KR20120006790A

KR20120006790A - System and method for computing fluorescence lifetime

Info

Publication number: KR20120006790A
Application number: KR1020100067446A
Authority: KR
Inventors: 원영재; 김덕영
Original assignee: 광주과학기술원
Priority date: 2010-07-13
Filing date: 2010-07-13
Publication date: 2012-01-19
Also published as: KR101147487B1

Abstract

PURPOSE: A system and a method for computing fluorescence lifetime are provided to enable fast dynamic analysis due to high data acquisition speed. CONSTITUTION: A system for computing fluorescence lifetime comprises a filtering part(110), a center measuring part(120), and a fluorescence lifetime calculation part(130). The filtering part filters fluorescence signals and reference signals, respectively. The fluorescence signal and the reference signal pass through a sample. The center measuring part measures the first center of the filtered fluorescence signal and the second center of the filtered reference signal. The fluorescence lifetime calculation part compares the first center and the second center to calculate the fluorescent lifetime using time difference.

Description

Fluorescence lifetime calculation system and its method {System and method for computing fluorescence lifetime}

The present invention relates to a system and method for calculating fluorescence lifetime. More particularly, the present invention relates to a fluorescence lifetime calculation system and method for fluorescence lifetime imaging.

Conventional fluorescence lifetime measurement using an analog signal is a method of extracting fluorescence lifetime as a difference between the average delay time of a pulse signal representing a system response function and the average delay time of a distorted fluorescent pulse signal. In this case, in order to calculate an average delay time of two pulse signals, an analog signal is stored in a data storage medium using a data acquisition device and then data processed. Data processing for fluorescence lifetime imaging includes data sharing for imaging and calculation of average delay time for fluorescence lifetime extraction. When data is processed by pre-save post-processing, the data processing time takes at least tens of seconds to several minutes, making real-time fluorescence lifetime imaging difficult.

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and calculates a fluorescence lifetime using the difference between the center point of the pulse signal representing the system device response function and the center point of the fluorescent pulse signal distorted by the system device response function. It is an object of the present invention to provide a system and a method thereof.

The present invention has been made in order to achieve the above object, the filtering unit for filtering each of the fluorescent signal and the reference signal passing through the sample; A center point measuring unit measuring a first center point of the filtered fluorescent signal and a second center point of the filtered reference signal; And a fluorescence lifetime calculation unit for calculating fluorescence lifetime using a time difference obtained by comparing the first center point and the second center point.

Preferably, the fluorescence lifetime calculator calculates the time difference, and includes a time difference calculator that calculates a time difference between two signals when the signal by the first center point and the signal by the second center point are input to different points; And a voltage intensity output unit configured to output the time difference as a voltage intensity. More preferably, the fluorescence lifetime calculation unit calculates the fluorescence lifetime, and converts the voltage intensity in the form of an analog signal into a digital signal; A storage unit which stores the converted digital signal as data; An imaging unit for imaging the stored data; And a calculator configured to calculate the fluorescence lifetime by applying a predetermined calibration factor to a difference value between the first center point and the second center point during the imaging. Still more preferably, the fluorescence lifetime calculation unit calculates the fluorescence lifetime, and further includes a synchronization unit for synchronizing the data storage address where the data is stored with the scanning mirror driving circuit.

Preferably, the center point measuring unit may include: a derivative unit for differentiating the filtered fluorescence signal to obtain a first differential value and differentiating the filtered reference signal to obtain a second differential value; And a zero crossing discrimination unit configured to measure the first center point and the second center point by distinguishing zero crossings from the first derivative value and the second derivative value, respectively.

Preferably, the fluorescence lifetime calculation system is to obtain the fluorescence signal, and to detect the fluorescence signal causing portion for causing the fluorescence signal by irradiating excitation light by a light source to the sample, and detecting the caused fluorescence signal. A first signal acquisition unit including a fluorescence signal detection unit; And a second signal obtaining unit that obtains the reference signal and includes a reference signal detecting unit detecting the reference signal from the excitation light that has not passed through the sample. More preferably, in consideration of the time taken until the fluorescence signal is acquired, the second signal acquisition unit delays the time taken for the excitation light not passing through the sample to be detected as the reference signal, The apparatus further includes a time delay unit that delays the time taken until the detected reference signal is filtered in consideration of the time taken until the fluorescent signal is filtered. Alternatively, the fluorescence lifetime calculation system further includes a light distribution unit for distributing excitation light by the light source to the first signal acquisition unit and the second signal acquisition unit, respectively.

Preferably, the filtering unit uses a Gaussian low pass filter to filter the fluorescent signal and the reference signal.

In addition, the present invention comprises the steps of filtering each of the fluorescence signal and the reference signal passing through the sample; (b) measuring a first center point of the filtered fluorescence signal and a second center point of the filtered reference signal; And (c) calculating a fluorescence lifetime using the time difference obtained by comparing the first center point and the second center point.

Preferably, the step (c) comprises: (ca) calculating a time difference between the two signals when the signal by the first center point and the signal by the second center point are input to different points; (cb) outputting the time difference at a voltage intensity; (cc) converting the voltage strength in the form of an analog signal into a digital signal; (cd) storing the converted digital signal as data; (ce) imaging the stored data; And (cf) calculating the fluorescence lifetime by applying a predetermined calibration factor to a difference value between the first center point and the second center point during the imaging. More preferably, as an intermediate step between the step (cd) and the step (ce), synchronizing the data storage address where the data is stored with the scanning mirror driving circuit.

Preferably, the step (b) comprises: (ba) differentiating the filtered fluorescence signal to obtain a first derivative value, and differentiating the filtered reference signal to obtain a second derivative value; And (bb) measuring the first center point and the second center point by distinguishing zero crossings from the first derivative value and the second derivative value, respectively.

Preferably, before the step (a), the step of irradiating the sample with excitation light by a light source to cause the fluorescent signal; And (a ') detecting the reference signal from the excitation light that has not passed through the sample. More preferably, as a previous step of the step (a '), in consideration of the time taken until the fluorescence signal is acquired, the time taken for the excitation light not passing through the sample to be detected as the reference signal is delayed. Delaying the time taken until the detected reference signal is filtered in consideration of the time taken until the fluorescence signal is filtered as a step between (a ') and (a). It comprises the step of.

The present invention can obtain the following effects by calculating the fluorescence lifetime value by using the difference between the center point of the system device response pulse signal and the fluorescent pulse signal. First, it enables real-time ultrafast fluorescence lifetime imaging, which was difficult to perform with conventional methods. Second, the fluorescence lifetime can be extracted directly from the analog signal containing the system device response function, resulting in faster data acquisition and faster dynamic phenomena analysis. Third, the fluorescence lifetime calculation formula can be simplified, and the configuration of the imaging circuit can be easily made using an analog circuit.

1 is a block diagram schematically illustrating a fluorescence lifetime calculation system according to a preferred embodiment of the present invention.
Figure 2 is an exemplary view of a fluorescence lifetime calculation system according to a preferred embodiment of the present invention.
3 is a graph for explaining a fluorescence lifetime measurement method according to the present embodiment.
4 is a graph showing the difference between the center point of the fluorescence pulse and the center point of the system device response function according to the fluorescence lifetime.
5 is a flowchart illustrating a fluorescence lifetime calculation method according to a preferred embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. First, in adding reference numerals to the components of each drawing, it should be noted that the same reference numerals are assigned to the same components as much as possible even if displayed on different drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. In addition, the following will describe a preferred embodiment of the present invention, but the technical idea of the present invention is not limited thereto and may be variously modified and modified by those skilled in the art.

1 is a block diagram schematically illustrating a fluorescence lifetime calculation system according to a preferred embodiment of the present invention. According to FIG. 1, the fluorescence lifetime calculation system 100 according to the present exemplary embodiment includes a filtering unit 110, a center point measurement unit 120, a fluorescence lifetime calculation unit 130, and a main control unit 140.

The fluorescence lifetime calculation system 100 extracts fluorescence lifetime for real-time fluorescence lifetime imaging, and extracts fluorescence lifetime as the difference between the peaks of two pulse signals. In this embodiment, a fluorescence lifetime calculation method for extracting fluorescence lifetime values using the center point difference between the system device response pulse signal and the fluorescence pulse signal through the fluorescence lifetime calculation system 100 is proposed. This simplifies the data processing procedure for fluorescence lifetime extraction, enabling the construction of an analog circuit for fluorescence lifetime extraction. In addition, since the extracted fluorescence lifetime value is directly stored in a data storage medium, data processing speed for imaging is finally increased, thereby enabling real-time fluorescence lifetime imaging.

The filtering unit 110 performs a function of filtering the fluorescence signal and the reference signal that have passed through the sample, respectively. The filtering unit 110 uses a Gaussian low pass filter to filter the fluorescent signal and the reference signal.

The center point measurer 120 measures a first center point of the filtered fluorescent signal and a second center point of the filtered reference signal.

The center point measurer 120 includes a derivative 121 and a zero crossing distinguisher 122. The derivative unit 121 performs a function of obtaining a first derivative value by differentiating the filtered fluorescent signal and a second derivative value by differentiating the filtered reference signal. The zero crossing distinguishing unit 122 performs a function of measuring the first center point and the second center point by distinguishing the zero crossing from the first derivative value and the second derivative value, respectively.

The fluorescence lifetime calculator 130 calculates a fluorescence lifetime using a time difference obtained by comparing the first center point and the second center point.

The fluorescence lifetime calculator 130 calculates a time difference, and includes a time difference calculator 131 and a voltage intensity output unit 132. The time difference calculator 131 calculates a time difference between the two signals when the signal by the first center point and the signal by the second center point are input to different points. The voltage strength output unit 132 outputs the time difference as voltage strength.

The fluorescence lifetime calculator 130 calculates a fluorescence lifetime and includes a converter 133, a storage 134, an imaging unit 135, and a calculator 136. The converter 133 performs a function of converting the voltage strength in the form of an analog signal into a digital signal. The storage unit 134 stores the converted digital signal as data. The imaging unit 135 performs a function of imaging the stored data. The calculator 136 calculates a fluorescence lifetime by applying a predetermined calibration factor to the difference between the first center point and the second center point at the time of imaging.

The fluorescence lifetime calculator 130 may calculate a fluorescence lifetime and may further include a synchronization unit 137. The synchronization unit 137 synchronizes the data storage address in which data is stored with the scanning mirror driving circuit.

The main controller 140 controls the overall operation of each unit constituting the fluorescence lifetime calculation system 100.

The fluorescence lifetime calculation system 100 may further include a first signal acquirer 150, a second signal acquirer 160, and a light distributor 170.

The first signal acquisition unit 150 acquires a fluorescence signal, and includes a fluorescence signal generating unit 151 and a fluorescence signal detection unit 152. The fluorescence signal generating unit 151 performs a function of causing a fluorescence signal by irradiating excitation light by the light source to the sample. The fluorescence signal detector 152 performs a function of detecting the caused fluorescence signal.

The second signal acquirer 160 acquires a reference signal and includes a reference signal detector 161. The reference signal detector 161 detects the reference signal from the excitation light that has not passed through the sample.

The second signal acquisition unit 160 may further include a time delay unit 162. The time delay unit 162 delays the time taken for the excitation light not passing through the sample to be detected as the reference signal in consideration of the time taken until the fluorescent signal is acquired, or the time taken until the fluorescent signal is filtered. In consideration of this, it performs a function of delaying the time taken until the detected reference signal is filtered.

The light distribution unit 170 distributes the excitation light by the light source to the first signal acquisition unit 150 and the second signal acquisition unit 160, respectively.

Next, the fluorescence lifetime calculation system 100 will be implemented as an example. Figure 2 is an exemplary view of a fluorescence lifetime calculation system according to a preferred embodiment of the present invention.

The fluorescence lifetime calculation system 100 is for ultrafast fluorescence lifetime imaging. The fluorescence lifetime calculation system 100 is a method for obtaining fluorescence lifetime using a difference between a center point of a pulse signal representing a system device response function and a center point of a fluorescence pulse signal distorted by the system device response function. will be. Fluorescent signals with periodic functional characteristics are convolved with the system device response function and collected as distorted signals. At this time, the highest point of the pulse is shifted to the right from the system device response function as the fluorescence lifetime value is larger. The peak shift becomes larger as the system device response function is wider, which has a low cutoff frequency after the photodetector, and the device response function is Gaussian with respect to time. -It can be realized by using Pass Filter. Although it does not change linearly according to the fluorescence lifetime value, it is possible to extract the fluorescence lifetime value for the center point travel time by applying a calibration factor. When the fluorescence lifetime calculation system 100 is applied, the fluorescence lifetime can be directly extracted from an analog signal including a system device response function, thereby enabling fast dynamic phenomenon analysis due to a high data collection speed. In addition, the fluorescence lifetime calculation is also simplified, allowing analog circuits to easily organize imaging circuits, enabling real-time monitoring of fluorescence lifetime images.

3 is a graph for explaining a fluorescence lifetime measurement method according to the present embodiment. In FIG. 3, reference numeral 300 denotes a system device response function, and reference numeral 310 denotes a fluorescence pulse of a fluorescent material having a fluorescence lifetime of 2 ns. Reference numeral 320 denotes a fluorescence pulse of the fluorescent material having a fluorescence lifetime of 6 ns. As shown in FIG. 3, the longer the width of the system device response function is, the more the peak of the fluorescence pulse is shifted to the right than the peak of the system device response function. The width of the system unit response function can be widened using a Gaussian low pass filter, in which case the fluorescence pulse is shifted to the right while maintaining its Gaussian shape, which is the system unit response function. As the movement width of the center point along the fluorescence lifetime length becomes larger, the fluorescence lifetime measurement by the center point shift value becomes more accurate. In addition, since the frequency component of the signal is lowered, the use of expensive equipment for high frequency signal processing is not required, and equipment selection is more free.

4 shows the difference between the center point of the fluorescence pulse and the center point of the system device response function according to the fluorescence lifetime. In FIG. 4, the horizontal direction value and the vertical direction value represent the fluorescence lifetime and the center point difference, respectively. Reference numeral 400 denotes an ideal case, and 410 denotes a case where the median width of the system device response function is 2.5 ns. Reference numeral 420 and 430 denote the case where the median width of the system device response function is 24ns and 42ns, respectively. The wider the median width of the system device response function, the closer to the ideal case. Therefore, if the calibration factor between the fluorescence lifetime and the center point difference is calculated in consideration of this, the fluorescence lifetime value can be easily extracted from the measured center point difference.

Fluorescence lifetime extraction method using the correction factor is as follows. The final fluorescence lifetime value to be calculated according to this method is a graph corresponding to 400 of FIG. 4. However, the center point difference value is measured as shown in the graphs 410 to 430 according to the intermediate width thereof. For example, for a fluorescent material having a fluorescence lifetime of 5 ns, the center point difference is measured to be less than 5 ns. By using a glass mirror instead of the fluorescent material, however, the response function 300 of the system in FIG. 3 can be measured. In addition, the ideal fluorescence curves 310 and 320 can be obtained by convolutional operation of this response function and an ideal exponential function (for example, an exponential function having fluorescence lifetime values of 2 ns and 6 ns). From this, as shown in reference numerals 410 to 430 of FIG. 4, the center point difference value can be obtained from the original fluorescence lifetime, and when the center point difference is obtained, the value of the center point difference must be added to correct the original fluorescence lifetime value. You can also determine the value (calibration factor). Accurate fluorescence lifetime can be measured by adding the correction value (correction factor) to the final center point difference.

This embodiment can be executed as shown in FIG. A description with reference to FIG. 2 is as follows.

First, the excitation light source 200 is divided into two, one is incident with the confocal fluorescence microscope 210 setup, and the other is incident with the first light detector 221. The confocal fluorescence microscope 210 has a configuration corresponding to the fluorescence signal generating unit 151 of FIG. 1, and the first light detector 221 has a configuration corresponding to the reference signal detection unit 161 of FIG. 1.

The excitation light source incident on the first photodetector 221 is used as a system device response function for fluorescence signal extraction. In order to divide the excitation light source 200 into two, an optical fiber coupler using an optical fiber or a beam splitter may be used in space. The optical coupler or light splitter is a configuration corresponding to the light splitter 170 of FIG. 1.

The excitation light source incident on the confocal fluorescence microscope 210 causes a fluorescence signal from the sample (sample), and the fluorescence signal is incident on the second light detector 222 through a path different from the incident path of the excitation light source. The second photo detector 222 has a configuration corresponding to the fluorescent signal detector 152 of FIG. 1.

Separate time delay devices may be used to match the timing of the two signals incident to the different photodetectors 221, 222, namely the excitation light source and the fluorescence signal for the system device response function. This is possible by using long optical fibers or by using an electrical time delay device after the light detector. The time delay device is a configuration corresponding to the time delay unit 162 of FIG.

Each signal detected from the photodetector is widened from the Gaussian low pass filters 231 and 232, and is divided into differentiators 241 and 242 and zero crossing discriminator to measure the center point of the two signals. 251, 252). The Gaussian low pass filters 231 and 232 correspond to the filtering unit 110 of FIG. 1, and the differentiators 241 and 242 correspond to the derivative 121 of FIG. 1. The zero crossing discriminator 251, 252 is a configuration corresponding to the zero crossing discriminating unit 122 of FIG. 1.

The output signal of the zero crossing discriminator 251, 252 appears as a short TTL signal at the center of the Gaussian pulse signal. The TTL signal for the system unit response function is input to the start point of the time-to-amplitude converter (TAC) 260 and the TTL signal for the fluorescence signal is input to the stop point of the TAC 260 so that the voltage is equal to the time difference between the two TTL signals. Appears as the strength of. The TAC 260 corresponds to a combination of the time difference calculator 131 and the voltage intensity output unit 132 of FIG. 1.

The strength of the voltage is detected by an analog digital converter 270 and converted into a digital signal, which is stored in the memory 280. The analog-to-digital converter 270 corresponds to the converter 133 of FIG. 1, and the memory 280 corresponds to the storage 134 of FIG. 1.

Synchronizing the address of the stored memory with the scanning mirror driving circuit of the confocal fluorescence microscope 210 may synchronize with the pixels of the image. The scanning mirror driving circuit has a configuration corresponding to the synchronization unit 137 of FIG. 1.

Finally, data stored in memory 280 is imaged using computer 290. At this time, the fluorescence lifetime value is extracted by applying a correction factor to the measured center point difference. Since the data operation is implemented by analog circuit, the processing speed is high, and the computer 290 takes the information from the memory 280 and outputs it to the monitor without any additional calculation work, and finally, the fluorescence lifetime imaging speed is increased. Can be. The computer 290 is a configuration corresponding to the combined configuration of the imaging unit 135 and the calculation unit 136 of FIG. 1.

Next, the fluorescent lifetime calculation method of the fluorescent lifetime calculation system 100 is demonstrated. 5 is a flowchart illustrating a fluorescence lifetime calculation method according to a preferred embodiment of the present invention. The following description refers to FIG. 5.

First, the filtering unit 110 filters the fluorescent signal and the reference signal that have passed through the sample, respectively (S500).

Thereafter, the center point measurer 120 measures the first center point of the filtered fluorescent signal and the second center point of the filtered reference signal (S510). The center point measurement process may be performed as follows in this embodiment.

In a first step, the differential unit 121 differentiates the filtered fluorescence signal to obtain a first differential value, and differentiates the filtered reference signal to obtain a second differential value. The differential unit 121 differentiates both the filtered fluorescent signal and the filtered reference signal until there is a zero crossing. Thereafter, in the second step, the zero crossing distinguishing unit 122 measures the first center point and the second center point by distinguishing the zero crossing from the first derivative value and the second derivative value, respectively.

Thereafter, the fluorescence lifetime calculation unit 130 calculates the fluorescence lifetime using the time difference obtained by comparing the first center point and the second center point (S520). The fluorescence lifetime calculation process may be performed as follows in this embodiment.

In the first step, when the time difference calculator 131 receives the signal by the first center point and the signal by the second center point to different points, calculates the time difference between the two signals. In the second step, the voltage intensity output unit 132 outputs the time difference as the voltage intensity. Then, in the third step, the converter 133 converts the voltage strength in the form of an analog signal into a digital signal. Thereafter, in the fourth step, the storage unit 134 stores the converted digital signal as data. Thereafter, in the fifth step, the imaging unit 135 images the stored data. Thereafter, in the sixth step, the calculator 136 calculates the fluorescence lifetime by applying a predetermined calibration factor to the difference between the first center point and the second center point at the time of imaging. Meanwhile, after storing the digital signal as data, the synchronization unit 137 may synchronize the data storage address where the data is stored with the scanning mirror driving circuit.

Meanwhile, in the present embodiment, the first signal acquirer 150 and the second signal acquirer 160 may obtain the first signal and the second signal, respectively, before filtering the fluorescent signal and the reference signal.

The fluorescence signal acquisition process of the first signal acquisition unit 150 may be performed as follows. First, the fluorescent signal generating unit 151 irradiates a sample with excitation light including fluorescent molecules to cause a fluorescent signal. Thereafter, the fluorescence signal detector 152 detects the generated fluorescence signal.

Meanwhile, the reference signal acquisition process of the second signal acquisition unit 160 is performed by the reference signal detection unit 161. The reference signal detector 161 detects the reference signal from the excitation light that has not passed through the sample.

It is also possible to delay the time before and after acquiring the reference signal so that the fluorescent signal and the reference signal can be simultaneously input to the TAC 260. At this time, the time delay unit 162 delays the time taken for the excitation light not passed through the sample to be detected as the reference signal in consideration of the time taken until the fluorescence signal is obtained. Alternatively, the time delay unit 162 delays the time taken until the detected reference signal is filtered in consideration of the time taken until the fluorescent signal is filtered.

The above description is merely illustrative of the technical idea of the present invention, and various modifications, changes, and substitutions may be made by those skilled in the art without departing from the essential characteristics of the present invention. will be. Accordingly, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical spirit of the present invention but to describe the present invention, and the scope of the technical idea of the present invention is not limited by the embodiments and the accompanying drawings. . The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

Analytical techniques using fluorescence lifetime provide more advanced information than the fluorescence intensity method and have been widely used for quantitative analysis of the amount or acidity of various inorganic ions or oxygen in living cells. Recently, it is widely used in research on important life mechanisms that could not be identified in combination with Fluorescence Resonance Energy Transfer (FRET) technique.

However, the conventional fluorescence lifetime imaging technique is significantly slower, which makes it difficult to analyze fast dynamic phenomena in cells. The present invention overcomes these shortcomings and aims to analyze fast dynamic phenomena in cells by fluorescence lifetime imaging techniques. According to the present invention, it can be very helpful in identifying basic biological mechanisms such as the identification of the correlation between proteins and the correlation between the protein and DNA that have not been revealed. It is also expected to be of great help in the development of innovative new drugs in the pharmaceutical field.

The present invention can be applied to three-dimensional nano light imaging technology, in particular three-dimensional nano light imaging technology using optical tomography and phase microscope technology.

100: fluorescence lifetime calculation system 110: filtering unit
120: center point measuring unit 121: differential part
122: zero crossing discrimination unit 130: fluorescence lifetime calculation unit
131: time difference calculator 132: voltage intensity output unit
133: converter 134: storage unit
135: imaging unit 136: calculation unit
137: synchronization unit 140: main control unit
150: first signal acquisition unit 151: fluorescent signal causing unit
152: fluorescent signal detection unit 160: second signal acquisition unit
161: reference signal detector 162: time delay unit
170: light distribution unit

Claims

A filtering unit for filtering the fluorescent signal and the reference signal passing through the sample, respectively;
A center point measuring unit measuring a first center point of the filtered fluorescent signal and a second center point of the filtered reference signal; And
A fluorescence lifetime calculator for calculating fluorescence lifetime using a time difference obtained by comparing the first center point and the second center point.
Fluorescence lifetime calculation system comprising a.

The method of claim 1,
The fluorescence lifetime calculator calculates the time difference,
A time difference calculator configured to calculate a time difference between the two signals when the signal by the first center point and the signal by the second center point are input to different points; And
Voltage intensity output unit for outputting the time difference in voltage intensity
Fluorescence lifetime calculation system comprising a.

The method of claim 2,
The fluorescence lifetime calculation unit calculates the fluorescence lifetime,
A converter for converting the voltage strength in the form of an analog signal into a digital signal;
A storage unit which stores the converted digital signal as data;
An imaging unit for imaging the stored data; And
A calculator for calculating the fluorescence lifetime by applying a predetermined calibration factor to the difference between the first center point and the second center point during the imaging.
Fluorescence lifetime calculation system comprising a.

The method of claim 3, wherein
The fluorescence lifetime calculation unit calculates the fluorescence lifetime,
A synchronization unit for synchronizing the data storage address where the data is stored with the scanning mirror driving circuit
Fluorescence lifetime calculation system characterized in that it further comprises.

The method of claim 1,
The center point measuring unit,
A derivative that obtains a first derivative by differentiating the filtered fluorescent signal and a second derivative by differentiating the filtered reference signal; And
A zero crossing distinction unit for measuring the first center point and the second center point by distinguishing zero crossings from the first derivative value and the second derivative value, respectively.
Fluorescence lifetime calculation system comprising a.

The method of claim 1,
Acquiring the fluorescence signal, a first signal acquisition unit including a fluorescence signal generating unit for causing the fluorescence signal by irradiating excitation light by a light source to the sample, and a fluorescence signal detection unit for detecting the induced fluorescence signal ; And
A second signal acquisition unit that obtains the reference signal and includes a reference signal detection unit that detects the reference signal from excitation light that has not passed through the sample
Fluorescence lifetime calculation system further comprises.

The method according to claim 6,
The second signal acquisition unit,
In consideration of the time taken until the fluorescence signal is acquired, the time taken for the excitation light not passing through the sample to be detected as the reference signal is delayed or in consideration of the time taken until the fluorescence signal is filtered. A time delay unit for delaying the time taken until the detected reference signal is filtered
Fluorescence lifetime calculation system further comprises.

The method according to claim 6,
An optical distribution unit configured to distribute excitation light by a light source to the first signal acquisition unit and the second signal acquisition unit, respectively
Fluorescence lifetime calculation system further comprises.

The method of claim 1,
And the filtering unit uses a Gaussian low pass filter to filter the fluorescence signal and the reference signal.

(a) filtering the fluorescence signal and the reference signal respectively through the sample;
(b) measuring a first center point of the filtered fluorescence signal and a second center point of the filtered reference signal; And
(c) calculating a fluorescence lifetime using a time difference obtained by comparing the first center point and the second center point
Fluorescence lifetime calculation method comprising a.

The method of claim 10,
In step (c),
(ca) calculating a time difference between the two signals when the signal by the first center point and the signal by the second center point are input to different points;
(cb) outputting the time difference at a voltage intensity;
(cc) converting the voltage strength in the form of an analog signal into a digital signal;
(cd) storing the converted digital signal as data;
(ce) imaging the stored data; And
(cf) calculating the fluorescence lifetime by applying a predetermined calibration factor to the difference between the first center point and the second center point during the imaging;
Fluorescence lifetime calculation method comprising a.

The method of claim 11,
As an intermediate step between the (cd) step and the (ce) step,
Synchronizing a data storage address storing the data with a scanning mirror driving circuit;
Fluorescence lifetime calculation method comprising a.

The method of claim 10,
In step (b),
(ba) differentiating the filtered fluorescence signal to obtain a first derivative value, and differentiating the filtered reference signal to obtain a second derivative value; And
(bb) measuring the first center point and the second center point by distinguishing a zero crossing from the first derivative value and the second derivative value, respectively;
Fluorescence lifetime calculation method comprising a.

The method of claim 10,
As a previous step of step (a),
Irradiating the sample with excitation light by a light source to cause the fluorescent signal; And
Detecting the caused fluorescence signal
Contains;
(a ') detecting the reference signal from excitation light that has not passed through the sample
Fluorescence lifetime calculation method comprising a.

The method of claim 14,
As a previous step of the step (a '),
Delaying the time taken for the excitation light not passing through the sample to be detected as the reference signal in consideration of the time taken until the fluorescence signal is obtained;
Contains;
As an intermediate step between the step (a ') and the step (a),
Delaying the time taken until the detected reference signal is filtered in consideration of the time taken until the fluorescent signal is filtered;
Fluorescence lifetime calculation method comprising a.