Fluorescence lifetime measuring device

Abstract

The invention relates to a fluorescence lifetime measuring device which is characterized in that a modulation instruction is generated by a computer, and a signal generator is driven to generate an alternating-current modulation signal; the first biasing circuit superposes the alternating-current modulation signal onto direct-current biasing current output by the current source to form a composite driving signal to be sent to the excitation light source, and the generated excitation light sequentially passes through the short-pass filter and the dichroic mirror and irradiates a sample to be detected; a fluorescence signal emitted after the sample to be detected is excited sequentially passes through the band-pass filter and the photomultiplier, a direct-current component and an alternating-current component are separated through the second biasing circuit, phase comparison is carried out according to the alternating-current component and an alternating-current modulation signal serving as a reference, phase delay is calculated, and the fluorescence lifetime is deduced. Compared with the prior art, the method has the advantages that the basic brightness of the light source can be maintained, the light intensity modulation is realized, the measurement result is accurate, the anti-interference capability is strong, and the like.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence lifetime measurement, and in particular to a fluorescence lifetime measurement device. Background Art

[0002] There are many methods for measuring fluorescence lifetime. The most widely used method is time-correlated single-photon counting (TCSPC). By measuring the delay time between the excitation pulse and the detection fluorescence signal, the distribution time curve of the fluorescence photons is recorded to calculate the fluorescence lifetime. However, for samples containing multiple fluorophores, the decay curve may show multiple peaks superimposed, and traditional single exponential fitting is difficult to accurately separate the lifetimes of each component.

[0003] Patent application number CN100510716C discloses a method for measuring fluorescence lifetime using periodic arbitrary waveform excitation. A fluorescent sample is excited by a frequency-tunable excitation light source with a certain modulation index. A computer performs Fourier series expansion on both the excitation and emission waveforms. By extracting the phase delay between the fundamental frequency components, an accurate fluorescence lifetime value can be calculated based on the relationship between the phase delay and the fluorescence lifetime.

[0004] However, the above scheme uses a light source whose luminous intensity changes with time in a sinusoidal waveform period as the excitation light source to directly excite the sample to be tested. The energy is dispersed throughout the entire period, making it difficult to ensure the average fluorescence intensity. The baseline stability of the excitation light intensity is poor, and the light intensity may drift due to current fluctuations, resulting in poor quality of the excited fluorescence signal and affecting the prediction results of the fluorescence lifetime value. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorescence lifetime measurement device in order to overcome the above-mentioned defects in the prior art and improve the accuracy of the measurement results.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] A fluorescence lifetime measurement device includes a computer, a signal generator, a first bias circuit, a current source, an excitation light source, a lock-in amplifier, an amplifier, a second bias circuit, a short-pass filter, a photomultiplier tube, a band-pass filter, a dichroic mirror, and a sample to be measured;

[0008] The computer is used to send modulation instructions to the signal generator;

[0009] The signal generator is used to generate an AC modulation signal according to a modulation instruction and send it to a lock-in amplifier and a bias circuit;

[0010] The first bias circuit superimposes the AC modulation signal on the DC bias current output by the current source to form a composite driving signal that is sent to the excitation light source;

[0011] The excitation light source generates excitation light which passes through the short-pass filter and the dichroic mirror in sequence and irradiates the sample to be tested;

[0012] The fluorescence signal emitted by the sample to be tested after being excited passes through a bandpass filter and a photomultiplier tube in sequence, is converted into an electrical signal, and is separated into a DC component and an AC component by a second bias circuit. The AC component is transmitted to an amplifier for amplification, and then the DC component and the amplified AC component are transmitted to a computer through a phase-locked amplifier. The phase is compared with the AC modulation signal received by the phase-locked amplifier as a reference to obtain a phase delay signal that matches the target fluorescence lifetime, so as to deduce the fluorescence lifetime.

[0013] Furthermore, the short-pass filter is used to filter out long-wavelength light from the excitation light and allow excitation light of a specific wavelength to pass through.

[0014] Furthermore, the dichroic mirror is used to reflect the excitation light output by the short-pass filter onto the sample to be tested, and to isolate the excitation light from the light beam reflected by the sample to be tested, and to transmit the fluorescence signal.

[0015] Furthermore, the bandpass filter is used to filter out interference from non-target wavelength signals in the fluorescence signal and focus the signal onto the photomultiplier tube.

[0016] Furthermore, the photomultiplier tube is used to convert the fluorescent signal into an electrical signal and amplify it.

[0017] Furthermore, the lock-in amplifier performs a real-time phase comparison between the reference AC modulated signal and the amplified AC component, adjusts the phase angle, and selectively extracts the phase-delayed signal that matches the target fluorescence lifetime, thereby calculating the phase difference and deriving the fluorescence lifetime.

[0018] Furthermore, the lock-in amplifier modulates the excitation light source to an optimal frequency by adjusting the phase angle, so that the fluorescence signal is modulated at the same frequency.

[0019] Furthermore, the fluorescence lifetime is calculated as follows:

[0020] tanθ=2πfτ

[0021] Where θ is the phase difference, f is the modulation frequency, and τ is the fluorescence lifetime.

[0022] Furthermore, the current source is a direct current power supply.

[0023] Furthermore, the current source ensures baseline stability of the excitation light intensity by providing a DC bias current, thereby avoiding light intensity drift caused by current fluctuation.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) The present invention provides a stable DC bias current through a current source, which is then superimposed with an AC modulation signal to form a "DC + AC" composite driving signal. The excitation light source generates excitation light for stimulating the fluorescence signal of the sample to be tested. The setting of the DC bias current can provide a stable baseline light intensity and improve the average excitation efficiency. The superimposed AC modulation signal can enhance the instantaneous excitation capability, while maintaining the basic brightness of the light source, achieving light intensity modulation, close to the pulse effect, and ensuring the reliability of the fluorescence signal. For the collected fluorescence signal, the DC component and the AC component are also separated by the bias circuit, and the fluorescence lifetime of the sample is predicted based on the AC component carrying the fluorescence lifetime information.

[0026] (2) A combination of a dichroic mirror, a two-stage filter and a phase-locked detection method is used. The dichroic mirror first physically isolates the excitation light from the fluorescence to prevent the excitation light from directly entering the detector and causing interference; the short-pass filter filters out long-wavelength light from the excitation light and allows the excitation light of a specific wavelength to pass; the band-pass filter further filters out non-target wavelength interference from the fluorescence signal, significantly improving the anti-interference ability in complex environments; the phase-locked detection has strong dynamic adaptability and can flexibly adapt to the needs of fluorescence lifetime detection from nanoseconds to microseconds by adjusting the modulation frequency and phase angle.

[0027] That is, the combination of optical path isolation and phase-locked amplifier can effectively eliminate the interference of stray light, suppress tributary noise and high-frequency electromagnetic interference, and ensure accurate measurement results.

[0028] (3) It has strong anti-interference ability, and the fluorescence decay time is not affected by changes in the optical properties of the sample, including turbidity, refractive index and coloration.

[0029] (4) High sensitivity. The high gain characteristic of the photomultiplier tube (PMT) has low distortion and is suitable for detecting low-intensity fluorescence signals. It has a fast response speed and nanosecond time resolution. It has a wide dynamic range and can adapt to different light intensity scenes. It has low noise, low dark current, and high signal-to-noise ratio.

[0030] (5) The phase-locked loop technology has high precision and strong noise resistance, and can accurately control the phase and frequency of the input signal, thereby achieving high-quality digital signal processing; the phase-locked loop technology can also be adaptively adjusted without temperature compensation to adapt to different measurement environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic structural diagram of a fluorescence lifetime measurement device provided in an embodiment of the present invention;

[0032] In the figure, 1. computer, 2. signal generator, 3. first bias circuit, 4. excitation light source, 5. lock-in amplifier, 6. amplifier, 7. second bias circuit, 8. short-pass filter, 9. photomultiplier tube, 10. band-pass filter, 11. dichroic mirror, 12. sample to be tested. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0034] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort shall fall within the scope of protection of the present invention.

[0035] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0036] In the description of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, or are the orientation or position relationship in which the product of the invention is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they should not be understood as limiting the present invention.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0038] Furthermore, terms such as "horizontal" and "vertical" do not necessarily mean that a component must be absolutely horizontal or overhanging, but rather that it can be slightly tilted. For example, "horizontal" simply means that its direction is more horizontal than "vertical," and does not mean that the structure must be completely horizontal, but rather that it can be slightly tilted.

[0039] Example 1

[0040] like Figure 1As shown, this embodiment provides a fluorescence lifetime measurement device, including a computer 1, a signal generator 2, a first bias circuit 3, a current source, an excitation light source 4, a lock-in amplifier 5, an amplifier 6, a second bias circuit 7, a short-pass filter 8, a photomultiplier tube 9, a band-pass filter 10, a dichroic mirror 11, and a sample to be measured 12;

[0041] Computer 1 is used to send modulation instructions to signal generator 2;

[0042] The signal generator 2 is used to generate an AC modulation signal according to the modulation instruction and send it to the lock-in amplifier 5 and the bias circuit;

[0043] The first bias circuit 3 superimposes the AC modulation signal on the DC bias current output by the current source to form a composite driving signal that is sent to the excitation light source 4;

[0044] The excitation light source 4 generates excitation light which passes through the short-pass filter 8 and the dichroic mirror 11 in sequence and is irradiated onto the sample 12 to be tested;

[0045] The fluorescence signal emitted by the sample 12 to be tested after being excited passes through the bandpass filter 10 and the photomultiplier tube 9 in sequence, is converted into an electrical signal, and is separated into a DC component and an AC component by the second bias circuit 7. The AC component is transmitted to the amplifier 6 for amplification, and then the DC component and the amplified AC component are transmitted to the computer 1 through the phase-locked amplifier 5. The phase is compared with the AC modulated signal received by the phase-locked amplifier 5 as a reference to obtain a phase delay signal that matches the target fluorescence lifetime, so as to deduce the fluorescence lifetime.

[0046] Specifically, the above scheme includes the following processes:

[0047] The computer 1 sends an instruction to the signal generator 2, so that the signal generator 2 generates a modulated signal and sends it to the lock-in amplifier 5;

[0048] At the same time, when the current source provides a stable DC bias current, the baseline stability of the excitation light intensity is ensured to avoid light intensity drift caused by current fluctuations;

[0049] The modulation signal passes through the first bias circuit 3, and the AC modulation signal is superimposed on the DC bias current to form a "DC + AC" composite drive signal, which is sent to the excitation light source 4, and the excitation light source 4 generates excitation light;

[0050] The excitation light passes through a short-pass filter 8, which filters out long-wavelength light and allows excitation light of a specific wavelength to pass through;

[0051] The fluorescence signal of the sample to be tested 12 is reflected by the dichroic mirror 11, and the excitation light and the fluorescence are physically isolated to prevent the excitation light from directly entering the detector and causing interference; that is, the dichroic mirror 11 is used to reflect the excitation light output by the short-pass filter 8 onto the sample to be tested 12, and isolate the excitation light from the light beam reflected by the sample to be tested 12, and transmit the fluorescence signal.

[0052] The fluorescence signal emitted by the sample 12 to be tested after being excited passes through the bandpass filter 10 to further filter out the interference of non-target wavelengths and is focused to the photomultiplier tube 9;

[0053] The photomultiplier tube 9 detects the fluorescent signal and converts it into an electrical signal, which is then separated into a direct current (DC) component and an alternating current (AC) component via the second bias circuit 7 .

[0054] The AC component of the modulated signal carrying the fluorescence lifetime information is transmitted to the amplifier 6 for amplification and then input to the lock-in amplifier 5 for phase detection.

[0055] The lock-in amplifier 5 receives the reference signal from the signal generator 2 and performs a real-time phase comparison with the AC component of the fluorescence signal. By adjusting the detector's phase angle, the system selectively extracts the phase-delayed signal that matches the target fluorescence lifetime. By solving for the phase difference, tanθ = 2πfτ, the fluorescence lifetime can be accurately derived.

[0056] This scheme detects fluorescence lifetime by detecting the time-dependent decay of fluorescence intensity after fluorescence excitation. The excitation light source is modulated at an optimal frequency so that the fluorescence signal is modulated at the same frequency, but with a phase delay (phase shift) compared to the excitation modulation. The alternating current (AC) component of the fluorescence signal and its phase information are extracted through a lock-in amplifier or digital signal processing technology, and the fluorescence lifetime is derived by calculating the phase difference.

[0057] Working principle:

[0058] Fluorescence lifetime refers to the time required for a fluorescent substance's intensity to decay to 1 / e of its initial value after being excited. This fluorescence lifetime measurement device uses the fluorescence decay time-frequency domain method to modulate the frequency of the excitation light and analyze the phase delay of the fluorescence signal. By solving the phase difference, tanθ = 2πfτ, the fluorescence lifetime can be accurately derived.

[0059] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A fluorescence lifetime measurement device, characterized in that: The device comprises a computer (1), a signal generator (2), a first bias circuit (3), a current source, an excitation light source (4), a lock-in amplifier (5), an amplifier (6), a second bias circuit (7), a short-pass filter (8), a photomultiplier tube (9), a band-pass filter (10), a dichroic mirror (11), and a sample to be measured (12); The computer (1) is used to send a modulation instruction to the signal generator (2); The signal generator (2) is used to generate an AC modulation signal according to a modulation instruction and send the signal to a lock-in amplifier (5) and a bias circuit; The first bias circuit (3) superimposes the AC modulation signal on the DC bias current output by the current source to form a composite driving signal that is sent to the excitation light source (4); The excitation light source (4) generates excitation light which passes through a short-pass filter (8) and a dichroic mirror (11) in sequence and is irradiated onto a sample to be tested (12); The fluorescence signal emitted by the sample to be tested (12) after being excited passes through a bandpass filter (10) and a photomultiplier tube (9) in sequence, is converted into an electrical signal, and is separated into a DC component and an AC component by a second bias circuit (7). The AC component is transmitted to an amplifier (6) for amplification, and the DC component and the amplified AC component are transmitted to a computer (1) through a phase-locked amplifier (5). The phase is compared with the AC modulation signal received by the phase-locked amplifier (5) as a reference to obtain a phase delay signal that matches the target fluorescence lifetime, so as to deduce the fluorescence lifetime.

2. The fluorescence lifetime measurement device according to claim 1, characterized in that: The short-pass filter (8) is used to filter out long-wavelength light from the excitation light and allow excitation light of a specific wavelength to pass through.

3. The fluorescence lifetime measurement device according to claim 1, wherein: The dichroic mirror (11) is used to reflect the excitation light output by the short-pass filter (8) onto the sample to be tested (12), and to isolate the excitation light from the light beam reflected by the sample to be tested (12), thereby transmitting the fluorescence signal.

4. The fluorescence lifetime measurement device according to claim 1, wherein: The bandpass filter (10) is used to filter out interference from non-target wavelength signals in the fluorescence signal and focus the signal onto the photomultiplier tube (9).

5. The fluorescence lifetime measurement device according to claim 1, characterized in that: The photomultiplier tube (9) is used to convert the fluorescent signal into an electrical signal and amplify it.

6. The fluorescence lifetime measurement device according to claim 1, characterized in that: The lock-in amplifier (5) performs real-time phase comparison between the AC modulated signal as a reference and the amplified AC component, adjusts the phase angle, and selectively extracts the phase delay signal that matches the target fluorescence lifetime, thereby calculating the phase difference and deriving the fluorescence lifetime.

7. The fluorescence lifetime measurement device according to claim 6, characterized in that: The lock-in amplifier (5) modulates the excitation light source (4) to the optimal frequency by adjusting the phase angle, so that the fluorescence signal is modulated at the same frequency.

8. The fluorescence lifetime measurement device according to claim 1, characterized in that: The fluorescence lifetime is calculated as follows: tanθ=2πfτ Where θ is the phase difference, f is the modulation frequency, and τ is the fluorescence lifetime.

9. The fluorescence lifetime measurement device according to claim 1, characterized in that: The current source is a direct current power supply.

10. The fluorescence lifetime measurement device according to claim 1, characterized in that: The current source ensures baseline stability of the excitation light intensity by providing a DC bias current, thereby avoiding light intensity drift caused by current fluctuation.

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