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Abstract
Biological delayed luminescence (DL) refers to the emission of photons of 200-800 nm after an organism is exposed to a beam of light. At present, DL research is mostly conducted on plants and cells. Most human research uses low-temperature photomultiplier tubes (PMT) with poor performance. This paper introduces a newly developed dual-channel, coincident counting DL acquisition system, as well as preliminary experimental results with acupuncture meridians and acupoints in humans. The system employs two low-noise SPAD (single photon avalanche diode) modules to achieve not only a time resolution as low as 1 μs, but also a spatio-temporal correlation relation between two simultaneous measurements. Using a laser excitation of 400 nm and 532 nm, we measured the DL emission for different parts of the human body and found that the corresponding DL curves follow the hyperbolic law of $I = {I_0}{\left( {1 + \frac{t}{\tau }} \right)^{ - \gamma }}$. The emission curves are sensitive to many factors including the current health state of the participants. We hypothesize that DL emission is a quantum process involving a variety of functional biomolecules in the human body, and that various DL curve parameters could reflect information about human health and disease. We predict that quantitative analysis, based on accurately measured DL emission, will provide a powerful new tool for monitoring human health in the future.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Biophotons are ultra-weak photons emitted by organisms in the wavelength range from 200 - 800 nm [1] which can be divided into two categories: spontaneous emission, also called ultraweak photon emission (UPE), and delayed luminescence (DL) following excitation by light [2]. After excitation, organisms will emit photons with a wide spectral distribution and whose intensity attenuation is non-exponential, Furthermore, the luminescence time is on the order of milliseconds or seconds indicating that cooperative radiation occurs between biomolecules [1].
DL was first observed in 1951 by Bernard et al. who observed very weak photon radiation in Chlorella, Scenedesmus, and Stickococcus which grows in Knop's solution [3]. In the past few decades, researchers have mostly focused on plants and cells [1–4], and as such there have been only limited reports of measurements of DL in humans [5–7]. In humans, the DL time is relatively shorter and weaker than plants and cells. Biophoton emission is a complex and multifactorial biological process which carries information about the molecular composition and structure of the organism. It is highly sensitive to physiological changes and the influence of the external environment and therefore might be useful for the assessment of the health status [2,8,9]. Since previous studies measuring luminescence and delayed luminescence from skin have limited sensitivity and time resolution, we developed new and improved methodology.
In principle, the rapidly developed fluorescence lifetime measurement system can be used to measure DL. However, the spectral response range of PMT (photomultiplier tube) is very limited, and the effective photosensitive area of SPAD (single photon avalanche diode) is very small. Therefore, it is difficult to obtain accurate DL data because of weak DL and wide spectrum. So far, most of the reported DL measurement systems have been constructed using PMT in counting mode. For example, Professor Han et al. used PMT and cooling device to measure the DL of Chinese medicinal materials [10]. Others including Wijk [11,12] et al., Zhang [13] et al., Yang [14] et al., all adopt similar device to construct the measurement system. Because the PMT circuit gain in the single photon counting state is very high, a small amount of excited light may damage the PMT and the circuit. Therefore, an electronic shutter is generally added to the optical path of the system (response time >1 ms), which causes these systems to be unable to distinguish the faster. Tudisco S et al. constructed a measuring device by transforming the high-voltage power supply circuit of PMT to measure the DL of human body, which can enter the measuring state by adding normal voltage 10 μs after excitation [15]. Another system for realizing human body DL measurement is ICCD (intensified charge coupled device) camera. Due to its extremely accurate time control (Princeton, PI-MAX4: timing resolution and timing jitter can reach 10 ps and 35 ps rms) device, ICCD is widely used in fluorescence spectral imaging and other research, and is a good method for imaging of large area such as human body for DL. However, the above methods are not suitable for accurately measuring the DL process of the human body: firstly, the system structured with PMT needs to be cooled to reduce the system noise. The scheme proposed by Tudisco [15] needs to design an extremely complex circuit, and it is difficult to control the shutter voltage. In addition, the sensitivity of ICCD is much lower than that of optical detector with single photon count, so it is difficult to obtain images with high SNR (signal to noise ratio). The fluorescence is ubiquitous included the sample box, that may produce the great noise, these noises should be removed in the calculation. There are also a lot of reports about the luminescence of the sample box containing the experimental samples [16].
With the rapid development of semiconductor optical devices, the APD (avalanche photodiode detector) in counting mode can be used as a time-dependent single-photon counter. At 400-900 nm, it has the advantages of a high quantum efficiency, small size and is easily controlled. Currently, a SPAD device with an APD has been commercialized and has a dark count up to 20 cps (photon counts per second) and a quantum efficiency as high as 80% (compared to 30% for PMT) in visible light. The technology is widely used in LIDAR, ToF (time-of-flight) 3D imaging [17], PET scanning, single-photon physics research, fluorescence lifetime microscopy and for optical communication (quantum key distribution) [18].
This manuscript introduces a fast measurement device which is composed of SPAD and a two-channel coincidence counter. It is suitable for DL imaging of the human body using photon collection and photo excitation, as well as for control and coincident counting. It can measure simultaneously the DL emission from two related parts of human body so that the time correlation of biophotons can be analyzed, which provides a good method for studying the spatio-temporal correlation of bioluminescence. The system can acquire signals with a time accuracy of 1 μs, a stable acquisition speed of 1000 times per second, a maximum of 4000 times per second, and a minimum acquisition interval of 100 μs. For the collection area with a diameter of 8 mm on the human body, a photon count of 80000 cps can be collected following an initial 10 mW excitation light, with a background fluorescence below 400 cps, and a very high SNR. By repeating the acquisition interval, the time resolution is improved and the noise is reduced. Therefore, using this high-resolution device, we have been able to accurately measure the close relationship between the health status of the human body and the photon emission parameters.
2. Materials and methods
2.1 Device and instrumentation
The schematic diagram of the system is shown in Fig. 1. The excitation wavelength of laser is 400 nm, and the detection wavelength of the detector is 400-1200 nm. The two channels are synchronized. A biological system (such as the left and right fingers of the person) is placed on the acquisition holes RD1 and RD2 respectively, and then the acquisition command of the acquisition software is started. The controller regulates the laser (L1 and L2 in Fig. 1) and the SPAD (D1 and D2 in Fig. 1) through the pulse signals, according to the parameters set by the software to acquire the emitted biophotons. The collected DL data is sent back to the PC. After collection, the software calculates and displays the DL decay curve and the decay curve parameters are calculated.
Fig. 1. The schematic diagram of DL system. MO1, MO2: micro-objective; FC1, FC2: coupling fiber; FE1, FE2: fiber optical excitation; FR1, FR2: liquid optical fiber for collecting photons (diameter 3 mm); L1, L2: pulsed laser; D1, D2: SPAD; RD1, RD2: portable for collecting the biological photon excitation.
2.2 Data acquisition and processing
DL data acquisition involves sending the pulse timing sequence (a typical timing sequence is shown in Fig. 2) from the controller to insure an accurate biophoton collection process. According to the predefined parameters, the controller output generates four pulses, two of which control the lasers and their high-level outputs, (otherwise the laser is turned off). The two channels output the SPAD gate control signal so that when the power is high the SPAD begins to collect data. When the power level is too low, the data collection is turned off. In Fig. 2, the signal from the single laser and the SPAD is plotted, and the signal of the other channel is synchronized with it.
The data acquisition is periodic, and the period in the example in Fig. 2 is 50 ms. The excitation period is set according to the DL time characteristic of the sample, and it is recommended to be twice or than the delay time. In Fig. 2, the wavelength of the laser is 400 nm and the average power of the pulse is 2 mW /cm2. It is divided into 4 acquisition gates, a total of 200 cycles are collected, and each gate collects 50 cycles. The same acquisition gate means that the delay and gate width of the relative excitation pulse back edge and SPAD gate control signal front edge are the same. In an acquisition gate of 20 ms, it is also divided into 50 times equally, that is, the controller collects data every 0.4 ms. In order to get a better time resolution, continue the work of the second acquisition gate in the next 50 cycles, and then delay the gate control signal by 0.1 ms. The third acquisition gate has a delay of 0.1 ms again, and the fourth has a delay of 0.1 ms based on the third acquisition gate.
Figure 3 shows the emitted photons for a typical DL decay from one channel using the left index finger of healthy people, according to the method of Chen et al. [19]. The four colors represent the delay data obtained from the four acquisition gates, red represents the first acquisition gate, yellow represents the second acquisition gate, green represents the third acquisition gate, and blue represents the fourth acquisition gate. It has been reported that for a great variety of biological system hyperbolic function can be used to model the DL temporal trend [9]. In contrast, our measured DL curve is well aligned with this model with higher SNR and wavelength sensitivity.
The maximum number of photons in Fig. 3 is 45850 cps where the raw data was converted to photon counts per second (cps). Normally, increasing the frequency of cup timer improves the time resolution. It can also increase the number of acquisition gates without increasing the clock frequency to improve the time resolution. Because the collection times in one gate is limited by the CPU speed, the gate width / frequency cannot be less than 200 μs. However, the delay time can be controlled within 15 ns. Therefore, increasing the number of gates with different delay time can improve the time resolution, which is equivalent to shortening the measurement interval. Because of overlapping collections, an integral fitting algorithm is used to fit the data, which generates more data points on the curve. This acquisition interval method can improve the time resolution and reduce the shot noise.
2.3 Experimental study details
The system is composed of two PCM-AQRH-16X fiber-coupling, single photon counting modules which were provided by Excelitas Technology. The parameters of SPCM-AQRH-16X are as follows: linear counting rate < 40 MkHz, dark count less than 25 cps (times per second), quantum efficiency of 70% (at 700 nm wavelength), effective spectral response range from 450-900 nm, effective photosensitive diameter of 150 μ m, working temperature range from 278-343 K and a gating control accuracy of less than 2 μs. The detector uses optical fiber coupling and has a built-in coupling lens, which can image a light spot with a diameter of 400 μm using a wavelength of 550 nm.
In order to collect biophotons in a circular region with a diameter of 6 mm, the system uses an inverted microscopic objective to focus the image of the bioluminescent surface into the effective aperture of the coupled fiber. The corresponding Abbe sine condition is:
where, n0 and ni are the refractive index of object and image, respectively and both are 1 in the air. Therefore, the aperture angle of the biological sample should be: where, h0 = 6 mm and hi = 0.4 mm, so we can get: u0 ≈ 0.4*0.3/6 = 0.02 rad, which is the half cone angle. The full cone angle of 2u0, should be 2.3°. Therefore, the collection area is increased by reducing the solid angle of the collected photons. In this example, the reduction rate is h0/hi = 15. The numerical aperture and magnification have been taken into account when selecting the microscopic objective lens.Many DL systems use LEDs (light emitting diode) as an excitation source, which allows for multi-wavelength excitation. However, the impulse response of LEDs is poor and therefore not suitable to achieve good time resolution. Hence, our system uses a semiconductor laser. When designing the excitation subsystem, factors such as excitation wavelength, intensity and pulse time of the laser were considered. It is particularly important that the fluorescence photons generated by the biological system should not enter the photon collection subsystem to avoid excess noise.
For practical applications, the head of the excitation subsystem can be added before the microscopic objective. As shown in Fig. 1, the excitation subsystem includes L1(L2), FE1(FE2), RD1(RD2). For the first and second paths, the beam of the semiconductor laser L1 is coupled to the excitation head and the RD1 collection head through the fiber FE1, and is directed to the biological sample. The rear end of RD1 is connected to a 5 mm diameter liquid core fiber (Nanjing Chunhui Science and Technology Industrial Co. Ltd) to collect the excited biophotons. Its transmittance is >80% in the 380-900 nm spectral range. An imaging lens with a short focal length is added to the RD1 head to image the photon of the 6 mm diameter round hole at the front end of RD1 to the lower end of the optical fiber. During operation, the controller first gives L1 and L2 short pulses (10 ms in this example). The pulsed laser illuminates the biological samples in front of RD1 and RD2, and 5 ∼ 10 μs after the excitation ends the SPAD collection starts.
The control system is employed to achieve the pulse synchronization which triggers the gate control function of the laser and the SPAD, and at the same time has the function of counting the pulses generated by the SPAD. The main controller uses a STM32F103ZET6 32-bit micro-controller. The main frequency and timer of this controller can be up to 72 MHz after PLL (phase locked loop) frequency multiplication, thus ensuring the time accuracy of the measurement (clock cycle is 15 ns). The data transmission between the controller and the host computer through the serial port provides the functions of the single photon counting module and the pulse laser, respectively. In a typical human DL acquisition sequence (Fig. 2), within a 20 ms period, during which the SPAD module is enabled, one can set the timer to send the count value to the host computer.
2.4 Controller
Since DL photons are closely related to biological processes, it is of great significance to explore the spatio-temporal correlation between photons. The system is equipped with a dual-channel coincidence counter, which is provided by the high-brightness PPKTP (periodically poled KTP) entangled light source system (KP-PE-ZY-001 from Qasky Quantum Technology Co. Ltd, Anhui, China). It can measure the number of coincident photons of the two channels at the set time interval by setting the coincidence gate width and delay time of the two channel pulses, thereby generating the time correlation of photons emitted from the same and different parts of human body.
The timer and counting module of the controller are both composed of a 16-bit automatic loading counter (CNT). After an acquisition cycle, the data is sent to the host computer for processing by DMA (direct memory access). The excitation source is a highly stable, semiconductor laser which can switch between 532 nm and 400 nm (replace the fiber plug) with the following characteristics; its wavelength drift is <0.1 nm, power stability is <1%, the maximum sampling rate reaches 4000/sec, the stable acquisition speed is 1000/sec and the minimum acquisition time interval is 100 μs.
The key to obtain high quality DL curve data is to improve the SNR. The sources of noise in our DL system are either due to fluorescent emissions from nearby excited molecules or quantum fluctuations in the biological system. However, the SNR remained relatively stable throughout the duration of a typical experimental reading. Nonetheless, fluctuation of photon number during the measurement process were observed and even able to form shot noise.
3. Discussion
3.1 Measurement results
When human DL is measured using the acquisition parameters shown in Fig. 2, the system can detect single photons on average within an initial photon counting period of 0.1 ms. The blue curve in Fig. 4 shows the change of stimulated emission photons with time for human finger, which has a hyperbolic decreasing trend. Compared with the previous reports, we measured by using multiple excitations to increase the photon number were more in line with the attenuation and had higher SNR [9]. Assuming that, the photon emission follows a Poisson distribution, and knowing the fluctuation of the photon number is $\sqrt N $, then the resulting measurement error is 1/$\sqrt N $. Therefore, for these measurements, the longer time interval increases the number of photons, and the delayed acquisition gate improves the time resolution. The acquisition time interval is 0.4 ms, but the time resolution is up to 0.1 ms, and the SNR is increased by $\sqrt {200} $ ≈ 14 times due to 50 consecutive repetitions. The number of photons in the first 0.4 ms of the first gate is 45,850 with a value of 917 for each measurement. Taking into account the interleaved acquisition, the data interval was increased to 0.1 ms (data requires post-processing). After one excitation with a 0.1 ms sampling interval, on average five photons could be measured.
During the measurement process, the incident light will excite substances other than the biological sample to be measured, and their fluorescent emissions will enter the measurement optical path within a certain time after the excitation light is turned off. This is one of the main sources of noise since the SNR is greatly reduced. Figure 4(a) is the result of RD1 acquisition using a circular hole with strong fluorescence: the blue is the DL curve of the index finger, and the red is the DL curve measured after the finger is removed.
The total SNR of the curve can be expressed as:
Figure 4(b) is the measured result of replacing the circular hole with a material with very low fluorescence (black photographic paper). It can be seen that the SNR is much improved, and the total SNR value was calculated to be 90.56. Therefore, to construct a system with a high SNR value should not only improved the collection efficiency of the emitted photons, but also significantly suppress the excitation fluorescence of irrelevant materials. It is possible to select biological samples with a very fast or very low excitation fluorescence at 532 nm or 400 nm and use appropriate high-quality filters for these specific wavelengths.
3.2 Hyperbolic delay in DL photons
The mechanisms associated with delayed luminescence and fluorescence are very different, although both can be calculated according to the following photon emission rate equation is [1]:
This equation represents the population of excited states. The equation shows that the reduction in population of high-level molecules exhibits a nonlinear relationship with its population number. It can be calculated that the number of DL photons (intensity I∝-dN) meets the following hyperbolic equation:
where γ = 1/(β-1). I0 is the initial photon number, τ is the DL time parameter, β is not 1, ie. it is nonlinear. All these three parameters are related to the measurement start time. In order to solve the fitting problem caused by interleaved data collection, Eq. (5) is expressed as an integral equation:Applying the acquisition parameters shown in Fig. 2, and using the software we developed to fit the decay curve, we obtain the results which are shown in Fig. 5 These results match very closely those obtained from the hyperbola curve obtained from Eq. (5), with an R-Square of 0.982. The software uses the nonlinear square method and uses two heuristic approximation algorithms: particle swarm optimization and the flower pollination algorithm. The three parameters in Eq. (5) are I0 = 48776 cps, τ = 2.65 ms, γ = 1.38. If the sampling times overlap, one can use Eq. (6) to find the theoretical photon number in Δt = 0.4 ms, compare it with the measured value in the same interval, calculate the loss function, and then iterate and approximate the fitting operation to get the minimum loss function. A curve with a high adjusted R-Square, indicates hyperbola-like behavior. and it can be seen that the relationship between the emitted DL photons and the original population number is nonlinear.
4. Conclusion
The DL system used in the present study, which utilizes SPAD technology, has a high-resolution time and gated control interface, so it can easily achieve accurate control of the acquisition time and furthermore, the SPAD can collect a very wide DL spectrum. In order to solve the problem of a small photosensitive area and avoid the fluorescent contributions from other substances, we used multiple excitations to increase the number of collected photons and to improve the SNR, and used multi-gate delay methodology to improve the data acquisition time accuracy. Our system can measure the delay time of 1 μs after the excitation. In addition, previous devices generally collect data in time bins in the ms level, our device is much faster and can achieve a 50 μs time bin.
It is worth mentioning that we have also carried out some preliminary experiments on the human body using our new DL system. Some experimental results show that in different health conditions, such as cold and high uric acid, where the photon counts and decay parameters are different from healthy individuals. We have continued more research in these areas. There are also reports about using DL to measure photon emission from cells [20], blood [21] to discriminate cancer cells, using DL to measure human skin to correlate with ages and seasonal variations [9]. We assume that DL will be sensitive to changes in human health and disease, and hope that our new methodology for measuring photon emission can be used for the health assessment of sub-healthy people in the future.
Funding
ENN Research Fund (EIST202008).
Acknowledgement
This project is supported by ENN Research Fund (Grant No. EIST202008). At the same time, we thank Chenhao Li for providing the fitting of the measured data, and Glen Rein for helpful discussion.
Disclosures
The authors declare no competing interests.
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