the specimen5. In other words, when the FRET analysis of the specimen5is performed, the specimen5is disposed so as to face the objective lens system18. The objective lens system18receives the light exiting from the illuminator section1to be reflected at the dichroic mirror DM1or DM2, and condenses and applies the received light to the specimen5. Moreover, the objective lens system18receives the fluorescence emitted from the specimen5, and transmits it to the dichroic mirror switcher16.

The total reflection mirror20is disposed on the opposite side of the dichroic mirror switcher16from the objective lens system18. The total reflection mirror20receives the fluorescence emitted from the specimen5through the objective lens system18and the dichroic mirror switcher16. The total reflection mirror20reflects the received fluorescence at a high reflectance toward the detector section3.

The detector section3is composed of a triple CCD camera22. The triple CCD camera22is an imaging device that detects fluorescence emitted from the specimen5and takes images of the detected fluorescence. The triple CCD camera22receives the fluorescence emitted from the specimen5and reflected at the total reflection mirror20, and generates output electric signals corresponding to the intensities of the fluorescence. The output signal is representative of a spatial distribution image of the fluorescence (hereinafter, referred to as “fluorescent image”) on the specimen5. The output signals are sent to the processing section4.

The triple CCD camera22has a prism23and three CCD chips24cto24e. The prism23is disposed on the light incident portion of the CCD camera22. The light from the total reflection mirror20enters the incident surface of the prism23. The CCD chips24cto24eface the exit surface of the prism23. The prism23is a spectroscope. When receiving the light from the total reflection mirror20, the prism23disperses the light in the directions corresponding to the wavelengths of the light. The spectral characteristic of the prism23is determined so that lights in different wavelength regions λem1to λem3enter the CCD chips24cto24e, respectively. These wavelength regions do not substantially overlap with one another. Each of the CCD chips24cto24eis the photodetector that generates an output electric signal corresponding to the intensity of the incident light. Accordingly, the CCD camera22is capable of independently detecting the components, in the different wavelength regions λem1to λem3, of the incident light. In the present embodiment, λem1is a wavelength region not less than 460 nm and less than 500 nm, λem2is a wavelength region not less than 500 nm and less than 565 nm, and λem3is a wavelength region not less than 565 nm and not more than 600 nm. The wavelength regions λem1to λem3are determined in consideration of the fluorescence spectra of both the donor ECFP and the acceptor EYFP.

The fluorescence spectrum of each of ECFP and EYFP is shown inFIG. 2. InFIG. 2, the solid line52shows the fluorescence spectrum of ECFP, and the solid line54shows the fluorescence spectrum of EYFP. The solid line “c” shows the wavelength region λem1, the solid line “d” shows the wavelength region λem2, and the solid line “e” shows the wavelength region λem3. The wavelength region λem1overlaps with the fluorescence spectrum52of ECFP, and includes the peak wavelength of the fluorescence spectrum52. The wavelength region λem1hardly overlaps with the fluorescence spectrum54of EYFP. The wavelength region λem2overlaps with the fluorescence spectrum54of EYFP, and includes the peak wavelength of the fluorescence spectrum54. The wavelength region λem2also overlaps with the fluorescence spectrum52of ECFP. The wavelength region λem3overlaps with both the fluorescence spectra52and54.

As shown inFIG. 2, the overlap between the wavelength region λem1and the fluorescence spectrum54of EYFP is extremely small. Consequently, the output of the CCD chip24creceiving the fluorescent component in the wavelength region λem1is substantially representative of the intensity of the fluorescence of ECFP. Thus the wavelength region λem1is determined so that the output of the detector4associated with the wavelength region λem1is representative of the fluorescence intensity of ECFP. Generally, when the overlap between the wavelength region λem1and the fluorescence spectrum52of ECFP is sufficiently larger than the overlap between the wavelength region λem1and the fluorescence spectrum54of EYFP, the output of the wavelength region λem1, (the output of the CCD chip24c) can be treated as being representative of the fluorescence intensity of ECFP. In this specification, the “overlap between the wavelength region and the spectrum” means the integral of the spectrum intensity over the wavelength region.

The wavelength region λem2has a larger overlap with the fluorescence spectrum54of EYFP than with the fluorescence spectrum52of ECFP. The overlap between the wavelength region λem2and the fluorescence spectrum54of EYFP is sufficiently larger than the overlap between the wavelength region λem2and the fluorescence spectrum52of ECFP. Consequently, the output of the detector4associated with the wavelength region λem2(the output of the CCD chip24d) can be treated as being representative of the fluorescence intensity of EYFP. Thus the wavelength region λem2is determined so that the output of the detector4associated with the wavelength region λem2is representative of the fluorescence intensity of EYFP.

The wavelength region λem3also has a larger overlap with the fluorescence spectrum54of EYFP than with the fluorescence spectrum52of ECFP. Moreover, the wavelength region λem3does not overlap with the wavelength region λex2of the light for acceptor bleaching. The overlap between the wavelength region λem3and the fluorescence spectrum54of EYFP is sufficiently larger than the overlap between the wavelength region λem3and the fluorescence spectrum52of ECFP. The wavelength region λem3is used for measuring the fluorescence intensity of EYFP during bleaching the EYFP. As mentioned later, when EYFP is bleached, light in the wavelength region λex2not including the absorption wavelength of ECFP and widely including the absorption wavelength of EYFP is used to illuminate the specimen5. Accordingly, most of the fluorescence detected in the wavelength region λem3is the fluorescence emitted from EYFP. Therefore, the output of the detector4associated with the wavelength region λem3(the output of the CCD chip24e) can be treated as being representative of the fluorescence intensity of EYFP during its bleaching.

The processing section4is a computer system. The processing section4acts both as a controller for controlling the measurement of the FRET efficiency and as a calculator for calculating the FRET efficiency using the output of the detector section3. The processing section4controls operations of the shutter12, the ND filter switcher13, the wavelength switcher14, the dichroic mirror switcher16and the CCD camera22to perform the donor excitation and acceptor bleaching of the specimen5. Moreover, the processing section4performs the FRET analysis process by use of the output signal of the CCD camera22. The processing section4includes a display unit. The processing section4displays the result of the FRET analysis on the screen of the display unit.

The procedures of the FRET analysis using the analyzer100will now be described with reference toFIGS. 3 and 4, which are flowcharts showing the analysis procedures.

When starting the FRET analysis, the operator sets the image obtaining parameters by operating the processing section4(step S310). This includes the setting for measuring the donor and acceptor fluorescence and the setting for monitoring the acceptor bleaching. The number of images to be obtained by the CCD camera22, the time interval of the image obtaining and the like are set as parameters for the fluorescence measurement. The time interval of the fluorescence intensity measurement by the CCD camera22, the intensity or attenuation rate of the fluorescence at which the monitoring of the acceptor bleaching is to stop and the like are set as parameters for the monitoring of the bleaching.

Then, the analyzer100constructs an optical system for measuring the fluorescence from the donor and acceptor (step S320). At this step, the processing section4sends a control signal to the wavelength switcher14so that the transmission wavelength region of the wavelength switcher14is set to λex1. Moreover, the processing section4sends a control signal to the ND filter switcher13so as to select one of the ND filters and sets the selected filter on the optical path. Further, the processing section4sends a control signal to the dichroic mirror switcher16so as to set the dichroic mirror DM1on the optical path.

The sensitivity of the CCD camera22is adjusted (step S330). The operator sets the exposure time and gain of the CCD camera22by operating the processing section4.

Then, a dark image is obtained using the CCD camera22(step S340). The data of the dark image is sent from the CCD camera22to the processing section4. The dark image data is used for correcting the data of the fluorescent images of the specimen5.

The operator sets measurement regions (imaging regions) on the specimen5by operating the processing section4(step S350). This includes the setting of the region where the intensities of the fluorescence of the donor and acceptor are measured and the setting of the region where the bleaching of the acceptor is monitored.

When the above-described preparations are completed, the analyzer100obtains the fluorescent image of each of the donor and the acceptor under the set condition (step S360). This step will be described in detail hereinafter.

The processing section4repeatedly opens and closes the shutter12at the time intervals set at step S310. When the shutter12is opened, the white light emitted from the light source10passes through the ND filter in the ND filter switcher13to enter the wavelength switcher14. The intensity of the light is decreased by the ND filter. The wavelength switcher14transmits only the component in the wavelength region λex1, and blocks the other wavelength components. The light for donor excitation is generated in this way. The light for donor excitation exits from the wavelength switcher14to be reflected at the dichroic mirror DM1, passes through the objective lens system18, and is then applied to the specimen5. Consequently, the donor, or ECFP, in the specimen5is excited to cause fluorescence.

The fluorescence emitted from the specimen5includes the one emitted from EYFP (the acceptor) having received excitation energy from ECFP through the FRET as well as the one emitted from ECFP itself. As shown by reference numerals52and54inFIG. 2, the fluorescence of ECFP and the fluorescence of EYFP have different wavelength regions. The fluorescence of ECFP and EYFP passes through the objective lens system18to enter the dichroic mirror switcher16.

As mentioned above, the dichroic mirror DM1transmits light with wavelengths not less than 455 nm, and reflects light with wavelengths less than 455 nm. Therefore, most of the fluorescence emitted from ECFP and EYFP passes through the dichroic mirror DM1. This fluorescence is reflected by the total reflection mirror20to enter the CCD camera22.

On the other hand, the donor excitation light reflected at the specimen5is blocked by the dichroic mirror DM1. This is because the donor excitation light has the wavelength region λex1, of 430 to 450 nm. The donor excitation light is prevented from entering the CCD camera22in this way.

The prism23in the CCD camera22disperses the incident fluorescence and sends it to the CCD chips24c,24dor24ein accordance with the wavelength regions of the incident fluorescence. This enables the fluorescent images of ECFP and EYFP to be obtained. As mentioned above, the fluorescence of ECFP is mainly detected by the CCD chip24c, and the fluorescence of EYFP is mainly detected by the CCD chip24d.

The processing section4subtracts the dark component of the CCD chip24cfrom the output of the CCD chip24c, and converts the obtained value into the fluorescence intensity of ECFP. Moreover, the processing section4subtracts the dark component of the CCD chip24dfrom the output of the CCD chip24d, and converts the obtained value into the fluorescence intensity of EYFP. This calculation is performed pixel by pixel. The fluorescent images of ECFP and EYFP are determined in this way. The processing section4calculates the fluorescence intensity ratio expressed by (the fluorescence intensity of EYFP)/(the fluorescence intensity of ECFP). The processing section4displays the calculated value of the fluorescence intensity ratio on the display unit.

The processing section4repeats the above-described fluorescent image obtaining the number of times that is set at step S310. Each time the fluorescent images are obtained, the fluorescence intensity ratio is displayed. The fluorescence intensity of ECFP at each measurement time is the Fd′ (see the expression (1) shown above) at that time.

When the fluorescent image obtaining is finished, the analyzer100constructs an optical system for monitoring the acceptor bleaching (step S370). The processing section4sends a control signal to the wavelength switcher14so as to change the transmission wavelength region thereof from λex1to λex2. Moreover, the processing section4sends a control signal to the ND filter switcher13so as to remove the ND filter from the optical path. Further, the processing section4sends a control signal to the dichroic mirror switcher16so as to place the dichroic mirror DM2on the optical path instead of the dichroic mirror DM1.

Then, the monitoring of the acceptor bleaching is started (step S380). The wavelength switcher14transmits only the component in the wavelength region λex2and blocks the other wavelength components. The light for acceptor bleaching is generated in this way. The bleaching light exits from the wavelength switcher14to be reflected at the dichroic mirror DM2, passes through the objective lens system18, and is then applied to the specimen5. Consequently, the acceptor, or EYFP, in the specimen5starts bleaching. The bleaching light for illuminating the specimen5has a high intensity because it is not attenuated by the ND filter. Therefore, the light is capable of efficiently bleaching the acceptor.

As shown by the solid line “b” inFIG. 2, the wavelength region λex2of the bleaching light does not include the absorption wavelength of ECFP, and widely includes the absorption wavelength of EYFP. Consequently, most of the fluorescence emitted from the specimen5is the fluorescence generated from EYFP. This fluorescence passes through the objective lens system18to enter the dichroic mirror switcher16.

As mentioned above, the dichroic mirror DM2transmits light with wavelengths not less than 560 nm and reflects light with wavelengths less than 560 nm. Therefore, as is apparent from the fluorescence spectrum54of EYFP shown inFIG. 2, only part of the fluorescence of EYFP passes through the dichroic mirror DM2. This fluorescence is reflected by the total reflection mirror20to enter the CCD camera22.

On the other hand, the acceptor bleaching light reflected at the specimen5is blocked by the dichroic mirror DM2. This is because the acceptor bleaching light has the wavelength region λex2of 502.5 to 547.5 nm. The acceptor bleaching light is prevented from entering the CCD camera22in this way.

Most of the fluorescence incident on the CCD camera22is detected by the CCD chip24ehaving the wavelength region λem3not less than 565 nm and not more than 600 nm. This is because the dichroic mirror DM2transmits fluorescence of wavelengths not less than 560 nm. Since the wavelength region λex2of the bleaching light does not overlap with the absorption spectrum of ECFP as mentioned above, ECFP hardly emits fluorescence during the bleaching. Moreover, since the detection wavelength region λem3does not overlap with the wavelength region λex2of the bleaching light, even if the bleaching light leaks from the dichroic mirror DM2to enter the detector section3, the leakage light is not detected in the wavelength region λem3. Therefore, the output of the CCD chip24eis representative of the fluorescence intensity of EYFP during its bleaching.

The bleaching light which is not attenuated by the ND filter has a high intensity, and the intensity of the fluorescence of EYFP is high accordingly. If fluorescence having an excessive intensity enters the CCD chip24e, the CCD chip24eis saturated, so that the fluorescence intensity cannot be measured. However, the detection wavelength region λem3of the CCD chip24eoverlaps with only the foot of the fluorescence spectrum54of EYFP at its longer wavelength side. The fluorescence intensity is low at the foot of the fluorescence spectrum54. Therefore, the CCD chip24eis capable of detecting EYFP fluorescence with an appropriate intensity that does not saturate the CCD chip24e. Thus the CCD chip24eis capable of appropriately detecting the fluorescence of EYFP and obtaining the fluorescent images while the specimen5is illuminated by the bleaching light.

The processing section4subtracts the dark component of the CCD chip24efrom the output of the CCD chip24e, and converts the obtained value into the fluorescence intensity of EYFP. This calculation is performed pixel by pixel. The fluorescent image of EYFP during the bleaching is obtained in this way. The processing section4displays the obtained value of the fluorescence intensity on the display unit.

The processing section4repeats the above-described obtaining of the EYFP fluorescent image during bleaching at the time intervals that is set at step S310. Each time the EYFP fluorescent image is obtained, the fluorescence intensity is displayed. The processing section4also calculates the attenuation rate of the fluorescence intensity each time the image is obtained. The attenuation rate A is calculated by the following expression:
A=1−It/It−1(2),
where Itis the fluorescence intensity of the acceptor at the time of the current image obtaining, and It−1is the fluorescence intensity of the acceptor obtained at the time of the previous image obtaining. The attenuation rate A approaches zero as the bleaching of the acceptor advances.

Each time the fluorescent image is obtained, the processing section4compares the calculated fluorescence intensity or attenuation rate with the value that is set at step S310. When the fluorescence intensity or the attenuation rate is lower than the value set at step S310, the processing section4determines that the bleaching of EYFP is completed, and stops the monitoring of the bleaching. Thus the processing section4determines whether the bleaching of the acceptor is completed or not according to the fluorescence intensity of the acceptor during its bleaching.

When determining that the bleaching of the acceptor is completed, the processing section4constructs an optical system for measuring the fluorescence of ECFP, which is the donor (step S410). The processing section4sends a control signal to the wavelength switcher14so as to return the transmission wavelength region thereof from λex2to λex1. Moreover, the processing section4sends a control signal to the ND filter switcher13so as to place the same ND filter as that used at step S320on the optical path. Further, the processing section4sends a control signal to the dichroic mirror switcher16so as to again place the dichroic mirror DM1on the optical path instead of the dichroic mirror DM2.

Then, the light in the wavelength region λex1is again applied from the illuminator section1to the specimen5, and the fluorescent image of the donor is obtained by the CCD chip24cin the CCD camera22(step S420). This is performed in a similar manner to the above-described step S360. The processing section4subtracts the dark component from the output of the CCD chip24c, and converts the obtained value into the fluorescence intensity value of the donor ECFP. This value is representative of the donor fluorescence intensity Fd when no FRET occurs. This calculation is performed pixel by pixel.

Then, the processing section4stores the data of the obtained fluorescent images and the created graphs (step S430). These data are stored in a storage device provided in the processing section4. The brightness of each pixel in the fluorescent image is representative of the intensity of the fluorescence emitted from the position, corresponding to the pixel, on the specimen5.

Then, the processing section4performs image calculation to generate a fluorescence intensity ratio image and an FRET efficiency image at each measurement time (step S440). The fluorescence intensity ratio image is an image having the fluorescence intensity ratio, or (the fluorescence intensity of EYFP)/(the fluorescence intensity of ECFP), calculated at step S360as the brightness for each pixel. The FRET efficiency image is an image having the FRET efficiency calculated pixel by pixel as the brightness for each pixel. As described above, the processing section4calculates the FRET efficiency Et according to the following expression:
Et=1−Fd′/Fd(1).
The values calculated at steps S360and S420are substituted into Fd′ and Fd, respectively. The calculation of the expression (1) is performed pixel by pixel. By doing this, the spatial distribution of the FRET efficiency on the specimen5can be represented as an image.

The processing section4stores the calculated data of each of the fluorescence intensity ratio image and the FRET efficiency image data (step S450). These data are stored in the storage device provided in the processing section4.

Then, the processing section4starts the analysis of the fluorescence intensity ratio image and the FRET efficiency image (step S460). In this analysis, the spatial distribution and the variations with time of the FRET efficiency on the specimen5are analyzed. The result of the analysis may be shown using pseudo colors, graphs or numerical values on the screen of the display unit of the processing section4.

Advantages of this embodiment will now be described. The FRET analyzer100is capable of continuously monitoring the bleaching process of the acceptor. That is, the analyzer100is capable of measuring in real time the fluorescence intensity of the acceptor during its bleaching while illuminating the specimen5with the light for acceptor bleaching. This is because the detector section3has the detection wavelength region λem3suitable for the measurement of the fluorescence intensity of the acceptor. Hence, it is unnecessary to stop the illumination by the bleaching light and reconstruct the optical system, like in the prior art, in order to measure the fluorescence intensity of the acceptor during the bleaching. According to the analyzer100, it is possible to find the time of completion of the acceptor bleaching based on the output in the wavelength region λem3of the detector section3while continuously illuminating the specimen5with the bleaching light. It is possible to omit a complicated operation of switching the wavelengths of the light for illuminating the specimen and the optical systems over and over again during the bleaching in order to check the degree of advance of the bleaching. Consequently, according to the analyzer100, the quantitative measurement of the FRET efficiency can be quickly performed by an easy operation.

Moreover, since the analyzer100measures the fluorescence intensity of the acceptor during its bleaching in real time, the time of illuminating the specimen5with the intense light for the bleaching can be minimized. Accordingly, it is possible to minimize influences on the specimen5such as formation abnormalities and breakage of cells of the specimen5. For example, if the thickness of the specimen5changes as the formation of the cell changes, a change in the fluorescence intensity not dependent on FRET is caused. Consequently, the FRET efficiency cannot be precisely determined. In the present embodiment, this problem is prevented, and the FRET efficiency can be measured precisely.

Second Embodiment

A second embodiment of the present invention will now be described.FIG. 5is a block diagram showing the structure of an FRET analyzer200according to this embodiment. The FRET analyzer200has a detector section3ainstead of the detector section3of the analyzer100of the first embodiment. Except for this, the structure is the same as that of the analyzer100.

The detector section3ahas a filter switcher30and a photodetector32. The filter switcher30is optically coupled to the photodetector32and the total reflection mirror20. The filter switcher30and the photodetector32are electrically connected to the processing section4.

The filter switcher30includes three bandpass filters31c,31dand31e. The filter switcher30selectively places one of these filters on the optical path. The fluorescence from the specimen5enters one of the filters. Which filter is placed on the optical path is determined on an instruction from the processing section4. The filters31cto31ehave the wavelength regions λem1to λem3, respectively. Therefore, the filter switcher30transmits the component, in one of the wavelength regions λem1to λem3, of the incident fluorescence.

The fluorescence, in the wavelength region λem1, λem2or λem3, exiting from the filter switcher30enters the photodetector32. The photodetector32is, for example, an imaging device such as a cooled CCD camera, or a photomultiplier. The photodetector32generates output electric signals corresponding to the intensities of the incident fluorescence. The output signals are transmitted to the processing section4. Thus the detector section3aindependently detects the fluorescent components in the wavelength regions λem1, λem2and λem3by filtering the fluorescence from the specimen5using the filter switcher30. Hence, in the present embodiment, the FRET efficiency can also be measured by the procedures shown inFIGS. 3 and 4. When the fluorescent images of the donor and the acceptor are obtained at step S360, switching between the filters31cand31dis made at high speed.

The FRET analyzer200has the same advantages as the analyzer100of the first embodiment. Since the apparatus200has the detection wavelength region λem3, it is capable of measuring the fluorescence intensity of the acceptor during its bleaching in real time, similarly to the analyzer100. Accordingly, the quantitative measurement of the FRET efficiency can be quickly performed by an easy operation. Moreover, by minimizing the time of illuminating the specimen5with the intense light for the bleaching to reduce the influences on the specimen5, the FRET efficiency can be measured precisely.

Third Embodiment

A third embodiment of the present invention will now be described.FIG. 6is a block diagram showing the structure of an FRET analyzer300according to this embodiment. The FRET analyzer300has a detector section3binstead of the detector section3of the analyzer100of the first embodiment. Except for this, the structure is the same as that of the analyzer100of the first embodiment.

The detector section3bhas three dichroic mirrors35cto35e, three bandpass filters36cto36eand three photodetectors37cto37e. The dichroic mirror35c, the bandpass filter36cand the photodetector37care optically coupled with each other. The dichroic mirror35cis also optically coupled with the total reflection mirror20and the dichroic mirror35d. The dichroic mirror35d, the bandpass filter36dand the photodetector37dare optically coupled to each other. The dichroic mirror35dis also optically coupled with the dichroic mirror35e. The dichroic mirror35e, the bandpass filter36eand the photodetector37eare optically coupled to each other. Moreover, the photodetectors37cto37eare electrically connected to the processing section4.

The dichroic mirror35ctransmits light with wavelengths not less than 565 nm and reflects light with wavelengths less than 565 nm. The dichroic mirror35dtransmits light with wavelengths not less than 500 nm and reflects light with wavelengths less than 500 nm. The dichroic mirror35etransmits light with wavelengths not less than 565 nm and reflects light with wavelengths less than 565 nm. The bandpass filters36cto36ehave the wavelength regions λem1to λem3as the transmission regions thereof, respectively. Consequently, in the fluorescence traveling from the total reflection mirror20to the detector section3b, the component in the wavelength region λem1enters the photodetector37d, the component in the wavelength region λem2enters the photodetector37e, and the component in the wavelength region λem3enters the photodetector37c. Instead of the dichroic mirror35e, a total reflection mirror may be used.

The photodetectors37cto37emay be imaging devices such as cooled CCD cameras, or photomultipliers. The photodetectors37cto37eeach generate output electric signals corresponding to the intensities of the incident fluorescence. The output signals are sent to the processing section4.

Since the fluorescence from the specimen5is split into three beams of the wavelength regions λem1to λem3using the dichroic mirrors35cto35eand the bandpass filters36cto36e, the detector section3bindependently detects the fluorescence in these wavelength regions. Consequently, in the present embodiment, the FRET efficiency can also be measured by the procedures shown in.FIGS. 3 and 4.

The FRET analyzer300has the same advantages as the apparatuses of the above-described embodiments. Since the apparatus300has the detection wavelength region λem3, it is capable of measuring the fluorescence intensity of the acceptor during its bleaching in real time. Accordingly, the quantitative measurement of the FRET efficiency can be quickly performed by an easy operation. Moreover, by minimizing the time of illuminating the specimen5with the intense light for the bleaching to reduce the influences on the specimen5, the FRET efficiency can be measured precisely.

Fourth Embodiment

A fourth embodiment of the present invention will now be described. An FRET analyzer of the fourth embodiment has the structure shown inFIG. 1, and performs an FRET analysis by the procedures shown inFIGS. 3 and 4. However, in the present embodiment, the method of determining the end of the monitoring of the acceptor bleaching at step S380ofFIG. 3is different from that of the first to third embodiments. This difference will be described in the following:

In the present embodiment, the end time of the monitoring of the bleaching is obtained by calculation based on the fluorescence brightness of the acceptor measured during the monitoring of the bleaching. Generally, the fluorescence brightness of the acceptor attenuates exponentially with time during its bleaching. Therefore, the brightness I of the light detected by the CCD chip24eduring the monitoring of the bleaching is expressed as:
I=a·e−bt+c(3),

where the first term a·e−btrepresents the fluorescence intensity of the acceptor, a and b are constants, and t is the elapsed time from the start of bleaching. The second term c represents the intensity of the light, other than the fluorescence of the acceptor, detected by the CCD chip24e. Examples of such light include stray light produced in the FRET analyzer and self-fluorescence of the cells themselves of the specimen5. When c is sufficiently low compared to the fluorescence intensity of the acceptor, the brightness I can be regarded as:
I=a·e−bt(4).

It is considered that c is negligibly low compared to the fluorescence of the acceptor in the initial stage of the bleaching. Therefore, in the present embodiment, the fluorescence brightness is measured twice by the CCD chip24ein the initial stage of the acceptor bleaching process. The constants a and b in the expression (3) are determined from the result of the measurement.

More specifically, at step S380, the processing section4obtains the EYFP fluorescent image at a time t0at which a predetermined time has elapsed since the start of the bleaching. Further, the processing section4again obtains the EYFP fluorescent image at a time t1at which a predetermined time has elapsed since the time t0. In order that c in the expression (3) can be ignored, these times t0and t1are set in the initial stage of the bleaching. Therefore, the brightness values I0and I1measured at the times t0and t1are expressed as:
I0=a·e−bt0(5),
I1=a·e−bt1(6).
The constants a and b can be calculated from these two expressions. This enables the fluorescence intensity of EYFP at a given time during the bleaching to be estimated based on the expression (4).

The processing section4uses the expression (4) to calculate the time when the value of the fluorescence intensity or the attenuation rate set at step S310is obtained. At this step, the attenuation rate set at step S310is treated as the attenuation rate of the fluorescence intensity from I0, that is, 1−I/I0. The processing section4calculates, according to the expression (4), the time necessary to obtain the value that is set at step S310, and adds the calculated time to the start time of the monitoring of the bleaching to determine the end time of the monitoring. The processing section4stops the monitoring of the bleaching at the determined end time, and performs the processes of step S410and succeeding steps.

The FRET analyzer of the this embodiment has not only the same advantages as those of the first embodiment but also the following advantage. In this embodiment, it is necessary to measure the fluorescence intensity of the acceptor only twice to determine the end time of the monitoring of the bleaching. Accordingly, the FRET efficiency can be measured more quickly.

However, according to the method of this embodiment, when c in the expression (3) is unignorably high, the precision of the determination of the bleaching monitoring end time is lowered. Moreover, there are cases where the expression (3) does not hold for some reason. In these cases, it is desirable to determine the end time of the monitoring of the bleaching by the method adopted in the first to third embodiments.

The method of determining the acceptor bleaching time adopted in this embodiment is also adoptable to the FRET analyzers having the structures shown inFIGS. 5 and 6.

The present invention has been described in detail with respect to the embodiments. However, the present invention is not limited to the above-described embodiments. The present invention may be modified in various manners without departing from the gist thereof.

In the above-described embodiments, ECFP and EYFP are used as the donor and acceptor pair. However, the present invention is applicable to other combinations of fluorescent dyes. Examples of such combinations include EGFP (donor) and RFP (acceptor), and EYFP (donor) and RFP (acceptor). When a combination of dyes different from that in the above embodiments is used, the same advantages as in the above embodiments can be obtained by setting the wavelength characteristics of the filter, the dichroic mirror or the prism according to the dyes.