KR20220106298A - Apparatus and method for in vitro diagnostic device by measuring fluorescence lifetime - Google Patents

Abstract

Disclosed are a method and a device for invitro diagnosis using fluorescence lifetime. An embodiment of the device comprises: a light generation unit generating excite light; a control unit controlling a path of the excite light; a detection unit outputting a first signal and a second signal having a plurality of second signal level ranges; and a signal processing unit changing the plurality of second signal level ranges to enable a fluorescence signal of a sample to be a saturation value or less by using the first and second signals. The first signal is a signal converted by detecting the excite light, and the second signal is the signal converted by detecting a fluorescence photon generated by radiating the excite light to the sample.

Description

In vitro diagnostic method and device using fluorescence lifetime

The embodiment relates to an in vitro diagnostic method and apparatus using fluorescence lifetime.

Molecules absorb incident light by electronic transition, and in some molecules, excited electrons recover to a ground state, causing a fluorescence phenomenon that emits light. In addition, each molecule has a unique absorption wavelength and fluorescence emission wavelength, and there is a difference in wavelength of several tens of nanometers, also called Stoke's shift, between them. That is, light with a wavelength slightly longer than the wavelength of the absorbed light is emitted through the fluorescence phenomenon. Fluorescent materials can be characterized by the absorption and emission wavelengths of light, and these properties become the principles of classical fluorescence microscopy.

In a fluorescence microscope, excitation light of a wavelength to be absorbed by a target fluorescent material is irradiated to a sample, and a Stokes-shifted fluorescence signal with a wavelength longer than this excitation light is collected at a specific point of the sample by film or CCD (Charge- An image is obtained by detecting using a photo-detector of an array type such as a coupled device.

In the application of fluorescence microscopy, an image is obtained by either the sample itself contains a molecule having a specific fluorescence (autofluorescence) or by injecting a fluorescent substance from the outside and labeling a specific site, mainly biological samples such as cells. It is used to study the molecular distribution of Recently, a confocal microscope, a multi-photon excitation fluorescence microscope, and the like have been developed to obtain a three-dimensional image.

In contrast to conventional fluorescence microscopes that only compose images based on the intensity of fluorescence light emitted from a fluorescent material, methods for composing images by collecting more advanced spectroscopic information other than the intensity of fluorescence light have been recently developed and have. In particular, information on fluorescence lifetime, which is an intrinsic optical property of a fluorescent material, is important because it provides more detailed information about the environment in which the fluorescent material is placed. In a fluorescent molecule, after being excited by an excitation light, electrons stay in an excited state for a certain period of time and then probabilistically transition to the ground state, and at this time, a fluorescent photon is generated. The transition probability of electrons in time, that is, the generation probability of fluorescent photons, draws an exponential decay curve with the peak at the time when the electrons are excited. The characteristic decay time of this exponential decay curve is called the fluorescence lifetime, and it can be measured by examining the generation time of a large number of fluorescence photons.

The original fluorescence lifetime is a characteristic value of each fluorescent molecule if there is no external interference, but the fluorescence lifetime may vary depending on the surrounding environment in which the fluorescent material is placed. That is, the value changes depending on the concentration of a specific ion, oxygen concentration, acidity (pH), etc. according to the characteristics of each fluorescent material. Accordingly, a fluorescence lifetime imaging microscope (FLIM), which is a method of examining the spatial distribution of the above-described variables through the information on the fluorescence lifetime, is being developed. Such a fluorescence lifetime imaging microscope has the strength to accurately obtain environmental information that could not be obtained or inaccurate through a conventional fluorescence microscope. In addition, confocal FLIM combined with confocal microscopy makes it possible to obtain information based on the fluorescence lifetime at a three-dimensional resolution, enabling so-called “four-dimensional imaging”.

On the other hand, in a confocal microscope including a multiphoton excitation fluorescence microscope, a measurement is made at a point in space every moment and the measurement point is scanned, whereby image information is sequentially acquired. In a confocal microscope, measurement is made at the focus of the objective lens of the microscope, and this focus is spatially scanned by the direction of the beam incident on the objective lens or the movement of the sample itself. And, in the case of a confocal microscope, it has a spatial filter such as a pinhole so that only light re-incident from the focal point to the objective lens can reach the sensing unit. In the case of multiphoton excitation fluorescence microscopy, the same effect can be achieved without a pinhole, thanks to the fact that multiphoton excitation is a nonlinear optical phenomenon that only effectively occurs at a focus with high light intensity.

Because of these characteristics of confocal microscopy, FLIM's fluorescence lifetime measuring device based on confocal microscopy is not significantly different from that of time-resolved spectroscopy. In classical time-resolved spectroscopy, a time-correlated single photon counting (TCSPC) or phase fluorometer is mainly used for measurement of fluorescence lifetime.

In addition, the measurement of the fluorescence lifetime may be performed by targeting a plurality of photons generated by a plurality of fluorescent molecules having the same fluorescence lifetime or a plurality of photons generated by excitation of a single fluorescent molecule multiple times. Conceptually, this is the process of analyzing a fluorescence waveform with an exponential decay shape on the time axis. If near-infinite fluorescence photons were collected, the resulting fluorescence waveform would be the same as the probability distribution function of fluorescence with an exponential decay shape. When excitation light irradiation with a very short pulse width is performed at time t=0, the intensity of the generated fluorescence light or the density of fluorescence photons, IF(t), has a distribution of . In this case, I0 is the initial value, τ means the fluorescence lifetime, and the u(t) function represents a step function of 0 when t<0 and 1 when t≥0. That is, the fluorescence lifetime means the time during which the emission probability of a fluorescence photon decreases by 1/e compared to the initial value.

In addition, the TCSPC detects a response by a single photon using a sensing unit having a high signal gain, such as a PMT or an avalanche photo diode (APD). Regardless of how long the shape of the response pulse by a single photon has on the time axis, the arrival time of a single photon can be precisely measured. Using this, it is possible to measure the fluorescence lifetime at the level of 0.1 nanoseconds.

However, due to the "single photon condition", there is a problem in time that the measurement of the fluorescence lifetime in TCSPC is completed only after multiple excitation light pulses are incident and photon counts are performed. In addition, the time interval between excitation light pulses is There is a limit in that it must be sufficiently longer than the fluorescence lifetime of the fluorescent material. If the time interval between the excitation light pulses becomes similar to the fluorescence lifetime, there is a problem in that the waveforms of two adjacent fluorescence emission in time overlap each other and an accurate value cannot be obtained. There is a limiting condition that must be more than 5 times the fluorescence lifetime τ. That is, even under the most ideal conditions, a very long measurement time is required to obtain an image by fluorescence lifetime measurement using TCSPC, and in particular, 3D imaging requires almost an hour or more. This processing time is a big limitation when examining a specific substance in a living organism with dynamic activity or in vitro.

Furthermore, there is a problem in that it is difficult to accurately calculate a fluorescence signal or a fluorescence lifetime because there are various fluorescence photons generated according to the state and region of the organism or sample.

The embodiment provides an in vitro diagnostic method and apparatus using a fluorescence lifetime that can accurately measure a fluorescence signal and a fluorescence lifetime from fluorescence photons generated in various sizes according to the state and region of a sample in a faster time.

In addition, the present invention provides an in vitro diagnostic method and apparatus using a fluorescence lifetime suitable for a fluorescence lifetime imaging microscope that constructs an accurate image based on the spatial distribution of the measured fluorescence lifetime.

The problem to be solved in the embodiment is not limited thereto, and it will be said that the purpose or effect that can be grasped from the solving means or embodiment of the problem described below is also included.

An in vitro diagnostic apparatus using a fluorescence lifetime according to an embodiment includes: a light generator for generating excitation light; a control unit for controlling a path of the excitation light; a sensing unit outputting a first signal and a second signal having a plurality of second signal level ranges; and a signal processing unit configured to change a range of the plurality of second signal levels so that a fluorescence signal of a sample becomes less than or equal to a saturation value by using the first signal and the second signal, wherein the first signal emits the excitation light A signal converted by sensing, and the second signal is a signal converted by detecting a fluorescence photon generated when the excitation light is irradiated to the sample.

The first signal may have a plurality of first signal level ranges, and the signal processing unit may calculate the fluorescent signal of the sample using the same first signal level range and the second signal level range.

The fluorescent signal of the sample may have a maximum value, and the signal processing unit may change the range of the second signal level such that a difference between the maximum value and the saturation value is smallest.

The controller may adjust the excitation light into first and second excitation light branches that are branched through different paths.

The first signal may be generated by the first excitation light, and the second signal may be generated by the second excitation light.

It may further include a shutter unit that blocks the first excitation light while sensing the fluorescent photon.

The shutter unit may block the fluorescent photon while receiving the first excitation light.

The signal processing unit may calculate the fluorescence lifetime of the sample by using a difference between the average time of the first signal and the average time of the second signal.

The signal processing unit may change the range of the second signal level for each area of the sample.

The signal processing unit may calculate the fluorescence signal of the sample or the fluorescence lifetime of the sample by using a second signal having a different second signal level range for each area of the sample.

According to the embodiment, there is provided an in vitro diagnostic method and apparatus using fluorescence lifetime that can measure the fluorescence signal and fluorescence lifetime from fluorescence photons generated in various sizes according to the state and region of the sample in a faster time with accuracy and precision. can be implemented

In addition, it is possible to implement an in vitro diagnostic method and apparatus using a fluorescence lifetime suitable for a fluorescence lifetime imaging microscope and the like.

Various and advantageous advantages and effects of the present invention are not limited to the above, and will be more easily understood in the course of describing specific embodiments of the present invention.

1 is a block diagram of an in vitro diagnostic apparatus using fluorescence lifetime according to an embodiment of the present invention;
2 is a view for explaining an operation of a signal processing unit according to an embodiment of the present invention;
3 is a view for explaining a first signal and a second signal of a sensing unit according to an embodiment of the present invention;
4 is a diagram illustrating a first signal having a plurality of first signal levels according to an embodiment of the present invention;
5 is a diagram illustrating a second signal having a plurality of second signal levels according to an embodiment of the present invention;
6 is an exemplary view before changing the range of the second signal level in the in vitro diagnostic apparatus using the fluorescence lifetime according to an embodiment of the present invention;
7 is an exemplary view showing after changing the range of the second signal level in the in vitro diagnostic apparatus using the fluorescence lifetime according to an embodiment of the present invention;
8 is a flowchart of an in vitro diagnostic method using fluorescence lifetime according to an embodiment of the present invention.

Since the present invention may have various changes and may have various embodiments, specific embodiments will be illustrated and described in the drawings. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

Terms including an ordinal number such as second, first, etc. may be used to describe various elements, but the elements are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the second component may be referred to as the first component, and similarly, the first component may also be referred to as the second component. and/or includes a combination of a plurality of related listed items or any of a plurality of related listed items.

When a component is referred to as being “connected” or “connected” to another component, it is understood that the other component may be directly connected or connected to the other component, but other components may exist in between. it should be On the other hand, when it is said that a certain element is "directly connected" or "directly connected" to another element, it should be understood that the other element does not exist in the middle.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Hereinafter, the embodiment will be described in detail with reference to the accompanying drawings, but the same or corresponding components are assigned the same reference numerals regardless of reference numerals, and overlapping descriptions thereof will be omitted.

1 is a block diagram of an in vitro diagnostic apparatus using fluorescence lifetime according to an embodiment of the present invention, FIG. 2 is a diagram for explaining the operation of a signal processing unit according to an embodiment of the present invention, and FIG. 3 is an embodiment of the present invention It is a diagram for explaining the first signal and the second signal of the sensing unit according to FIG.

1 to 3 , the in vitro diagnostic apparatus according to the embodiment may include a light generating unit 100 , a control unit 200 , a sensing unit 300 , a signal processing unit 400 , and a shutter unit 500 . can

First, the light generator 100 is a module that generates excitation light to be irradiated to a sample S containing fluorescent molecules, and collects the excitation light and the excitation light source 110 for generating pulsed excitation light. It consists of a first lens 120 for irradiating the sample (S).

The controller 200 may adjust the path of the excitation light emitted from the light source 110 . For example, the controller 200 may branch the excitation light into different paths. The controller 200 may branch the excitation light into a first excitation light and a second excitation light according to a path. The first excitation light may travel through a first path. The second excitation light may travel through a second path.

In an embodiment, the first excitation light may be provided to the sensing unit 300 without passing through the sample S. The second excitation light may be provided to the sample S. In addition, fluorescent photons may be generated in the sample S by the second excitation light. The generated fluorescence photons may be provided to the sensing unit 300 . However, the second path includes a movement path of a fluorescent photon generated by the second excitation light. Furthermore, a first signal, which is an electrical signal generated by the sensing unit by the first excitation light, is also shown to correspond to the first path, and a second signal which is an electrical signal generated by the sensing unit by the second excitation light (or fluorescent photon) is shown. A signal is also shown corresponding to the second path.

The sensing unit 300 may collect a plurality of fluorescence photons and excitation light generated by irradiating the sample S with excitation light.

In an embodiment, the sensing unit 300 may include a plurality of photodetection elements. As the sensing unit 300 , for example, a photo-multiplier tube (PMT) or an avalanche photo diode (APD) may be used.

Furthermore, the sensing unit 300 may include at least one sensing unit 300 and the sensing unit 300 . Hereinafter, one sensing unit will be described, and thus, in the in vitro diagnostic apparatus according to the embodiment, it is possible to minimize the variation of the intrinsic response function by the plurality of sensing units.

The sensing unit 300 may convert the excitation light that has not passed through the sample into a first signal that is an electrical signal. The sensing unit 300 may adjust the magnification of the first signal by the power and voltage applied to the sensing unit.

Also, the first signal may have a plurality of first signal level ranges. The plurality of first signal level ranges may be adjusted by power and voltage applied to the sensing unit by adjusting the magnification of the first signal. For example, the first signal may have a voltage level range. In addition, the plurality of first signal level ranges may be 0 to 1 [V], 0 to 3 [V], 0 to 5 [V], and the like.

The first signal may be output before and after outputting a second signal to be described later. For example, a plurality of first signals may be output before the second signal is output by the sensing unit 300 . The first signals having different first signal level ranges may be output by excitation light having different phases or time-shifted applied from the light source.

The sensing unit 300 may receive a fluorescence photon generated when the excitation light is irradiated to the sample, and convert the received fluorescence photon into a second signal that is an electrical signal.

The sensing unit 300 may convert fluorescent photons collected by a predetermined lens to be described later and passed through a filter (not shown) into an electrical signal (corresponding to the second signal). In this case, the magnification (eg, amplification) of the second signal may be adjusted by the applied power.

Also, the second signal may have a plurality of second signal level ranges. Similarly, the plurality of second signal level ranges may be adjusted by power and voltage applied to the sensing unit by adjusting the magnification of the second signal. For example, the second signal may have a voltage level range. In addition, the plurality of second signal level ranges may be 0 to 1 [V], 0 to 3 [V], 0 to 5 [V], and the like. Also, the second signal level range may be the same as or different from the first signal level range. For example, the second signal level range may be greater than the first signal level range. With this configuration, it is possible to easily measure fluorescence photons generated in different amounts depending on the state, region, and the like of the sample.

In addition, the sensing unit 300 may further include a lens, a filter, and the like. The lens may collect fluorescence photons from the sample S. And the filter can remove the excitation light. Accordingly, accuracy in collecting fluorescent photons can be improved.

The signal processing unit 400 may calculate a fluorescence signal and a fluorescence lifetime by using the first signal and the second signal converted by the sensing unit 300 . For example, the signal processing unit 400 may digitize or quantize the second signal output from the sensing unit 300 and then calculate the fluorescence signal function as a graph with respect to time or a time domain.

In addition, the signal processing unit may change the plurality of second signal level ranges by using the first signal and the second signal so that the fluorescent signal or the fluorescent signal function of the sample is equal to or less than a saturation value.

Accordingly, the signal processing unit 400 may adjust the range of the second signal level of the second signal to calculate an accurate fluorescent signal function without affecting the intensity of the fluorescent photon.

Furthermore, the signal processing unit 400 may digitize or quantize the first signal output from the sensing unit 300 and then calculate the device response function as a graph with respect to time or a time domain. In addition, the signal processing unit 400 may calculate the fluorescence lifetime using the device response function and the fluorescence signal function.

Also, the shutter unit 500 may include a plurality of shutters. For example, the shutter unit 500 may include a first shutter 510 and a second shutter 520 . The second shutter 520 may be located at the front end of the sensing unit, and the second shutter 520 may be located at the front end of the sample S. Each shutter of the shutter unit 500 may block or pass light by partially turning on/off or opening/closing a region.

Accordingly, the shutter unit 500 may block the first excitation light while sensing the fluorescent photon through the first shutter 510 . Also, the shutter unit 500 may block fluorescent photons while receiving the first excitation light through the second shutter 520 .

Additionally, the in vitro diagnostic apparatus using fluorescence lifetime may further include a detector 300 at the front end of the signal processor 400 and a signal measuring unit measuring the electrical signal converted by the sensing unit 300 . The signal measurement unit (not shown) may be located inside/outside the signal processing unit 400 . For example, an oscilloscope may be used as the signal measuring unit (not shown).

The signal processing unit 400 may calculate the fluorescence lifetime by using a difference between the average time of the first signal and the average time of the second signal measured by a signal measuring unit (not shown). In this case, since the above-described digitized first signal and second signal are used, the accuracy of the device response function and the fluorescence signal function may be different depending on the signal level range. Since the signal processing unit 400 according to the embodiment changes the range of the second signal level so that the difference between the maximum value and the saturation value of the fluorescent signal function is smallest, it is possible to calculate a more accurate fluorescent signal function and furthermore, to calculate an accurate fluorescent lifespan. can

Accordingly, the first signal corresponds to or corresponds to I _M (t), the average time of I _M (t) corresponds to an average delay time to be described later, and the second signal corresponds to a device response function I _IRF '(t) to be described later. Corresponds to or corresponds to IRF, and the average time of _IRF '(t) corresponds to the device delay time to be described later. In this case, the first signal may also have a device delay time of I _IRF (t). And, according to an embodiment, since the sensing unit 300 is the same component and has different environmental factors according to the path difference of the response characteristics, some response characteristics may be different. Alternatively, when a plurality of sensing units are configured, response characteristics may be different as different components. In this case, the device response functions are also different. In other words, in the case of a plurality of sensing units, the device response function I _IRF (t) of the first signal and the device response function I _IRF '(t) of the second signal are different from each other.

Hereinafter, the measuring principle of the fluorescence lifetime according to the present invention will be described.

First, the in vitro diagnostic apparatus according to the embodiment may inject excitation light in a short pulse form into a sample in order to measure or calculate the light lifetime, and measure a time waveform of emitted fluorescence through a high-speed sensing unit.

Accordingly, the light source 110 may generate excitation light having a short pulse width. For example, the light source 110 may be formed of a pulsed laser.

Since the intensity of fluorescence and the intensity of excitation light are small, the detector 300 and the detector 300 may be formed of a photo-multiplier tube (PMT) or an avalanche photo diode (APD) having a relatively large signal amplification capability.

[Equation 1]

" 기호는 컨볼루션을 나타내며, I_M(t)는 감지부에서 얻어지는 파형(이하 '형광신호 함수' 또는 '형광신호'와 혼용함)으로 특히 제2 여기광에 대한 신호 또는 제2 여기광에 의해 시료로부터 발생된 형광광자에 대한 신호나 파형이고, I_E(t)는 여기광(예, 제1 여기광)의 파형으로 제1 여기광에 대한 신호 또는 파형이고, I_P(t)는 감지부의 임펄스 응답(impulse response) 파형이고, T₀는 여기광이 광원에서 제1 렌즈를 거쳐 감지부에 도착하는데 걸리는 거리에 따른 지연 시간을 나타낸다. 이러한 지연 시간은 형광 현상과 무관한 장치 고유 특성(응답 특성과 혼용) 값으로 사전에 측정될 수 있을 것이다. 본 명세서에서, 형광신호 함수는 디지털화된 제2 함수를 시간 영역 또는 시간에 대한 함수(또는 그래프)로서 아날로그화된 함수이고, 장치 응답 함수는 제1 함수를 시간 영역 또는 시간에 대한 함수(또는 그래프)로서 아날로그화된 함수이다. 본 명세서에서는 제1,2 신호를 디지털화한 후 시간 영역 또는 시간에 대한 함수로서 아날로그화한 함수(예, 형광신호 함수 또는 장치 응답 함수)를 디지털화 및 아날로그화로 도시한다. 나아가, 형광신호 함수가 디스플레이부(미도시됨)로 표시되며, 산출된 형광 수명도 디스플레이부를 통해 시료의 각 영역에 대해 도시되거나 정보 제공될 수 있다.here, '"

" symbol indicates convolution, and I _M (t) is a waveform obtained from the sensing unit (hereinafter, mixed with 'fluorescence signal function' or 'fluorescence signal'), especially the signal for the second excitation light or the second excitation light. is a signal or waveform for a _{fluorescence photon} generated from a sample by the It is an impulse response waveform of the detector, and T ₀ represents a delay time according to the distance it takes for the excitation light to arrive at the detector from the light source through the first lens. It may be measured in advance as a value (mixed with response characteristic) In this specification, the fluorescence signal function is a function that is analogized as a function (or graph) of a time domain or time with a digitized second function, and the device response The function is a function that is analogized as a function (or graph) of the first function in the time domain or time.In the present specification, the first and second signals are digitized and then analogized as a function (eg, in the time domain or time) . information can be provided.

I_P(t)를 장치 응답 함수(instrument response function, 이하 IRF)라 하고, I_IRF(t)로 표기하기로 한다. I_IRF(t)는 체외진단 장치의 전체 응답 함수를 의미하며 형광 현상과 무관한 장치 고유의 값일 수 있다. 이 때, 제1 여기광과 감지부에 대한 장치 응답 함수는 I_IRF(t)로, 제2 여기광과 감지부에 대한 장치 응답 함수는 I_IRF'(t)이나 제1 여기광과 감지부에 대한 장치 응답 함수는 I_IRF(t)과 일부 차이 또는 동일할 수 있다. 그리고 감지부에서 검출된 파형은 형광 본연의 지수함수 감쇠 함수인 I_F(t)와 I_IRF(t)의 콘볼루션이고 이것이 T0만큼 지연된 형태로 나타나게 된다. At this time, I _E (t)

I _P (t) will be referred to as an instrument response function (hereinafter, IRF), and will be expressed as I _IRF (t). I _IRF (t) refers to the overall response function of the in vitro diagnostic device and may be a device-specific value independent of fluorescence. In this case, the device response function for the first excitation light and the sensing unit is I _IRF (t), and the device response function for the second excitation light and the sensing unit is I _IRF '(t), or the first excitation light and the sensing unit The device response function for _IRF (t) may be the same or some difference from IRF(t). In addition, the waveform detected by the sensor is a convolution of I _F (t) and I _IRF (t), which are the natural exponential decay functions of fluorescence, and this is delayed by T0.

Based on this, the in vitro diagnostic apparatus according to the embodiment uses Fourier-domain deconvolution to remove the contribution of the IRF waveform from the measured or calculated analog waveform or the calculated device response function to fluorescence lifetime. can be detected.

That is, the signal processing unit 400 converts the measured analog waveforms _IM (t) and I _IRF (t) into _IM (f) and I _IRF (f) in the frequency domain through Fourier transform, respectively. And if the inverse Fourier transform is performed after calculating I _M (f)/I _IRF (f), only I _F (t) will remain due to the mathematical properties of the Fourier transform and convolution. This is because the convolutional relationship is transformed into a simple multiplication relationship in the Fourier-transformed frequency domain. Through this deconvolution method, the contribution of IRF can be minimized.

Also, the signal processing unit 400 may calculate the fluorescence lifetime directly in the frequency domain without performing the inverse Fourier transform. The signal processing unit 400 may calculate the fluorescence lifetime by analyzing the magnitude or phase component of the absolute value of IF ( _f ), which is the fluorescence lifetime in the frequency domain. That is, the signal processing unit 400 calculates the fluorescence lifetime by curve fitting the measurement result with an exponential decay curve in the time domain with a frequency domain expression curve of the exponential decay curve in the frequency domain. have. In other words, for an arbitrary frequency f, if the difference between the phase component of the complex function I _F (f) and the phase component of I _IRF (f) is θ, the fluorescence lifetime can be expressed by Equation 2 below.

[Equation 2]

Here, τ means the fluorescence lifetime.

Accordingly, accurate fluorescence lifetime can be calculated by curve-fitting the measurement result with the above function within the effective frequency range. And if the measured result is transformed into the frequency domain through Fourier transform, the contribution of IRF appears as a algebraic relationship such as simple multiplication (for absolute value) or addition (for phase), so the contribution of IRF can be easily removed. have.

In addition, in order to obtain high-speed measurement of fluorescence lifetime, the signal processing unit 400 according to an embodiment of the present invention receives an analog pulse electrical signal caused by multiple fluorescence photons rather than a single photon response as in TCSPC. It can also be processed to calculate the fluorescence lifetime.

As described above, the pulsed electrical signal measured from the sensing unit may be a convolution of an exponential decay curve unique to the fluorescence emission phenomenon and an IRF waveform.

In this case, the signal processing unit 400 according to the embodiment may measure the fluorescence lifetime by determining the fluorescence lifetime in the average time domain and subtracting the deconvolution (deriving the difference value). Accordingly, the in vitro diagnostic apparatus according to the embodiment can more easily measure the fluorescence lifetime.

In the signal processing unit 400, the pulse electrical signal generated by an infinite number of fluorescent photons is a pulse electrical signal for one individual fluorescent photon, that is, quantum-mechanically, the amount of unit charge (the amount of electrons) arrives at the signal processing unit to be described later. It can be solved with a probability distribution function (PDF) of And for a random signal, the convolution of the probability distribution function becomes the sum of individual random variables corresponding to each probability distribution function. Accordingly, the following Equation 3 corresponding to Equation 1 above is established.

[Equation 3]

Here, M, E, F, and P are temporal random variables corresponding to each of I _M (t), I _E (t), I _F (t), and I _P (t) as PDFs. T ₀ is a variable determined as a fixed delay time by the light path. Specifically, M is the arrival time of electrons arriving at the signal processing unit, E is the arrival time of excitation photons causing fluorescence photons among them, and P is the arrival of one photon-electron converted electron by fluorescent photons. Time, F, is the time it takes for one fluorescent molecule to emit one fluorescent photon. For one fluorescent photon and one signal electron caused by it, E, F, P are or can be considered as stochastic random variables. If an additive relationship is established between random variables, an additive relationship is also established with respect to the expected value of the random variable, that is, the average value. That is, if E[·] is an average operator (a subscript indicates a target excitation light in the sensing unit, the first excitation light is 1, and the second excitation light is 2), the average value for the time variable, that is, the average Time is expressed by the following Equation 4 for the integration period T.

[Equation 3]

Here, A(t) represents a probability distribution function for each temporal random variable. In addition, the integral period T theoretically needs to have an infinite value to obtain an ideal integral value. However, in a system that obtains the fluorescence lifetime using pulsed excitation light, integration can be performed within the limited cycle time of the signal. The integral in the exponential decay curve is 5 times the specific time constant τ, that is, the integral until the time e ^-5 of the maximum value of the decay curve is integrated, which is 99.3% of the area. Therefore, there may not be much difference from the integral for more time. Therefore, the integration interval T for obtaining the average time can be defined as, for example, an interval up to 5τ.

According to Equation 3, the following Equation 5 holds for the average time.

[Equation 5]

Equation 5 shows that the average delay time E[M] of the pulsed electrical signal waveform arriving at the signal processing unit, which will be described later, is the IRF delay E[E]+E[P] as the response characteristics of the excitation light source and the sensing unit and the optical path. It means that it is expressed as the sum of the delay T0 caused by the fluorescence phenomenon and the delay E[F] caused by the fluorescence phenomenon. Of these, if the sum of the delay due to the IRF delay and the light path is the device delay time, it is expressed as E[E]+E[P]+T0. And since the characteristic time constant τ in the exponential decay curve is equal to the average delay time value of the curve (τ=E[F]), the fluorescence lifetime τ is finally E[M]-{E[E]+E[P ]+T0}. Therefore, the fluorescence lifetime is a value obtained by subtracting the device delay time from the average delay time as described above.

2 and 3 , the average <t> _IRF of the device response function I _IRF (t) represents the device delay time for the above-described sensing unit, and the average of the waveform I _M (t) obtained from the sensing unit <t > _M represents the above-mentioned average delay time. In this case, the device response function and the starting point of the fluorescence signal waveform obtained from the sensing unit may be the same or different. Hereinafter, description will be made on the basis of different things. In addition, E[F], that is, the fluorescence lifetime τ, can be calculated by using the difference between the average times of the two waveforms as shown in FIG. 2 . That is, the signal processing unit may calculate the fluorescence lifetime in the above-described manner.

In addition, the average time E ₁ (t) of the first signal in the signal processing unit 400 may be calculated using Equation 6 below.

[Equation 6]

Here, A(t) represents the first signal or device response function, and T is an integration period, which may be set to a specific value according to the accuracy of fluorescence lifetime measurement.

Also, the average time E ₂ (t) of the second signal in the signal processing unit 400 may be calculated using Equation 7 below.

[Equation 7]

Here, B(t) represents the function of the second signal or fluorescence signal, and T is the same value as the integration period in the above equation.

The signal processing unit 400 compensates for the average time of the second signal, and as described above, the difference between the average time of the second signal and the average time of the first signal (E ₂ (t) - E ₁ (t)) can be calculated as the fluorescence lifetime.

By this compensation, the signal processing unit 400 may calculate the first excitation light that does not pass through the sample and the IRF delay E ₁ [E] + E ₁ [P] as a response characteristic of the sensing unit to the first excitation light (T ₀ ). is the delay time as described above, and the delay time is a device-specific characteristic (mixed with response characteristic) independent of the fluorescence phenomenon, which is excluded because it may be measured in advance).

More specifically, the signal processing unit 400 converts the first signal representing the response characteristics of the sensing unit into a compensated first signal representing the response characteristics of the sensing unit, and a compensation value stored by reflecting the unique fluorescence lifetime for a specific sample. is available.

In other words, the signal processing unit 400 may calculate the compensation value using Equation 8 below.

[Equation 8]

CV={(E ₂ [E]+E ₂ [P])}- {E ₁ [E]+E ₁ [P]}

Here, CV is the compensation value, E ₁ [E]+E ₁ [P] is the response characteristic of the excitation light (first excitation light) light source and the sensing unit, and E ₂ [E]+E ₂ [P] is the excitation light (Second excitation light) The response characteristics of the light source and the sensing unit, E ₁ [E]+E ₁ [P] and E ₂ [E]+E ₂ [P] are the intrinsic fluorescence lifetime of a specific sample in Equation 1 obtained by substituting Alternatively, when there are a plurality of sensing units, the compensation value may be calculated as a difference between response characteristics of the first excitation light with respect to the plurality of sensing units.

With this configuration, the in vitro diagnostic device according to the embodiment can accurately calculate the fluorescence lifetime while obtaining the second signal for the fluorescence photon and the first signal in which the excitation light does not pass through the sample without a time difference or with a minimized time difference. can

In particular, in the in vitro diagnostic apparatus according to the embodiment, the difference between the response characteristic (device response function) of the first signal measured by the sensing unit and the response characteristic (fluorescence signal function) to the fluorescent photon (second signal) collected by the sensing unit can be removed. That is, errors due to environmental factors such as path differences may be removed. Accordingly, the signal processing unit according to the embodiment compensates only the difference between the response characteristic (fluorescent signal) measured by the sensing unit and the response characteristic (device response function) of the sensing unit, and as described above, the response characteristic to the sensing unit and the fluorescent photon The fluorescence lifetime can be calculated using the signal for . According to this configuration, time can be saved as much as the overlapping time by receiving the excitation light having a response characteristic for a time that is at least partially overlapping with the time for receiving the fluorescence photon regardless of the path difference. Accordingly, the fluorescence lifetime can be calculated more quickly and accurately.

Furthermore, as shown in FIG. 3 , in the device according to the embodiment, a signal reception time difference ( Even if Δt) exists, it is a time difference according to the difference in application of the excitation light by the light source (time difference between the application of the first excitation light and the second excitation light), and thus it can be removed with a value obtained in advance. Accordingly, the signal processing unit according to the embodiment compensates for the difference between the response characteristics of the first signal and the response characteristics of the second signal measured by the sensing unit and the above-described time difference, and as described above, the response characteristics of the sensing unit and the fluorescence The fluorescence lifetime can be calculated using the signal for the photon.

4 is a diagram illustrating a first signal having a plurality of first signal levels according to an embodiment of the present invention, and FIG. 5 is a diagram illustrating a second signal having a plurality of second signal levels according to an embodiment of the present invention it is one drawing

4 and 5 , the first signal SG1 may have a plurality of first signal level ranges SSG1 to SSG3 , and the second signal may have a plurality of second signal level ranges SSG4 to SSG6 . have.

First, the first signal SG1 has a plurality of first signal level ranges SSG1 to SSG3 as described above. For example, the plurality of first signal level ranges SSG1 to SSG3 include a first sub-signal level range SSG1 , a second sub-signal level range SSG2 , and a third sub-signal level range SSG3 .

As described above, the first signal SG1 is a signal obtained by converting the first excitation light. That is, the first signal SG1 is a converted electrical signal of the first excitation light. The first signal SG1 may be an electrical signal, for example, a voltage.

In this case, the first signal SG1 may have the above-described various first signal level ranges by adjusting the power applied to the sensing unit. The plurality of first signal level ranges may include different electric signal ranges. For example, the first sub-signal level range SSG1 may be comprised of a 0 level to a first level A0. Also, the second sub-signal level range SSG2 may be in the range of 0 level to the second level A1. And the third sub-signal level range SSG3 may be in the range of 0 level to the third level A2. Here, the first level A0 may be smaller than the second level A1 , and the second level A1 may be smaller than the third level A2 .

Accordingly, in the first signal SG1 converted by the sensing unit, the above-described device response function I _IRF (t) may also appear differently according to a plurality of first signal level ranges. That is, it may be digitized or quantized based on the first signal SG1 having a specific first signal level range. The digitization or quantization may be performed by, for example, a plurality of bits in the signal processing unit. Then, the device response function I _IRF (t) is calculated using the digitized first signal SG1 and may be displayed as shown in a graph with respect to a time domain or time.

Therefore, in the same in vitro diagnostic device, even if the device itself has the same response function or eigenfunction, the device response function (I _IRF (t)) calculated by digitization or quantization may be partially different.

The first signal having the first sub-signal level range SSG1 may be calculated or expressed as a first device response function I _IRF1 (t). Also, the first signal having the second sub-signal level range SSG2 may be calculated or expressed as a second device response function I _IRF2 (t). Also, the first signal having the third sub-signal level range SSG3 may be calculated or expressed as a third device response function I _IRF3 (t).

The first device response function I _IRF3 (t) to the third device response function I _IRF3 (t) may be the same or at least partially different. For example, some errors may occur due to digitization or quantization.

In addition, the first sub-signal level range SSG1 to the third sub-signal level range SSG3 has a maximum level difference (a difference between the first level and the third level). Also, the first signal SG1 having the plurality of first signal level ranges may be output before or after the output of the second signal SG2 . Further, the excitation light is sequentially applied corresponding to the number of the plurality of first signal level ranges.

The second signal SG2 has a plurality of second signal level ranges SSG4 to SSG6 . For example, the plurality of second signal level ranges SSG4 to SSG6 include a fourth sub-signal level range SSG4 , a fifth sub-signal level range SSG5 , and a sixth sub-signal level range SSG6 .

As described above, the second signal SG2 is a signal obtained by converting the second excitation light. That is, the second signal SG2 is a converted electrical signal of the second excitation light. The second excitation light is a fluorescence photon generated after the excitation light is irradiated to the sample. Also, the second signal SG2 may be an electrical signal, for example, a voltage.

In this case, the second signal SG2 may have the above-described various second signal level ranges by controlling the power applied to the sensing unit. The plurality of second signal level ranges may include different electric signal ranges. For example, the fourth sub-signal level range SSG4 may include a 0 level to a fourth level A3. Also, the fifth sub-signal level range SSG5 may be in the range of the 0 level to the fifth level A4. In addition, the sixth sub-signal level range SSG6 may be in the range of the 0 level to the sixth level A5. Here, the fourth level A3 may be smaller than the fifth level A4 , and the fourth level A4 may be smaller than the sixth level A5 .

Accordingly, in the second signal SG2 converted by the sensing unit, the above-described fluorescent signal function I _M (t) may also appear differently according to a plurality of first signal level ranges. That is, it may be digitized or quantized based on the second signal SG2 having a specific second signal level range like the first signal. The digitization or quantization may be performed by, for example, a plurality of bits in the signal processing unit. In addition, the fluorescence signal function I _M (t) is calculated using the digitized second signal SG2 and may be displayed as shown in a graph with respect to a time domain or time.

Therefore, even if the fluorescence signal generated from the sample by the excitation light is the same, the fluorescence signal function I _M (t) calculated by digitization or quantization may be different according to the range of the second signal level.

Furthermore, the size of the fluorescence signal generated from the sample by the excitation light by the same in vitro diagnostic device may vary according to the state of the sample, etc. for each region. This will be described later.

In addition, the second signal having the fourth sub-signal level range SSG4 may be calculated or expressed as the first fluorescent signal function I _M1 (t). Also, it may be calculated or expressed as a second fluorescent signal function I _M2 (t) having a fifth sub-signal level range SSG5. Also, the second signal having the sixth sub-signal level range SSG6 may be calculated or expressed as a third fluorescent signal function I _M3 (t).

At least a part of the first fluorescent signal function I _M1 (t) to the third fluorescent signal function I _M3 (t) may be different. For example, some errors may occur due to digitization or quantization.

In addition, the fourth sub-signal level range SSG4 to the sixth sub-signal level range SSG6 has a maximum level difference (a difference between the first level and the third level).

6 is an exemplary view before the second signal level range is changed in the in vitro diagnostic apparatus using the fluorescence lifetime according to an embodiment of the present invention, and FIG. 2 It is an exemplary view after changing the signal level range.

Referring to FIG. 6 , the size of a fluorescence signal generated in a sample by excitation light by the same in vitro diagnostic apparatus may vary according to the state of the sample, etc. for each region. In addition, the signal processing unit may calculate a fluorescence signal function and a fluorescence lifetime by changing the range of the second signal level for each region of the sample. With this configuration, it is possible to calculate an accurate fluorescence signal function and fluorescence lifetime even if the level measured by the sensing unit is different depending on the state, position, or degree of fluorescence of the sample. Furthermore, it is possible to reduce power consumption in calculating an accurate fluorescence signal function and fluorescence lifetime.

In the in vitro diagnostic apparatus using the fluorescence lifetime according to the embodiment, the fluorescence signal function I _M (t) may be calculated or displayed based on a second signal having one of a plurality of second signal level ranges.

For example, the fluorescence signal function I _M (t) (corresponding to the second fluorescence signal function) for each region is calculated or can be displayed. Also, each fluorescence signal function may have a maximum value.

In the first area K1, the 2-1 fluorescence signal function I _MK1 (t) may be calculated or displayed. The 2-1 fluorescence signal function I _MK1 (t) may have a first maximum value V _M1- -. And in the second region K2, a 2-2 fluorescence signal function I _MK2 (t) may be calculated or displayed. The 2-2 fluorescence signal function I _MK2 (t) may have a second maximum value V _M2- -. In addition, in the third region K3 , a 2-3 th fluorescence signal function I _MK3 (t) may be calculated or displayed. The 2-3 fluorescence signal function I _MK3 (t) may have a third maximum value V _M3'- -.

In this case, the signal processing unit may change the range of the second signal level so that the difference between the maximum value of the fluorescent signal function and the saturation value is the smallest. Here, the saturation value may be a value obtained by digitizing the maximum level of the sensing unit. That is, the electric signal of the maximum level output from the sensing unit to which the maximum power is applied may be a digitized value. For example, the second signal may have a value of 0 to 5 [V], and the second signal may be digitized with 8 bits. In this case, a signal of 255 level (eg, voltage, etc.) may be a saturation value.

In the first region K1, the 2-1 fluorescence signal function I _MK1 (t) may be completely calculated or displayed. Referring further to FIG. 7 , the 2-1 fluorescence signal function I _MK1 (t) may be maintained and calculated or displayed in the first region K1.

On the contrary, in the second region K2, the 2-2 fluorescence signal function I _MK2 (t) may be calculated or displayed discontinuously. For example, there may be a plurality of second maximum values V _M2- - of the 2-2 fluorescence signal function I _MK2 (t). Furthermore, the second maximum value (V _M2- -) of the 2-2 fluorescence signal function (I _MK2 (t)) may be spaced apart from each other in time. In this case, the signal processing unit may change the range of the second signal level so that the difference between the maximum value of the fluorescent signal function and the saturation value is the smallest.

Referring further to FIG. 7 , a modified 2-2 fluorescence signal function I _MK2' (t) may be calculated or displayed in the second region K2 . Accordingly, the value of the fluorescence signal function having a value greater than the saturation value can be accurately calculated by changing the range of the second signal level. Accordingly, a fluorescence signal function more accurate than before the change of the second signal level range can be calculated or displayed. Furthermore, an accurate fluorescence lifetime can be calculated using the calculated or displayed fluorescence signal function and the device response function according to the first signal.

And in the third region K3, the 2-3 th fluorescence signal function I _MK2 (t) may be completely calculated or displayed. However, the third maximum value V _M3- may be smaller than a predetermined ratio of the saturation value. For example, the third maximum value V _M3- may be less than or equal to 50% of the saturation value. In this case, the signal processing unit may change the range of the second signal level so that the difference between the maximum value of the fluorescent signal function and the saturation value is the smallest.

Referring further to FIG. 7 , in the third region K3 , the modified 2-3 th fluorescence signal function I _MK3' (t) may be calculated or displayed. Therefore, it is possible to reduce the digitization error for a very small value of the fluorescence signal function. In other words, a fluorescence signal function more accurate than before the change of the second signal level range may be calculated or displayed. Accordingly, an accurate fluorescence lifetime can be calculated using the calculated or displayed fluorescence signal function and the device response function according to the first signal.

Furthermore, it is possible to accurately calculate the fluorescence signal function for the second signal by using the first signal having the plurality of first signal level ranges described above. For example, the signal processing unit may calculate a fluorescent signal or a fluorescent signal function of the sample by using the first signal level range and the second signal level range having the same signal level range. Accordingly, an accurate fluorescence lifetime with reduced error may be calculated by the signal processing unit.

In addition, the in vitro diagnostic apparatus using the fluorescence lifetime according to the embodiment may irradiate the excitation light to a predetermined area (corresponding to the sample measurement range) with respect to the sample through line scanning.

Accordingly, as described above, the signal processing unit may calculate the fluorescence signal of the sample or the fluorescence lifetime of the sample by using the second signal having a different second signal level range for each area of the sample.

8 is a flowchart of an in vitro diagnostic method using fluorescence lifetime according to an embodiment of the present invention.

Referring to FIG. 8 , it should be understood that the above description of the in vitro diagnostic apparatus shown in FIGS. 1 to 7 also applies to the method for measuring fluorescence lifespan according to the present embodiment, even if omitted below.

The fluorescence lifetime measurement method includes the steps of irradiating excitation light to the sample (S1010), receiving the first signal and the second signal (S1020), and using the first signal and the second signal, the fluorescence signal of the sample is less than or equal to the saturation value. It may include changing the range of the second signal level so as to be (S1030) and calculating the fluorescence lifetime (S1040).

First, in the step of irradiating the excitation light to the sample ( S1010 ), the in vitro diagnostic apparatus may generate the excitation light from the light source. Accordingly, the excitation light is irradiated to the sample according to the two paths described above, and the fluorescence photons generated from the sample may move to the sensing unit. In addition, the excitation light may move to the sensing unit without passing through the sample according to the first path described above. At this time, the excitation light and the fluorescence photon are received by the shutter unit to the sensing unit, and mutual influence can be suppressed.

In the step of receiving the first signal and the second signal ( S1020 ), the sensing unit may generate the first signal and the second signal.

Specifically, the first signal is an electrical signal converted from excitation light (first excitation light) that does not pass through the sample. Excitation light that has not passed through the above-described sample may be collected by the sensing unit. The second signal is an electrical signal converted from a fluorescence photon generated by the excitation light to be irradiated to the sample S including the fluorescent molecule. The above-described fluorescent photons may be collected by the sensing unit.

In the step (S1030) of changing the range of the second signal level so that the fluorescence signal of the sample is less than or equal to the saturation value by using the first signal and the second signal, the signal processing unit digitizes or quantizes the second signal generated by the sensing unit, , for example, it is possible to calculate and display the digitized second signal as a fluorescent signal function and the digitized first signal as a device response function.

In this case, as described above, the signal processing unit may calculate the fluorescence signal of the sample or the fluorescence lifetime of the sample by using the second signal having a different second signal level range for each area of the sample.

In addition, the signal processing unit may change the range of the second signal level so that the difference between the maximum value of the fluorescent signal function and the saturation value is smallest. Also, the signal processing unit may change the range of the second signal level so that the difference between the maximum value of the fluorescent signal function and the saturation value is the smallest. Furthermore, the signal processing unit may calculate the fluorescence signal or the fluorescence signal function of the sample by using the first signal level range and the second signal level range having the same signal level range. In this case, the first signal having a plurality of first signal level ranges may be measured in advance and stored in a database or the like.

And finally, calculating the fluorescence lifetime using the first signal and the second signal (S1040). In other words, it is possible to calculate the fluorescence signal function for the second signal obtained by changing the range of the second signal level. In addition, a more accurate and less error fluorescence lifetime can be calculated by using the fluorescence signal function for the first signal for the first signal level range having the same signal level range and the fluorescence signal function for the second signal obtained by changing the second signal level range. have.

For example, as described above, the difference between the average time of the first signal or the device response function based on the first signal and the average time of the fluorescence signal function for the second signal or the second signal obtained by changing the second signal level range can be used to calculate the fluorescence lifetime.

As described above, the in vitro diagnostic method and device using fluorescence lifetime according to the present invention eliminates the contribution of the device response function in a very short time through measurement of average delay time and device delay time, and easy calculation, while providing accuracy and precision. can be used to measure the fluorescence lifetime.

In addition, when the in vitro diagnostic method and device using the fluorescence lifetime according to the present invention, which has improved processing speed, is applied to a fluorescence lifetime imaging microscope, deterioration due to the photo-bleaching effect can be minimized, and a three-dimensional image can be obtained in real time. makes it possible to obtain

Meanwhile, the above-described embodiments of the present invention can be written as a program that can be executed on a computer, and can be implemented in a general-purpose digital computer that operates the program using a computer-readable recording medium. The computer-readable recording medium includes a magnetic storage medium (eg, ROM, floppy disk, hard disk, etc.), an optically readable medium (eg, CD-ROM, DVD, etc.) and a carrier wave (eg, Internet storage media such as transmission).

The term '~ unit' used in this embodiment means software or hardware components such as field-programmable gate array (FPGA) or ASIC, and '~ unit' performs certain roles. However, '-part' is not limited to software or hardware. '~unit' may be configured to reside in an addressable storage medium or may be configured to refresh one or more processors. Thus, as an example, '~' denotes components such as software components, object-oriented software components, class components, and task components, and processes, functions, properties, and procedures. , subroutines, segments of program code, drivers, firmware, microcode, circuitry, data, databases, data structures, tables, arrays, and variables. The functions provided in the components and '~ units' may be combined into a smaller number of components and '~ units' or further separated into additional components and '~ units'. In addition, components and '~ units' may be implemented to play one or more CPUs in a device or secure multimedia card.

In the above, the embodiment has been mainly described, but this is only an example and does not limit the present invention, and those of ordinary skill in the art to which the present invention pertains are not exemplified above in the range that does not depart from the essential characteristics of the present embodiment. It will be appreciated that various modifications and applications are possible. For example, each component specifically shown in the embodiment can be implemented by modification. And the differences related to these modifications and applications should be construed as being included in the scope of the present invention defined in the appended claims.

Claims (10)

a light generator generating excitation light;
a control unit for controlling a path of the excitation light;
a sensing unit outputting a first signal and a second signal having a plurality of second signal level ranges; and
a signal processing unit configured to change the range of the plurality of second signal levels using the first signal and the second signal so that the fluorescence signal of the sample becomes less than or equal to a saturation value;
The first signal is a signal converted by sensing the excitation light,
The second signal is a signal converted by detecting a fluorescence photon generated when the excitation light is irradiated to the sample.
According to claim 1,
The first signal has a plurality of first signal level ranges,
The signal processing unit calculates the fluorescence signal of the sample using the same first signal level range and the same second signal level range.
According to claim 1,
The fluorescence signal of the sample has a maximum value,
The signal processing unit changes the range of the second signal level so that the difference between the maximum value and the saturation value is smallest.
According to claim 1,
The control unit controls the excitation light into a first excitation light and a second excitation light branched in different paths.
5. The method of claim 4,
the first signal is generated by the first excitation light;
The second signal is an in vitro diagnostic device using a fluorescence lifetime generated by the second excitation light.
6. The method of claim 5,
The in vitro diagnostic apparatus using a fluorescence lifetime further comprising a shutter unit that blocks the first excitation light while sensing the fluorescent photon.
7. The method of claim 6,
The shutter unit blocks the fluorescent photons while receiving the first excitation light.
According to claim 1,
The signal processing unit calculates the fluorescence lifetime of the sample by using a difference between the average time of the first signal and the average time of the second signal.
According to claim 1,
The signal processing unit is an in vitro diagnostic device using a fluorescence lifetime to change the range of the second signal level for each region of the sample.
10. The method of claim 9,
The signal processing unit calculates the fluorescence signal of the sample or the fluorescence lifetime of the sample by using a second signal having a different second signal level range for each region of the sample.

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