KR20120001533A

KR20120001533A - A device for measuring fluorescence lifetime and a method thereof

Info

Publication number: KR20120001533A
Application number: KR1020100062363A
Authority: KR
Inventors: 원영재; 김덕영
Original assignee: 광주과학기술원
Priority date: 2010-06-29
Filing date: 2010-06-29
Publication date: 2012-01-04
Also published as: KR101209230B1

Abstract

An apparatus and method for measuring fluorescence lifetime are disclosed. An apparatus for measuring fluorescence lifetime according to the present invention includes: an excitation light generator for generating excitation light; A fluorescent photon collector for collecting fluorescent photons generated by irradiating the excitation light to the sample; A light detector for amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electrical signal; And an average time of the first electrical signal calculated based on the first reference signal, and the excitation light source calculated based on the second reference signal of the second electrical signal converted by the light sensing unit without passing through the sample. It includes a fluorescence lifetime extraction unit for calculating the fluorescence lifetime using the difference with the average time. According to the present invention, the fluorescence lifetime measurement error is reduced, and more accurate measurement is possible.

Description

A device for measuring fluorescence lifetime and a method

The present invention relates to an apparatus and method for measuring fluorescence lifetime, and more particularly, to an apparatus and method for measuring fluorescence lifetime, which can reduce fluorescence lifetime measurement errors and enable more accurate measurement.

The location of specific molecules or proteins in a cell can be effectively detected with the help of recently developed fluorescent probes and optical fluorescence imaging techniques that measure the intensity of light emitted from the fluorescent probes. However, people want to know not only the structural distribution of specific molecules in biological samples, but also the function of unspecified molecules or proteins. Functional imaging is the measurement of specific parameters such as pH, ion concentration, oxygen saturation, local pressure, thermal diffusivity, and the like. One approach is to use fluorescence probes whose emission spectra depend on the local environment and analyze the spectral resolution response of the medium. The fluorescence emission spectrum of the probe measured in the frequency domain is related to the relaxation lifetime of the fluorescent marker measurable in the time domain. Therefore, if the environment of the fluorescent probe such as electrical, chemical or mechanical properties near the probe in the sample is changed, it can be detected in the time domain by measuring the lifetime of the probe or in the frequency domain by spectral measurement of the probe. Since spectroscopic analysis of emission spectra of fluorescent probes is usually slow and time consuming, much effort has been made to obtain the local environmental state near the probe by measuring the fluorescence lifetime of the fluorescent probe. Such fluorescence lifetime imaging microscopy (FLIM) is considered a good tool for many biological and biophysical studies. Since the lifetime of the probe can be easily influenced by the environment of fluorescent molecules, FLIM images are used to obtain information about the electrical, chemical and biological properties of biological samples such as ions, pH and oxygen concentrations. Local concentrations of ions or pH are usually measured by confocal microscopy using specific fluorescent probes whose fluorescence intensity is proportional to the concentration of the target molecule. However, the fluorescence intensity can be easily influenced by other effects such as photo-bleaching and local concentration differences of the fluorescent probes that it detects. Unlike functional imaging techniques based on fluorescence intensity, functional imaging methods using fluorescence lifetime measurements are insensitive to photobleaching or irregular concentrations of fluorophores. A very major application of FLIM can be found in Forster resonance energy transfer (FRET) imaging. In the life sciences, FRET imaging is commonly used to find locations where one protein contacts another. The donor fluorescent probe is attached to one protein and the other protein is represented by an accepting fluorescent probe whose absorption spectrum overlaps with the emission spectrum of the donor fluorescent probe. If the two fluorescent probes are as close as several nanometers, the fluorescence emission from the receiving probe can be observed even though only the donor fluorescent probe can be excited with a light source having a spectrum that does not overlap with the absorption spectrum of the receiving probe. Since the fluorescent light emitted from the donor can be absorbed directly by the receiver, the receiver fluorescence is excited by the optical absorption of the light emitted from the donor, and the receiver can receive the resonance energy by the close contact on the donor and the receiver. It is difficult to distinguish from the case here. On the other hand, the lifetime of the donor fluorescent photon decreases only when the receiver receives energy from the donor by resonant energy transfer. Thus, life-time imaging can accurately locate donors and recipients within a few nanometers, and FLIM is considered a good tool in FRET imaging applications to study the interaction of specific proteins in cells.

Many methods are being developed for confocal and wide-field FLIM systems. A key advantage of the point scanning confocal FLIM system is its ability to three-dimensional imaging. In conventional confocal FLIM systems, single photon counting (SPC) measurement techniques, such as time-correlated SPC and multirate SPC, are widely used because of many advantages such as high time resolution and good photon economics. The critical disadvantage of SPC based FLIM systems is the slow photon detection speed. Although many new SPC-based technologies have been developed to achieve high photon detection rates of several tens of MHz, their application to techniques for observing dynamic phenomena within one second is still difficult. The main advantage of the wide field FLIM system is its fast image acquisition speed. By combining a charge-coupled device (CCD) with a gated intensifier or a modulated image intensifier, the high-band time signals of all CCD pixels can be processed simultaneously, resulting in a point of acquisition time. This can be significantly reduced compared to scanning confocal FLIM systems. The operating speed of these wide-field FLIM systems is fast enough to support today's video speed imaging. However, the 3D capability, lifetime accuracy, spatial resolution, and photon economics of the wide field FLIM system are not comparable with a confocal point scanning system.

We previously proposed an analog mean-delay (AMD) method for fast fluorescence lifetime measurements with fast measurement speeds and high lifetime accuracy. It was developed for point scanning video speed 3D confocal FLIM systems. Theoretically, the fluorescence lifetime can be accurately extracted by subtracting the average delay of the sampleless device response from the average delay of the fluorescence signal. This relationship is as shown in Equation 1 below.

Where i _e ( t ) is the measured temporal fluorescence intensity signal and i _irf ( t ) is the impulse response function (IRF) of the measurement system. < T _e > and < T _e ⁰ > are defined as the average delay of the fluorescence signal and the IRF, respectively. In order to extract the absolute fluorescence lifetime of fluorescence photons using the AMD method, the initial time of the time functions i _e ( t ) and _irf ( t ) in Eq. (1) or the point where t is zero must be accurately defined and perfectly matched. . In the experiment, i _e ( t ) and _irf ( t ) are measured by an electronic data acquisition (DAQ) board, and the zero point of each function is obtained by a trigger signal from a pulsed laser light source. . However, the temporal position of each excitation laser pulse may vary from trigger to trigger because of timing jitter in the pulsed laser light source.

In addition, timing errors due to incomplete electronic DAQ boards are another major problem in accurately defining the zero position of the time-strength function obtained experimentally. That is, incompleteness of semiconductor devices (diodes, transistors, etc.) that are frequently used in electronic circuits cause time delay jitter between the input and the output of the data acquisition device. This time delay jitter is especially severe when the equipment warm-up conditions are not met. Therefore, even when a stable trigger signal comes in, the start point of data collection may vary slightly each time data collection is performed. Therefore, when a system device response pulse signal and a fluorescence pulse signal that are to be collected for fluorescence lifetime measurement are asynchronously collected, the time point of data collection due to time delay jitter may be different, and this difference is an error in fluorescence lifetime extraction. Will be generated.

The technical problem to be achieved by the present invention is to reduce the fluorescence lifetime measurement error by reducing the timing errors associated with the timing error of the data acquisition device, and to a fluorescence lifetime measurement apparatus and method that enables more accurate measurement.

The present invention also relates to an apparatus and method for measuring fluorescence lifetime which eliminates the timing jitter effect of a pulsed excitation light source, thereby reducing fluorescence lifetime measurement error and enabling more accurate measurement.

In order to solve the above technical problem, an apparatus for measuring fluorescence lifetime according to an embodiment of the present invention includes: an excitation light generator for generating excitation light; A fluorescent photon collector for collecting fluorescent photons generated by irradiating the excitation light to the sample; A light detector for amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electrical signal; And an average time of the first electrical signal calculated based on the first reference signal, and the excitation light source calculated based on the second reference signal of the second electrical signal converted by the light sensing unit without passing through the sample. It includes a fluorescence lifetime extraction unit for calculating the fluorescence lifetime using the difference with the average time. Here, the fluorescence lifetime extracting unit may include a signal collecting unit collecting the first reference signal and the first electrical signal at the same time, and collecting the second reference signal and the second electrical signal at the same time. Each of the reference signal and the second reference signal is a pulse signal synchronized with the excitation light associated with the first electrical signal and the second electrical signal. In addition, each of the first reference signal and the second reference signal may be generated from a trigger signal associated with the first electrical signal and the second electrical signal. The signal collector may include a first channel collecting the first electrical signal and the second electrical signal and a second channel different from the first channel collecting the first reference signal and the second reference signal. Or a low pass filter for restoring the first reference signal and the second reference signal to a low frequency.

In order to solve the above technical problem, a fluorescence lifetime measuring method according to an embodiment of the present invention, generating an excitation light to be irradiated to the sample; Collecting fluorescent photons generated by irradiating the excitation light to the sample; Amplifying the collected fluorescent photons and converting them into a first electrical signal; An average time of the first electrical signal calculated based on a first reference signal, a second electrical signal converted by the light sensing unit without the sample being excited by the excitation light source calculated based on a second reference signal; Calculating the fluorescence lifetime using the difference in the average time of the. The fluorescence lifetime measuring method may further include collecting the first reference signal simultaneously with the first electrical signal and collecting the second reference signal simultaneously with the second electrical signal. Each of the reference signal and the second reference signal may further comprise generating from a trigger signal associated with the first electrical signal and the second electrical signal. The generating of the first reference signal and the second reference signal may further include passing a lowpass filter to restore the associated trigger signal to a low frequency.

According to another aspect of the present invention, there is provided an apparatus for measuring fluorescence lifetime, comprising: an excitation light generator for generating excitation light; A fluorescent photon collector for collecting fluorescent photons generated by irradiating the excitation light to the sample; A light detector for amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electrical signal; And a fluorescence lifetime extracting unit configured to calculate a fluorescence lifetime using a difference between the average time of the first electrical signal and the average time of the second electrical signal converted by the light sensing unit by delaying a part of the excitation light source. . Here, a part of the excitation light source is collected in a single mode optical fiber, and the fluorescent photon is collected in a multi mode optical fiber. Further, part of the excitation light source may be delayed to be obtained within the interval between the first electrical signals.

According to another aspect of the present invention, there is provided a method of measuring fluorescence lifetime, comprising: generating excitation light to be irradiated onto a sample; Collecting fluorescent photons generated by irradiating the excitation light to the sample; Amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electric signal; And calculating a fluorescence lifetime by using a difference between the average time of the first electrical signal and the average time of the second electrical signal converted by the photodetector due to a delay of a part of the excitation light source. The fluorescence lifetime measuring method may further include calculating a difference between an average time of the first electrical signal and an average time of the second electrical signal in the state in which the sample is removed, and reflecting the result to the excitation light. can do.

The apparatus and method for measuring fluorescence lifetime according to one embodiment of the present invention described above reduces the fluorescence lifetime measurement error and enables more accurate measurement.

For example, the fluorescence lifetime value of a commonly used fluorescent sample is several ns, whereas the error due to the variation of the start point of the data collection device may range from several hundred ps to several ns depending on the data collection device used. In this case, using the apparatus and method proposed in the present invention, the fluorescence lifetime measurement error can be reduced to several ps.

1A is a block diagram of an apparatus for measuring fluorescence lifetime according to an embodiment of the present invention.
1B is a block diagram of an apparatus for measuring fluorescence lifetime according to another embodiment of the present invention.
FIG. 2 illustrates a temporal waveform of electrical signals according to the embodiment of FIG. 1B.
3 is a diagram illustrating IRF waveforms according to an embodiment of the present invention.
FIG. 4 is a diagram illustrating a fluorescence lifetime measurement error calculated for each IRF signal of FIG. 3.
5A is a block diagram of an apparatus for measuring fluorescence lifetime according to another embodiment of the present invention.
5B is a block diagram of an apparatus for measuring fluorescence lifetime according to still another embodiment of the present invention.
5C is a flowchart of a fluorescence lifetime measuring method according to an embodiment of the present invention.
6 is a diagram illustrating a temporal waveform of electrical signals according to the embodiment of FIG. 5B.
FIG. 7 is a diagram illustrating a calculated fluorescence lifetime measurement error with respect to the IRF signal of FIG. 6.
8 is a block diagram of a fluorescence lifetime measuring apparatus according to another embodiment of the present invention.
9 is a flowchart illustrating a method of measuring fluorescence lifetime according to another embodiment of the present invention.
10 is a diagram illustrating a temporal waveform of electrical signals according to the embodiment of FIG. 8.
FIG. 11 is a diagram illustrating a calculated fluorescence lifetime measurement error with respect to the IRF signal of FIG. 10.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description and the accompanying drawings, substantially the same components are denoted by the same reference numerals, and redundant description will be omitted. In addition, in the following description of the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted.

First, a fluorescent lifetime measuring apparatus and a method for measuring fluorescent lifetime without a reference signal in the AMD method will be described with reference to FIGS. 1A and 1B.

1A is a block diagram of an apparatus for measuring fluorescence lifetime according to an embodiment of the present invention.

The excitation light source 1 is incident in space and parallel through the single mode fiber 2 and the collimating lens 3 in a pulse form with respect to time. The incident light source passes through the band pass filter 4 and is reflected from the dichroic filter 5 and is incident on the sample 7 through the objective lens 6. The incident light source causes fluorescence photons to be generated from the sample, and the generated fluorescence photons are collected through the objective lens 6 and then passed through the dichroic filter 5. The dichroic filter used is an optical filter for selectively passing the incident light source according to the wavelength. Here, the dichroic filter reflects the wavelength band corresponding to the excitation light source and passes the wavelength band corresponding to the fluorescent photon. Therefore, the fluorescent light source from which the excitation light source has been removed is concentrated from the collimating lens 3 after passing through another bandpass filter 8 and is incident on the multimode fiber 9. This fluorescent light source is converted into an electrical signal through the photodetector 10 and amplified from the amplifier 11. Finally, the fluorescent light source, which has been transferred and amplified to an electrical signal, is restored in the data collection device 12. The system device response function is the excitation light source itself undergoing the same path and can be obtained by using a mirror as the sample and removing the band pass filter 8.

Meanwhile, a filter (particularly, an electronic Gaussian low pass filter (GLPF)) may be further included between the photo detector 10 and the amplifier 11. The electronic Gaussian low pass filter considers a sampling frequency for signal reconstruction, which will be described later. When a filter is not provided in FIG. 1A, a high-performance digitizer capable of high frequency sampling for signal recovery in consideration of an excitation light source having a small pulse width is preferably provided.

1B is a block diagram of an apparatus for measuring fluorescence lifetime according to another embodiment of the present invention, which will be described with reference to specific components and / or product names used in experiments for proving an effect according to an embodiment of the present invention.

As the laser light source (Laser source) as the excitation light source 1, a half-width full width of the output pulse is approximately 800 ps, and a homemade gain switched semiconductor laser having a wavelength of 635 mm operating at a repetition rate of 5.0 MHz can be used. As the bandpass filter 4, in order to extract pure laser light with a narrow wavelength band of 10 nm, XB108, which is an Omega Optical laser line filter (LLF), is used as a beam splitter (BS) as an optical filter. ) In front of it. This functions to automatically extract a small amount of the emitted light of the spectrum corresponding to the fluorescent light signal from the fluorescent photon from the laser light source.

50% of the excitation light is reflected by the 50/50 beam splitter BS and is incident on the sample through the objective lens. Fluorescent signals emitted from the sample are collected through the objective lens and then passed through a beam splitter. Fluorescent signals passing through the beam splitter are injected into a photo-multiplier tube (PMT) that is part of a photodetector, for example, a multimode optical fiber (MMF) connected to a R7400U-20 manufactured by Hamamatsu. .

An optical low pass filter (LPF) as a bandpass filter 8 placed in front of a multimode fiber may use, for example, 3RD650LP from Omega Optical, which is an excitation pulse signal reflected from the beam splitter. Used to extract small amounts of. The spectrally filtered pure fluorescence signal is injected into the PMT and converted into an electrical pulse signal. The electrical pulse signal from the PMT is amplified in time by a homemade fifth order electronic Gaussian low pass filter (GLPF). This GLPF is the key to enabling fast fluorescence signal life measurements even with slow DAQ boards with a sampling rate of only 100MS / s. This ensures that electronic signals entering the DAQ do not have a frequency portion above 50 MHz to satisfy the Nyquist-Shannon sampling theory. By using GLPF, the size of DAQ board data storage can be significantly reduced without compromising the accuracy of life measurements. The cost of a 100MS / s DAQ board is lower than that of a 2-10GHz DAQ board, which can be reduced by using GLPF as well as the cost of an AMD system. The slow electrical signal through GLPF is amplified by a 16-circuit electrical amplifier (AMP), GALI-2 from Minicircuits.

The amplified signal is obtained by a high-speed digitizer PCI-5114, a National Instruments Corporation DAQ board with 8-bit voltage resolution and 100 MS / s sampling rate, which collects the electrical signals and based on the fluorescence It functions as part of the fluorescence life extracting section for calculating the lifetime. The clock signal of the DAQ board and the clock signal of the pulsed laser light source are fixed in phase by a coaxial cable. As in the description of FIG. 1A, the IRF of the AMP-FLIM system can be measured by replacing the sample with a glass mirror and removing the LPF. The fluorescence signal acquired by the 100MS / s DAQ board and the waveform data of the IRF are interpolated by spline interpolation to be 2GS / s data. To calculate the average delay of the waveform, we use an optimal integral window with the same magnitude as the full width at half maximum of the measured IRF. Five iterative processes are performed to find the optimal center position of the optimal integration window. In the experiment, the IRF of the system i _irf ( t ) first extracts the laser light source pulse as the excitation light source without the fluorescent photon, and then the fluorescent signal i _e ( t ) of the fluorescent photon to extract the fluorescence lifetime from equation (1). Measure

FIG. 2 shows the temporal waveforms of the system device impulse response function i _irf ( t ), the fluorescence signal i _e ( t ), and the trigger signal from the laser light source according to the embodiment of FIG. 1B. Where f _o is the repetition rate of the pulsed laser light source. Here, the fluorescence lifetime extraction process of collecting signals from the digitizer and calculating fluorescence lifetime is initiated by the trigger signal from the laser light source. The evenly spaced rectangular boxes shown in this figure are digitized scales and have the width t _o equal to the sampling interval of the DAQ board, which is a fluorescence lifetime extraction device. For example, for an experiment using a DAQ board with a sampling rate of 100 MS / s, t _o is 10 ns. t _trig represents the initial time for the trigger signal from the laser light source to reach the DAQ board. t _start indicates the start time of the waveform measured by the DAQ board. As mentioned above, the measurement apparatus used for IRF measurement is the same as that used for fluorescence signal measurement except for removing the LPF in FIG. 2. Since the optical path generated by the movement IRF car approximately 0.45mm, the difference between <T _e ^0> of the actual value and the measured value of <T _e ^0> is about 1.5ps. This is less than 0.1% of the lifetime of ordinary fluorescent photons used in FLIM and can be ignored.

Since the sampling interval t _o has a finite value, there is a delay time Δt between t _trig and t _start , which occurs in the temporal mismatch between the trigger signal from the laser light source and the sampling interval of the DAQ board. Because of this discrepancy, the average delay of IRF < T _e ⁰ > in Equation (1) needs to be replaced by the measured average delay of [< T _e ⁰ >-Δt ₂ ]. The mean delay of the fluorescence signal < T _e > should also be transformed into the measured mean delay of [ <T _e >-Δt ₁ ]. Here, Δt ₁ and Δt ₂ represent temporal mismatches of the fluorescence signals i _e ( t ) and IRF i _irf ( t ), respectively. When the clock signal of the DAQ board is synchronized with the clock signal of the pulsed laser light source, assuming that the temporal mismatch between the trigger signal from the laser light source and the sampling interval Δt of the DAQ board is constant at all times, the following equation can be obtained. .

However, due to incompleteness in the electronic components used in AMD systems, there is a large undetermined deviation in Δt . Fast pulse-to-pulse fluctuations of Δt arise from timing jitter in electronic components, and slow fluctuations may occur due to temperature changes in electronic and optical components. In the AMD system, the IRF i _irf ( t ) and the fluorescence pulse signal i _e ( t ) are obtained independently in sequence. If Δt changes during the measurement, the fluorescence lifetime measured in equation (1) needs to be corrected as follows.

An IRF signal unique to the fluorescence lifetime measuring device according to an embodiment of the present invention is repeatedly measured every 30 seconds for 30 minutes to measure an error caused by incompleteness in these electronic components. The measurement starts as soon as all of the electronics are turned on at the same time and averaged 50,000 pulses for each waveform measurement to eliminate other noise effects. The average number of photons of a single fluorescent pulse signal is approximately 40, so that the final pulse signal will acquire approximately 200,000 photons.

3 shows six IRF waveforms measured every 2.5 minutes for 15 minutes after the fluorescence lifetime measuring device according to an embodiment of the present invention is turned on. All of these waveforms were measured using a slow 100MS / s DAQ board, but were interpolated to obtain 2GS / s data, showing a very gentle shape. The IRF signals obtained after the first signal at t = 0 can be regarded as virtual fluorescence (V.F.) whose lifetime is assumed to be '0'. The reason for introducing the concept of a virtual fluorescence signal having a 'zero' average lifetime is to evaluate the accuracy of the measuring method according to an embodiment of the present invention.

The average delay or fluorescence lifetime calculated for each VF signal is shown in FIG. Referring to the figure, the measured fluorescence lifetime of the VF signal continues to increase in the region ① during the preheating time of the fluorescence lifetime measuring apparatus according to an embodiment of the present invention, and after 15 minutes, it is saturated to 2.2 ns in the region ② but continues. It can be seen that there is a slight variation without maintaining a constant value. Thus, it can be seen that Δt is not constant even if there is no special change in the hardware of the fluorescence lifetime measuring apparatus. The error range of the measured fluorescence lifetime in the region ① during the preheating time was 2.03 ns, whereas the error range of the measured fluorescence lifetime in the region ② after 12 minutes of preheating was 200 ps and the standard deviation of the fluorescence lifetime was 51 ps. Given that the lifetime of most fluorescent photons used in biology is several ns or less, the amount of error is relatively large for most FLIM applications. Therefore, for another embodiment that can effectively eliminate the deviation of Δt caused by the incompleteness of the electronic components used in the fluorescence lifetime measurement apparatus according to an embodiment of the present invention, in particular trigger-related error caused by the digitizer DAQ board This will be described with reference to FIGS. 5A to 7.

5A is a block diagram of an apparatus for measuring fluorescence lifetime according to another embodiment of the present invention.

Referring to FIG. 5A, the excitation light generator 50 is a module for generating excitation light to be irradiated onto a sample S including fluorescent molecules, and includes an excitation light source 52 for generating excitation light in the form of pulses, and And an objective lens 54 for condensing the excitation light to irradiate the sample S. A band pass filter may be further included between the excitation light source 52 and the objective lens 54.

The fluorescent photon collecting unit 60 is a module for collecting a plurality of fluorescent photons generated by irradiating the sample S. The fluorescent photon collecting lens 62 collects a plurality of fluorescent photons generated from the sample and an excitation light. It consists of the dichroic filter 64 as an excitation light removal filter for preventing the light reception part 70 from receiving this backward. Here, the collection lens 62 may be substituted for the objective lens 54.

The light detecting unit 70 is a module for amplifying and converting a fluorescent photon collected by the fluorescent photon collecting lens 62 and passing through the dichroic filter 64 into an electrical signal. , The photodetector 74 and the amplifier 76. The photodetector 74 and the amplifier 76 may include, for example, PMT, Avalanche Photo Diode (APD), and / or GLPF, AMP, or the like. Here, the light receiving lens 72 may be configured to further include a band pass filter.

The fluorescence life extracting unit 80 is a module for obtaining fluorescence life using the electric signal converted by the light detecting unit 70 and the electric signal synchronized to the excitation light source 52, and is converted by the light detecting unit 70. And a signal collecting unit 82 for collecting the electrical signal and the electrical signal synchronized to the excitation light source 52, and a fluorescence lifetime calculating unit 84 for calculating the fluorescence lifetime based on the collected result. Here, a signal acquisition unit 82 may be a DAQ board, and the first channel for collecting the electrical signal converted by the light detector 70 and the agent for simultaneously collecting the electrical signal synchronized with the excitation light source 52 It may be configured to have a second channel different from one channel.

The fluorescence lifetime calculator 84 calculates the average time of the first electrical signal calculated based on the electrical signal synchronized to the excitation light source 52 and the excitation calculated based on the electrical signal synchronized to the excitation light source 52. The light source calculates the fluorescence life using the difference in the average time between the sample and the second electrical signal converted by the light sensing unit without passing through the bandpass filter in front of the light receiving lens 72.

5B is a block diagram of an apparatus for measuring fluorescence lifetime according to still another embodiment of the present invention.

Referring to FIG. 5B, in order to reduce the fluorescence lifetime measurement error due to the variation of the data collection start time of the data collecting device 15, a pulse signal synchronized with the excitation light source 13 may be supplied to the second channel of the data collecting device 15. Except for collecting at (Ch2), the fluorescence lifetime measuring apparatus of this embodiment includes the same components as the fluorescence lifetime measuring apparatus of FIG. 1A. In addition, the method may further include a low pass filter LPF 16 to allow sampling for signal recovery to be applied in the same manner as the first channel Ch1. As the lowpass filter 16, for example, a minicircuit SLP-15 + having a 3 dB bandwidth of 17 MHz may be used, and before collecting a signal synchronized with the excitation light source from the second channel. It can be used to restore the trigger signal to a lower frequency. As a result, the smoothed trigger signal is digitized at a sampling rate of 100 MS / s.

5C is a flowchart of a fluorescence lifetime measuring method according to an embodiment of the present invention. The fluorescence lifetime measuring method according to the present embodiment includes steps processed in the fluorescence lifetime measuring apparatus shown in FIGS. 5A to 5B. Therefore, even if omitted below, the contents described above with respect to the fluorescence lifetime measurement apparatus shown in FIGS. 5A and 5B are also applied to the fluorescence lifetime measurement method according to the present embodiment.

In step 500, the fluorescence lifetime measuring device generates excitation light to irradiate the sample.

In step 510, the fluorescent light photons generated by irradiating the excitation light to the sample S are collected.

In operation 520, the fluorescent photon collected in operation 510 is amplified and converted into a first electrical signal using the light detector 70.

In operation 530, the first reference signal is collected simultaneously with the first electrical signal through different channels. Here, the first reference signal is a pulse signal synchronized with the excitation light associated with the first electrical signal, and is generated by amplifying a trigger signal associated with the first electrical signal.

In operation 540, the second reference signal is collected at the same time as the second electrical signal in a state where the sample is removed through different channels. Here, the second electrical signal is generated by irradiating the excitation light to a mirror, and the second reference signal is a pulse signal synchronized with the excitation light associated with the second electrical signal, and amplifies the trigger signal associated with the second electrical signal. To create. This step is an independent step from the steps 510 to 530, and may be performed before or after the step because there is no meaning in the order.

In operation 550, the average time of the first electrical signal calculated based on the first reference signal and the second excitation light source calculated based on the second reference signal are converted by the light detector without passing through the sample. The fluorescence lifetime is calculated using the difference from the average time of the electrical signal. A detailed description of the method for calculating the fluorescence lifetime by the method proposed in step 550 will be described with reference to FIG. 6.

6 is a diagram illustrating a temporal waveform of electrical signals according to the embodiment of FIG. 5B. Where i _e ( t ) and i _irf ( t ) represent the amplified signal from the fluorescence photon and the IRF signal of the system, respectively, and i _trig ( t ) represents the amplified trigger signal. As briefly mentioned above, two consecutive measurements are performed in this example. First, in the first measurement, i _e ( t ) and i _trig ( t ) are simultaneously collected by the first channel Ch1 and the second channel Ch2. Subsequently, in the second measurement, i _irf ( t ) and i _trig ( t ) are simultaneously collected by the first channel Ch1 and the second channel Ch2. Although signal collection is actually indicated by the t _trig signal, the actual signal collection starts after Δt ₁ from the t _trig signal by the time delay jitter. Δt ₁ is the temporal mismatch between the trigger signal from the laser light source and the sampling interval of the data acquisition device for the fluorescence signal i _e ( t ) and the amplified trigger signal i _trig ( t ) used in the first measurement. . That is, Δt ₂ is the temporal mismatch between the trigger signal from the laser light source and the sampling interval of the data acquisition device for IRF i _irf ( t ). Data acquisition on both channels is initiated simultaneously by an external trigger signal. Since the amplified trigger signal i _trig ( t ) is measured together with the fluorescence signal i _e ( t ) or IRF i _irf ( t ), the temporal mismatch of i _trig ( t ) is determined by i _e ( t for two consecutive measurements. ) Or i _irf ( t ). Therefore, the measurement errors due to the variation of Δt or the temporal mismatch between the trigger signal and the sampling interval of the data collecting device can be effectively eliminated, and this relationship can be seen from the following equation.

Here, < T _e >, < T _e ⁰ > and < T _e-sync > are the average delays of the fluorescence signal, the IRF, and the amplified trigger signal, respectively. [< T _e >-DELTA t ₁ ] and [< T _e-sync >-DELTA t ₁ ] are the measured mean delays of the fluorescence signal and the amplified trigger signal for the first measurement, respectively. [< T _e ⁰ > -Δt ₂ ] and [< T _e-sync > -Δt ₂ ] are the measured average delays of the amplified trigger signal for the IRF and the second measurement, respectively. Δt ₁ and Δt ₂ are the temporal inconsistencies between the trigger signal from the laser light source and the sampling interval of the data collection device for the first and second measurements. On the other hand, Equation 4 may be expressed as follows.

Here, t ₁ is the sum of the time from the start of the excitation light source to the incident to the sample and the time taken until the fluorescent light source emitted from the sample is collected by the data collection device. Similarly, t ₂ is the sum of the time taken by the pulse signal synchronized with the excitation light source to be collected by the data collection device. The two values represent a fixed value at which time the light source moves inside the device.

FIG. 7 is a diagram illustrating a calculated fluorescence lifetime measurement error for each IRF signal of FIG. 6. Referring to FIG. 7, a slow fluorescence lifetime change is observed in the region ① within 10 minutes after all the electronic components are turned on. However, the slow fluorescence lifetime error range in FIG. 3 was approximately 2.2ns, whereas the slow fluorescence lifetime error range in FIG. 7 was less than 100ps. After approximately 12 minutes, the measured fluorescence lifetime is in the range of 31 ps in the region ②. The standard deviation of fluorescence lifetime in this region is 9ps. This amount of error is negligible for almost all fluorescence lifetime applications. According to the results shown in this figure, the timing error associated with the temporal mismatch between the trigger signal and the digitization interval of the data acquisition device can be eliminated using the amplified trigger signal as the electronic reference signal.

On the other hand, since the timing error caused by the incompleteness of the electronic components, in particular the data collection device, has already been eliminated, this slow change in fluorescence life may be mainly due to the temperature difference of the electronic components of the pulsed laser light source during the preheating period. This can also be caused by temperature changes of the laser itself, drive circuits or other electronic devices such as PMT and AMP. The electronic signal path difference between the IRF and the fluorescence signal can of course also cause this error. Since the degree of fluorescence lifetime measurement error and the preheating time may vary depending on the electronic device and components used, the degree of fluorescence lifetime measurement error and the preheating time of the system need to be calibrated. For example, in FLIM applications using extremely short lifetime fluorescent photons, it may be problematic to have a fluorescence lifetime error range of about 100 ps. Another embodiment proposed to solve this problem will be described with reference to FIGS. 8 to 10.

8 is a block diagram of a fluorescence lifetime measuring apparatus according to another embodiment of the present invention.

Referring to FIG. 8, shown in FIG. 1B, except that a portion of the excitation pulse that has passed directly through the beam splitter BS is collected in a single mode optical fiber SMF and passed to the same PMT detector used for fluorescence signal detection. It includes the same components as the fluorescence lifetime measurement apparatus according to an embodiment of the present invention. In addition, the overall configuration is the same as that of Fig. 5A, but only the configuration of the fluorescence lifetime extraction unit for calculating the fluorescence lifetime is different. That is, in the fluorescent lifetime measuring apparatus according to the embodiment, instead of receiving a reference signal from the excitation light source, the average time of the first electrical signal generated in the same manner as in FIG. 5A and a part of the excitation light source are delayed to detect the light. It includes a fluorescence lifetime extraction unit for calculating the fluorescence lifetime using the difference in the average time with the second electrical signal converted in the negative. Here, the directly measured excitation laser pulse signal acts as the system's IRF. The length of the SMF is set to control the time delay of the IRF, allowing the IRF pulse to be placed between the fluorescent signals. Since these interlaced optical signals are detected in the same PMT and digitized by a single channel on a 100MS / s DAQ board, there is no trigger related timing error in this method. Since the intensity of the IRF signal is quite large and the optical path of the IRF must be longer than the fluorescence signal, the SMF is used to collect the IRF signal, and the length of the SMF is, for example, about 45 m. Since the intensity of the fluorescence signal is very weak, a 3m long multimode fiber (MMF) is used for fluorescence signal collection. Since the bandwidth of the MMF is 3 GHz.km, the modal dispersion introduced by the MMF is expected to be approximately 1 ps, and the bandwidth degradation caused by the MMF is negligible compared to the typical fluorescence lifetime used in most FLIM applications. It is enough. With the exception of these two other optical fibers, all the components used for the collection of IRF and fluorescent signals are identical.

9 is a flowchart illustrating a method of measuring fluorescence lifetime according to another embodiment of the present invention. The fluorescence lifetime measuring method according to the present embodiment includes steps processed in the fluorescence lifetime measuring apparatus shown in FIG. 8. Therefore, even if omitted below, the above description of the apparatus for measuring fluorescence lifetime shown in FIG. 8 is also applied to the method for measuring fluorescence lifetime according to the present embodiment.

In operation 900, the fluorescence lifetime measuring device generates excitation light for irradiating the sample.

In operation 910, the fluorescent light photons generated by irradiating the sample S with the excitation light are collected.

In step 920, a portion of the excitation light source is collected in the single mode optical fiber as a delayed IRF signal. This step may be performed simultaneously with step 910.

In operation 930, the photodetector 70 is used to amplify the fluorescent photons collected in operation 910 and convert the fluorescent photons into a first electrical signal.

In operation 940, the difference between the average time of the second electrical signal and the delayed IRF average time is calculated while the bandpass filter in front of the sample and the MMF is removed. This step is an independent step from the above steps 910 to 930. Since the order does not have a meaning, the step may be performed earlier or later. In order to eliminate the determined path difference between the optical path of the IRF signal and the first electrical signal, it is preferable to perform the operation before step 910 to reflect the result in the excitation light.

In step 950, the fluorescence lifetime is calculated using a difference between the average time of the IRF signal obtained in step 920 and the average time of the first electrical signal obtained in step 930. A detailed description of the method for calculating the fluorescence lifetime by the method proposed in FIG. 9 will be described with reference to FIG. 10.

10 is a diagram illustrating a temporal waveform of electrical signals according to the embodiment of FIG. 8. Where i _e ( t ) represents the measured fluorescence signal and i _irf ( t ) represents the delayed IRF signal. Make two separate measurements. In the first measurement, i _e ( t ) and i _irf ( t ) are obtained from a single waveform measurement (see the middle figure in FIG. 9). < T _e > and < T _e ⁰ > are defined as the average delay of the fluorescence signal and the delayed IRF, and [< T _e >-Δt ₁ ] and [< T _e ⁰ > -Δt ₁ ] are the fluorescence signal and This is the measured average delay of the delayed IRF. Δt ₁ is the temporal mismatch between the trigger signal from the laser light source and the sampling interval of the data acquisition device in the first measurement. Since i _e ( t ) and i _irf ( t ) signals are obtained within an excitation laser light source, in this embodiment 400 ns pulse interval, i _e ( t ) and i _irf ( t ) have the same temporal mismatch Δt Will experience ₁ Subtracting the measured average delay of the IRF from the measured average delay of the fluorescence signal, we obtain

Here, T _delay is the time delay between the IRF and the fluorescence signal due to the determined path difference between the two optical paths. From Eq. (5) it is necessary to take a second measurement to eliminate this established time delay T _delay . Using the method used to obtain the virtual fluorescence signal, the glass mirror is placed in the same step. Excitation laser pulses are detected by the same PMT after removing the LPF in front of the MMF of FIG. 8 to obtain the virtual fluorescence signal i _vir ( t ). In a second measurement, i _vir ( t ) and i _irf ( t ) are obtained from a single waveform measurement. i _irf ( t ) is exactly the same as i _vir ( t ), but delayed by a time delay T _delay determined by the path difference between the two signal paths (see first figure in FIG. 9). < T _e-sync > and < T _e ⁰ > are defined as the average delay of the virtual fluorescence signal and the delayed IRF, and [< T _e-sync >-Δt ₂ ] and [< T _e ⁰ > -Δt ₂ ] Is the measured mean delay of the fluorescence signal and the delayed IRF. Δt ₂ is the temporal mismatch between the trigger signal from the laser light source and the sampling interval of the DAQ system for the second measurement. Subtracting the measurement average delay of the IRF from the measurement average delay of the virtual fluorescence signal, the following equation is obtained.

The time delay T _delay between the IRF and the fluorescence signal of the fluorescence lifetime measuring apparatus as an example for obtaining the result of FIG. 10 was measured to be 199.32 ns. The total number of photons obtained by < T _e-sync > and < T _e ⁰ > is approximately 4 × 10 ^6, so that the signal-to-noise ratio (SNR) is not a factor that limits the accuracy of these measurements. IRF and fluorescence signals are to be obtained within the interval between pulses of the pulsed laser light source used in the fluorescence lifetime measurement device. To give a 400 ns pulse-to-pulse spacing, the repetition rate of the gain-changing laser was reduced to 2.5 MHz in this case. Measurements involving fluctuations at positions t = 0 in the two time functions i _e ( t ) and i _irf ( t ) of equation (1) by acquiring both signals within a defined time delay less than the interval between pulses The error can be effectively eliminated.

FIG. 11 is a diagram illustrating a calculated fluorescence lifetime measurement error with respect to the virtual fluorescence signal of FIG. 10.

For the accuracy of the fluorescence lifetime measurement method proposed in FIGS. 8 to 9, a virtual fluorescence signal was measured and fluorescence lifetime was repeatedly calculated every 30 seconds for 30 minutes. Approximately 200,000 photons were used for each waveform measurement. When t = 0, all the electronics were turned on at the same time and measurement was started. The slow 100MS / s DAQ board was used to acquire the fluorescent and reference signals. Unlike in the case illustrated in FIGS. 4 and 7, FIG. 11 has little error in measuring fluorescence lifetime during the preheating period of the electronic components. Because the measured IRF and fluorescence signals are measured within 200 ns intervals, timing errors slower than this time interval are all effectively eliminated. The error range of fluorescence lifetime was 23ps and the standard deviation of fluorescence lifetime was only 4ps. This degree of error can also be compared with the result of region ② after the 12-minute warm-up period of FIG. It is also a small enough error that can be applied to most FLIM applications. Although similar to the performance after the preheating period of FIG. 7, the embodiment may be more desirable in view of the preheating time of the fluorescence lifetime measurement device which depends on temperature, humidity, or other various surrounding environments.

The above-described embodiments of the present invention can be written as a program that can be executed in a computer, and can be implemented in a general-purpose digital computer which operates the program using a computer-readable recording medium. The computer-readable recording medium may be a magnetic storage medium (for example, a ROM, a floppy disk, a hard disk, etc.), an optical reading medium (for example, a CD-ROM, a DVD, etc.) and a carrier wave (for example, the Internet). Storage medium).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

The apparatus and method for measuring fluorescence lifetime according to the present invention reduces the timing errors associated with the timing error of the data acquisition device to reduce the fluorescence lifetime measurement error, and uses an fluorescence photon including a FLIM that requires more accurate measurement. Applicable to the field to produce. In addition, the apparatus and method for measuring fluorescence lifetime according to the present invention eliminates the timing jitter effect of a pulsed excitation light source to reduce fluorescence lifetime measurement error and generate an image using a fluorescence photon including FLIM which requires more accurate measurement. Applicable to the field.

1. excitation light source 2. single-mode fiber
3. Optical collimation lens 4. Band pass filter
5. Dichroic Filter 6. Objective Lens
7. Sample 8. Bandpass Filter
9. Multimode Fiber Optic 10. Photo Detector
11. Amplifier 12. Data Acquisition Device
13. Excitation light source 14. Remaining components
15. Data acquisition device 16. Low pass filter

Claims

An excitation light generator for generating excitation light;
A fluorescent photon collector for collecting fluorescent photons generated by irradiating the excitation light to the sample;
A light detector for amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electrical signal; And
An average time of the first electrical signal calculated based on a first reference signal and an average of the second electrical signal converted by the light sensing unit without the sample being excited by the excitation light source calculated based on the second reference signal Fluorescence Life Extraction Unit Calculates Fluorescence Lifetime Using Time Difference
Fluorescence lifetime measurement apparatus comprising a.

The method of claim 1,
The fluorescence lifetime extracting unit includes a signal collection unit for collecting the first reference signal and the first electrical signal at the same time, and collecting the second reference signal and the second electrical signal at the same time. .

The method according to claim 1 or 2,
Wherein each of the first reference signal and the second reference signal is a pulse signal synchronized with an excitation light associated with the first electrical signal and the second electrical signal.

The method of claim 3, wherein
And each of the first reference signal and the second reference signal is generated from a trigger signal associated with the first electrical signal and the second electrical signal.

The method of claim 2,
The signal collector may include a first channel collecting the first electrical signal and the second electrical signal, and a second channel different from the first channel collecting the first reference signal and the second reference signal. Fluorescence lifetime measuring apparatus, characterized in that.

The method of claim 5, wherein
The signal collection unit, the fluorescence lifetime measurement apparatus further comprises a low pass filter for low-pass filtering the first reference signal and the second reference signal.

Generating excitation light to irradiate the sample;
Collecting fluorescent photons generated by irradiating the excitation light to the sample;
Amplifying the collected fluorescent photons and converting them into a first electrical signal; And
An average time of the first electrical signal calculated based on a first reference signal and a second electrical signal converted by the light sensing unit without the sample being excited by the excitation light source calculated based on a second reference signal; Calculating Fluorescence Lifetime Using Mean Time Difference
Fluorescence lifetime measurement method comprising a.

The method of claim 7, wherein
Collecting the first reference signal simultaneously with the first electrical signal and collecting the second reference signal simultaneously with the second electrical signal.

The method according to claim 7 or 8,
And generating each of the first reference signal and the second reference signal from a trigger signal associated with the first electrical signal and the second electrical signal.

The method of claim 9,
The generating of the first reference signal and the second reference signal may further include low-pass filtering each associated trigger signal.

An excitation light generator for generating excitation light;
A fluorescent photon collector for collecting fluorescent photons generated by irradiating the excitation light to the sample;
A light detector for amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electrical signal; And
A fluorescence lifetime extracting unit calculates a fluorescence lifetime by using a difference between the average time of the first electrical signal and the average time of the second electrical signal converted by the light detector due to a delay of a part of the excitation light source.
Fluorescence lifetime measurement apparatus comprising a.

The method of claim 11,
And a portion of the excitation light source is collected in a single mode optical fiber, and the fluorescent photon is collected in a multi mode optical fiber.

The method according to claim 11 or 12,
And a portion of the excitation light source is delayed to be obtained within the interval between the first electrical signals.

Generating excitation light to irradiate the sample;
Collecting fluorescent photons generated by irradiating the excitation light to the sample;
Amplifying the collected fluorescent photons and converting the collected fluorescent photons into a first electric signal; And
Calculating a fluorescence lifetime using a difference between the average time of the first electrical signal and the average time of the second electrical signal converted by the light sensing unit due to a delay of a part of the excitation light source;
Fluorescence lifetime measurement method comprising a.

The method of claim 14,
And calculating a difference between the average time of the first electrical signal and the average time of the second electrical signal while the sample is removed, and reflecting the result to the excitation light.