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Discipline
Biological
Keywords
Ultra-Weak Photon Emission
Autoluminescence
Imaging
Biophotonics
Observation Type
Follow-up
Nature
Continuing the storyline
Submitted
Jan 25th, 2018
Published
Mar 16th, 2018
  • Abstract

    Biological tissue exhibits autoluminescence, i.e. a spontaneous ultra-weak photon emission (UPE) without photoexcitation. In this study, we report UPE measurements on human hands using an electron-multiplying CCD (EMCCD) camera. The images from left and right hands (2 subjects, 10 images in total) were taken on different dates. We observed the following spatio-temporal aspects of the hand UPE images: (i) all images show clearly that human hands exhibit autoluminescence; (ii) there was large variability in the UPE intensity with respect to the factors: time (time of day, date), anatomical location on the hand, and individual subject; (iii) images taken on the same date (but at different times of day) showed a stronger similarity than images of the same subject taken on different dates; (iv) the mean UPE intensity (averaged over 2 regions of interest at the right and left dorsum of the hands) correlated with the time of measurement (lower intensity at around noon). Our study documents the feasibility of UPE imaging of hands with an EMCCD camera and shows that the spatio-temporal variability of the UPE pattern harbours information that seems to be correlated to internal and external factors.

  • Figure
  • Introduction

    Whereas autofluorescence of cells and biological tissue is fluorescent light emission due to stimulated light reactions with endogenous fluorophores, tissue exhibits also autoluminescence, i.e. spontaneous ultra-weak photon emission (UPE) in the ultra-violet to visible/near-infrared spectral range (approx. 200–1300 nm) without photoexcitation. The photon emission associated with autoluminescence is magnitudes lower compared to the case of autofluorescence, challenging the technical detection of this phenomenon. The UPE intensity has generally only an intensity in the order of 101–104 photons/s cm2.

    The source of the UPE due to autoluminescence is mainly biochemical, i.e. the de-excitation of energetically excited species like atoms and/or molecules to a deeper energy level generating photons. The excited states are generally caused by oxidative reactions involving radical or non-radical reactive oxygen (ROS) and nitrogen (RNS) species that react with several biomolecules like lipids, proteins and nucleic acids. Also, the self-recombination of organic radicals is relevant. For example, triple-excited carbonyls are responsible for the emission in the wavelength range of 350–550 nm, singlet excited melanin with 360–560 nm, dimolar singlet oxygen with 634 nm and 703 nm, and monomolar singlet oxygen with 1270 nm.

    Autoluminescence has been detected from several living biological organisms like mice, rats, humans, cell cultures and plants.

    Due to its technical challenges, spatially-resolved (two-dimensional, 2D) UPE measurements on human body parts are currently done only by a few research groups worldwide. Initial work on human UPE imaging has been done since the 1990s by the Japanese research group of H. Inaba, demonstrating with a 2D photomultiplier setup that human hands show an inhomogeneous spontaneous UPE pattern with the highest intensity at the fingers and the lowest at the palm region. The group also found that subjects with hypothyroidism had lower UPE values of their hands compared to a healthy control group. It was also observed that the UPE intensity at the right index finger in humans is generally higher than at the left one. To obtain the UPE images, an exposure duration of 1 h has been used, illustrating clearly the extreme low intensity of the UPE detected. In the late 1990s, it was demonstrated for the first time that UPE images can be obtained from plants and animal tissue using a charge-coupled device (CCD) camera. Subsequent UPE imaging of human body parts with cryogenically cooled CCD cameras has been performed since the 2000s. Measurements on multiple body parts showed that a high UPE emission is detectable at the face and neck and that the hands exhibit a higher UPE intensity compared to the rest of the arm. The next step in improving the technical setups to image human UPE was the usage of electron-multiplying CCD (EMCCD) cameras; first results were published in 2013 by the research group of R. van Wijk, E.P.A. van Wijk and J. van der Greef. The UPE images of the hands showed a “high variability in emission intensity and pattern”. This study is a follow-up report on these measurements. Only a few reports of UPE EMCCD imaging have been published so far. A new development in UPE imaging is the realization of whole-body UPE counting and imaging of humans as well as the correlation of UPE images with metabolomics.

  • Objective

    Our objective was to document and analyze a series of UPE images of hands obtained in 2011 by EMCCD imaging.

  • Results & Discussion

    10 UPE hand images were obtained and analyzed in the present study (i.e. 8 images of subject #1, and 2 of subject #2). The following spatio-temporal aspects of the hand UPE images were noticed (Fig. 1): (i) all 10 images show clearly that human hands exhibit autoluminescence, i.e. spontaneous UPE; (ii) there was a large variability of the UPE intensity with respect to the factors time (time of day, date), anatomical location, and individual subject; (iii) images taken at the same date (but different times of day) showed a stronger similarity than images of the same subject taken on different dates.

    Regarding the dependence of the UPE hand image features on the anatomy, the following aspect was observed: (i) the strongest UPE was measured generally at the fingers with the nails as the regions of maximal intensity; (ii) the nails of the thumbs exhibited generally especially large intensity values.

    The mean intensity of the spontaneous UPE of the hands measured at regions of interest (ROIs) at the left and right hand were time-dependent. For subject #1, mean UPE values were quadratically correlated with the times of measurement (n = 16, r = 0.668, p = 0.0065), i.e. during noon minimal UPE values were recorded (Fig. 1N).

    The mean intensity values from the ROIs were also dependent on the date. The lowest values were measured on August 31st, 2011, the highest on August 30th, 2011 (Fig. 1M). The UPE hand images of subject #2 were comparable to all 4 images of subject #2 with respect to anatomical characteristics of the UPE.

    In 4 UPE images (Fig. 1A, B, F, G)) local increase in UPE intensity could be observed on the left hand at the skin between the fingers. At one instance a UPE “hot spot” at the left lateral side of the left hand can be seen (Fig. 1G) (the subject notices that this part of the skin was quite dry during the imaging); the right hand shows two “hot spots” too, but on the left lateral side (below the thumb).

    The finding that hand UPE images exhibit a quite large variability of the intensity patterns with respect to the anatomy, subject and time, has been published. Our finding in the current study that the nails and fingers exhibit the strongest UPE intensity of the hands, is in agreement with previous studies. The reason why the nails show a strong UPE is not fully understood. Possible explanations include differences in tissue, optical properties of tissue, microbial colonization or simply remaining dirt trapped in this area and causing delayed luminescence.

    That the UPE intensity pattern varies strongly from day to day is an important finding in the current study. The time-dependence of UPE value from human body parts has been noticed already by previous investigations. In particular, Kobayashi et al. reported that the mean UPE intensity measured from 5 subjects at the face and upper body parts showed a diurnal time-dependence with a peak in afternoon and the lowest values in the morning. This is partially in agreement with our observation of a peak of the hand UPE intensity in the afternoon. Kobayashi et al. noticed also that the diurnal UPE intensity variation showed a statistically significant correlation with diurnal cortisol concentration fluctuations but not with body temperature. Cifra et al. conducted 24 h measurements of spontaneous UPE of hands in humans and observed the same intensity pattern at the dorsum as we did: a decrease in intensity during noon and an increase in early afternoon. However, their inverse quadratic function fitted to the data is phase shifted by a few hours towards the evening as compared to our observed pattern.

    Concerning the inter-subject variability of UPE intensity from human hands, it was shown by Zhao et al. that it can be explained to a large degree by the age of the subject. Since only UPE hand images of 2 subjects were investigated in our study, we could not test a possible age-correlation.

  • Conclusions

    In this study, we presented examples of the feasibility of using an EMCCD camera to measure spontaneous ultra-weak photon emission (autoluminescence) from human hands. The image analysis revealed a large spatio-temporal variability of the UPE pattern correlations to anatomical locations and the time of measurement (especially the time of day).

    The imaging of spontaneous UPE from human hands offers a new window into the (metabolic, compositional, hemodynamic) state of the tissue as well as possibly the state of the whole human subject. UPE imaging of hands (or even the whole body) might be a useful technique not only for basic research in human physiology and medical applications.

    Since it is known that spontaneous UPE is correlated with the oxidative state of the tissue (oxidative stress), it would be very interesting for future studies to quantify the oxidative stress level at different parts of the hands and capture subsequently UPE images of them, enabling to analyze the correlation of both phenomena. Since there is also a correlation with local blood flow and tissue oxygenation with UPE, it would be of interest for future studies to analyze the correlation of the local microcirculatory state of hand tissue with UPE intensity.

  • Limitations

    There are three main limitations to this study. First, only 2 subjects and only a quite small number (n = 10) of UPE images were analysed in the present study. A larger number would have made it easier to statistically investigate the dependence of the spatio-temporal characteristics of the UPE hand image patterns with several factors like the time of measurement (time of day, date) or individual subject. Second, the parallel measurement of additional local and systemic physiological parameters (like cortisol concentration, thermal image of the hand, microcirculation, melanin concentration, body temperature, heart rate, partial pressure of CO2 in the blood, blood biomarkers of oxidative stress as well as O2, CO2 and CO concentration of the air in the darkroom in combination with temperature, humidity and pressure in the darkroom) would have given additional insights into the origin of the spatio-temporal characteristics of the UPE hand image patterns. It has been shown already that the temperature of the hand is positively correlated with UPE intensity. Third, a complete removal of cosmic ray artifacts in the UPE images was not conducted since there are no standard algorithms available to perform this task and an algorithm specifically dedicated to this task is currently still under development.

  • Methods

    Camera and lens

    An iXon 888EM+ (back-illuminated) EMCCD camera (Andor Technology Ltd, Belfast, Northern Ireland) with the following specifications was used: CCD201 (e2v technologies Ltd., Essex, UK), pixel format 1024×1024, pixel size 13 µm2, 16-bits at 1 MHz readout rate, system readout noise < 1e- at 16-bit 1 MHz readout rate, peak quantum efficiency (QE) 92.5% at 575 nm, and a dark current of <<1 electrons/pixel/s. The spectral sensitivity of the CCD is in the range of about 200–1000 nm.

    The detector was cooled to -100°C with thermoelectric cooling including extra cooling by a water-cooled chiller (OasisTM 160LT, Solid State Cooling Systems, NY, USA) which was set to 15°C to avoid condensation in the camera. When using water cooling, the internal fan of the camera can be turned off and exceptional cooling performance is maintained indefinitely while absolutely no errors will be introduced due to vibrations of the internal fan.

    A Xenon 0.95/25 mm C-mount lens (Jos. Schneider Optische Werke GmbH, Bad Kreuznach, Germany) was used. It covers a 1” format sensor with a 16 mm image circle. The high aperture (f/0.95) provides good image quality even under low light conditions. Xenon lenses are designed to cover the visible spectrum (400–700 nm) whereby the overall spectral sensitivity of the camera and lens system was reduced in the UV and the IR region. A lens with a slightly smaller image circle than the diagonal of the EMCCD detector (18.8 mm) was chosen as a trade-off for higher aperture since there are not many lenses with high aperture available on the market for a C-mount of 25 mm.

    The acquisition parameters for UPE imaging of two human hands were empirically determined by imaging a representative set of subjects with different acquisition settings. There is a trade-off between imaging time and resolution (as well as signal-to-noise ratio) when choosing acquisition parameters. As a long imaging time ensures a high signal-to-noise ratio and a good resolution, it is demanding for the subject that needs to sit motionless in a completely darkroom for the respective time. For our UPE images the following settings were found to be optimal and were used: 1800 s (= 30 min) exposure time, 4×4 binning, 1.7 µs vertical shift speed, 1 MHz pixel readout rate, ‘normal’ clock amplitude, Electron Multiplying ‘on’, EM gain level= 300, pre-amplifier gain= 5.2. Pilot tests revealed that shorter exposure times than 30 min did not give enough signal intensity for some subjects, resulting in a poor signal-to-noise ratio. This could not be satisfactorily compensated by larger binning (e.g. 8×8) because cosmic ray effects and other noise on the detector would, in case of larger binning, affect a larger area of the image at the same time. The readout rate was the slowest possible to introduce less noise; a fast readout was not required for this experiment. For the two-hand imaging setup, the camera was placed at a distance of 65 cm from the hands to achieve an adequate field of view encompassing both left and right hand.

    Measurement room

    A darkroom for applications up to whole body UPE imaging was utilized. It was designed to be absolutely dark for reliable and high-quality UPE imaging of different kinds of biosystems. The darkroom itself was positioned in a temperature controlled room. It was ensured that the darkroom was free from any phosphorescent objects (walls, surfaces, etc.) that would interfere with the UPE imaging. A light lock with 2 doors was installed for entering the room without introducing stray light. The inner walls of the darkroom were covered with a black graphite paint (RAL 9005) having no phosphorescence. Electricity cables were introduced into the room through a sophisticated light-tight access. The cables were zigzagged from the bottom to the top of a double wall with alternating light blocking beams such that no light was able to come through the hole in the wall. In the inner wall, a fan was installed to introduce fresh air in the room.

    For UPE imaging of two hands simultaneously, the hands were situated on a wooden plate with 2 wooden stubs designating the points between middle and ring finger of left and right hand. The plate and stubs were painted with black graphite. To avoid possible light reflections from one hand to the other, a wooden plate or black cardboard was introduced between the hands.

    The light-tightness of the room was checked utilizing 2 procedures: (i) As conventionally done, a photomultiplier tube (PMT) was placed in the darkroom at various locations and with different orientations to estimate the signal as compared to the dark count of the PMT. This method provided first insights into light leakages. (ii) Since a PMT may have different spectral sensitivity than the EMCCD camera, a second procedure was introduced. A (dark adapted) white ball that reflects light was imaged with the EMCCD camera itself to see if a part of the ball was illuminated by stray light. Light leakage into the room relevant to the field of view could thus be located using the orientation of light on the ball. The same acquisition parameters of the camera were used for this purpose. The room was said to be completely dark when the image had similar intensity to an image with the camera shutter closed. For the UPE images of the hands shown in this manuscript this was ensured, i.e. the room was completely dark.

    Experimental protocol

    2 subjects participated in this experiment. The study was conducted in accordance with the principles of the Declaration of Helsinki. Subject #1 was a female (age: 32 years) and subject #2 a male (age: 44 years). All subjects adequately understood the study procedure and gave their oral informed consent.

    On 5 different days, UPE hand images were taken twice (one in the morning and one in the afternoon), on 4 days with subject #1 (16th, 19th, 30th and August 31st, 2011) and on one day (August 27th, 2011) with subject #2. The time difference between the UPE images taken in the morning and the afternoon was 4.2 ± 2.5 h.

    Right and left hand were imaged simultaneously with an exposure duration of 30 min. The dorsum side of the hand was imaged. Before starting the imaging, the subject was out of bright daylight for 2 h and the hands were dark-adapted for 30 min using hand gloves and once inside the darkroom, a dark-adapted black cloak was used to cover clothing to ensure that no delayed luminescence but only spontaneous UPE was imaged.

    Image processing and statistical analysis

    The UPE images were processed with Matlab (version 2013b; Natick, MA, USA). Noise from cosmic rays was removed by thresholding the images at 2000 total photon counts. Adaptive noise-removal filtering was applied to each image using a pixel-wise adaptive low-pass Wiener filter with a 5×5 pixel kernel to estimate the local image mean and standard deviation. Mean values of regions of interest (ROI) from the dorsum side of each hand (right and left) were determined by a 150×150 pixel ROI. These ROI values were used for a correlation analysis with respect to the time of day the measurements were taken. A second-order polynomial function was fitted to the data and the goodness of the fit was determined as well as the statistical significance of the correlation (t-test).

  • Ethics statement

    According to Medical Research Involving Human Subjects Act, this study did not require a medical ethics review. All subjects adequately understood the study procedure and gave their oral informed consent. The subjects measured were persons that were executing the experiments and writing the paper, i.e. no external subjects.

  • References
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    Matters10/20

    Spatio-temporal dynamics of spontaneous ultra-weak photon emission (autoluminescence) from human hands measured with an EMCCD camera: Dependence on time of day, date and individual subject

    Affiliation listing not available.
    Continuing the storyline of

  • van Wijk, R., van Wijk, E.P.A., Schroen, Y., van der Greef, J.
    Imaging human spontaneous photon emission: Historic development, recent data and perspectives
    Trends in Photochemistry & Photobiology, 15/2013, pages 27-40 chrome_reader_mode
  • Abstractlink

    Biological tissue exhibits autoluminescence, i.e. a spontaneous ultra-weak photon emission (UPE) without photoexcitation. In this study, we report UPE measurements on human hands using an electron-multiplying CCD (EMCCD) camera. The images from left and right hands (2 subjects, 10 images in total) were taken on different dates. We observed the following spatio-temporal aspects of the hand UPE images: (i) all images show clearly that human hands exhibit autoluminescence; (ii) there was large variability in the UPE intensity with respect to the factors: time (time of day, date), anatomical location on the hand, and individual subject; (iii) images taken on the same date (but at different times of day) showed a stronger similarity than images of the same subject taken on different dates; (iv) the mean UPE intensity (averaged over 2 regions of interest at the right and left dorsum of the hands) correlated with the time of measurement (lower intensity at around noon). Our study documents the feasibility of UPE imaging of hands with an EMCCD camera and shows that the spatio-temporal variability of the UPE pattern harbours information that seems to be correlated to internal and external factors.

    Figurelink

    Figure 1. EMCCD images of spontaneous UPE of human hands.

    (A-J) UPE images of 2 subjects and 5 experiments (with 2 measurements per day). The information about the time of day when the images were made is given in the headers. Subject #1: A-C, F-H. Subject #2: E and J.

    (K) Visualization of the distribution of the dates and times of day when the UPE images were performed (5 different days, 10 different times of day).

    (L) ROIs used to calculate the mean UPE values for each of the 10 images. R: right, L: left.

    (M) Dependence of the mean UPE values (ROI, left and right hands) on experiment and subject. R: right, L: left, a: first measurement, b: second measurement.

    (N) Correlation of mean UPE values of subject #1 (n = 16) with the time of day (r = 0.668, p = 0.0065).

    Introductionlink

    Whereas autofluorescence of cells and biological tissue is fluorescent light emission due to stimulated light reactions with endogenous fluorophores[1], tissue exhibits also autoluminescence, i.e. spontaneous ultra-weak photon emission (UPE) in the ultra-violet to visible/near-infrared spectral range (approx. 200–1300 nm) without photoexcitation[2][3][4]. The photon emission associated with autoluminescence is magnitudes lower compared to the case of autofluorescence, challenging the technical detection of this phenomenon. The UPE intensity has generally only an intensity in the order of 101–104 photons/s cm2[5].

    The source of the UPE due to autoluminescence is mainly biochemical, i.e. the de-excitation of energetically excited species like atoms and/or molecules to a deeper energy level generating photons[3][6][7]. The excited states are generally caused by oxidative reactions involving radical or non-radical reactive oxygen (ROS) and nitrogen (RNS) species that react with several biomolecules like lipids, proteins and nucleic acids. Also, the self-recombination of organic radicals is relevant[3][6]. For example, triple-excited carbonyls are responsible for the emission in the wavelength range of 350–550 nm, singlet excited melanin with 360–560 nm, dimolar singlet oxygen with 634 nm and 703 nm, and monomolar singlet oxygen with 1270 nm[3].

    Autoluminescence has been detected from several living biological organisms like mice[8], rats[9], humans[10], cell cultures[11] and plants[12].

    Due to its technical challenges, spatially-resolved (two-dimensional, 2D) UPE measurements on human body parts are currently done only by a few research groups worldwide. Initial work on human UPE imaging has been done since the 1990s by the Japanese research group of H. Inaba, demonstrating with a 2D photomultiplier setup that human hands show an inhomogeneous spontaneous UPE pattern with the highest intensity at the fingers and the lowest at the palm region[13][14]. The group also found that subjects with hypothyroidism had lower UPE values of their hands compared to a healthy control group[13][14]. It was also observed that the UPE intensity at the right index finger in humans is generally higher than at the left one[9]. To obtain the UPE images, an exposure duration of 1 h has been used, illustrating clearly the extreme low intensity of the UPE detected. In the late 1990s, it was demonstrated for the first time that UPE images can be obtained from plants and animal tissue using a charge-coupled device (CCD) camera[9][15]. Subsequent UPE imaging of human body parts with cryogenically cooled CCD cameras has been performed since the 2000s. Measurements on multiple body parts showed that a high UPE emission is detectable at the face and neck and that the hands exhibit a higher UPE intensity compared to the rest of the arm[16]. The next step in improving the technical setups to image human UPE was the usage of electron-multiplying CCD (EMCCD) cameras; first results were published in 2013 by the research group of R. van Wijk, E.P.A. van Wijk and J. van der Greef[10]. The UPE images of the hands showed a “high variability in emission intensity and pattern”. This study is a follow-up report on these measurements. Only a few reports of UPE EMCCD imaging have been published so far. A new development in UPE imaging is the realization of whole-body UPE counting and imaging of humans[17][18] as well as the correlation of UPE images with metabolomics[19][8][20].

    Objectivelink

    Our objective was to document and analyze a series of UPE images of hands obtained in 2011 by EMCCD imaging.

    Results & Discussionlink

    10 UPE hand images were obtained and analyzed in the present study (i.e. 8 images of subject #1, and 2 of subject #2). The following spatio-temporal aspects of the hand UPE images were noticed (Fig. 1): (i) all 10 images show clearly that human hands exhibit autoluminescence, i.e. spontaneous UPE; (ii) there was a large variability of the UPE intensity with respect to the factors time (time of day, date), anatomical location, and individual subject; (iii) images taken at the same date (but different times of day) showed a stronger similarity than images of the same subject taken on different dates.

    Regarding the dependence of the UPE hand image features on the anatomy, the following aspect was observed: (i) the strongest UPE was measured generally at the fingers with the nails as the regions of maximal intensity; (ii) the nails of the thumbs exhibited generally especially large intensity values.

    The mean intensity of the spontaneous UPE of the hands measured at regions of interest (ROIs) at the left and right hand were time-dependent. For subject #1, mean UPE values were quadratically correlated with the times of measurement (n = 16, r = 0.668, p = 0.0065), i.e. during noon minimal UPE values were recorded (Fig. 1N).

    The mean intensity values from the ROIs were also dependent on the date. The lowest values were measured on August 31st, 2011, the highest on August 30th, 2011 (Fig. 1M). The UPE hand images of subject #2 were comparable to all 4 images of subject #2 with respect to anatomical characteristics of the UPE.

    In 4 UPE images (Fig. 1A, B, F, G)) local increase in UPE intensity could be observed on the left hand at the skin between the fingers. At one instance a UPE “hot spot” at the left lateral side of the left hand can be seen (Fig. 1G) (the subject notices that this part of the skin was quite dry during the imaging); the right hand shows two “hot spots” too, but on the left lateral side (below the thumb).

    The finding that hand UPE images exhibit a quite large variability of the intensity patterns with respect to the anatomy, subject and time, has been published[10]. Our finding in the current study that the nails and fingers exhibit the strongest UPE intensity of the hands, is in agreement with previous studies[16][21]. The reason why the nails show a strong UPE is not fully understood. Possible explanations include differences in tissue, optical properties of tissue, microbial colonization or simply remaining dirt trapped in this area and causing delayed luminescence.

    That the UPE intensity pattern varies strongly from day to day is an important finding in the current study. The time-dependence of UPE value from human body parts has been noticed already by previous investigations. In particular, Kobayashi et al.[22] reported that the mean UPE intensity measured from 5 subjects at the face and upper body parts showed a diurnal time-dependence with a peak in afternoon and the lowest values in the morning. This is partially in agreement with our observation of a peak of the hand UPE intensity in the afternoon. Kobayashi et al.[22] noticed also that the diurnal UPE intensity variation showed a statistically significant correlation with diurnal cortisol concentration fluctuations but not with body temperature. Cifra et al.[23] conducted 24 h measurements of spontaneous UPE of hands in humans and observed the same intensity pattern at the dorsum as we did: a decrease in intensity during noon and an increase in early afternoon. However, their inverse quadratic function fitted to the data is phase shifted by a few hours towards the evening as compared to our observed pattern.

    Concerning the inter-subject variability of UPE intensity from human hands, it was shown by Zhao et al.[24] that it can be explained to a large degree by the age of the subject. Since only UPE hand images of 2 subjects were investigated in our study, we could not test a possible age-correlation.

    Conclusionslink

    In this study, we presented examples of the feasibility of using an EMCCD camera to measure spontaneous ultra-weak photon emission (autoluminescence) from human hands. The image analysis revealed a large spatio-temporal variability of the UPE pattern correlations to anatomical locations and the time of measurement (especially the time of day).

    The imaging of spontaneous UPE from human hands offers a new window into the (metabolic, compositional, hemodynamic) state of the tissue as well as possibly the state of the whole human subject. UPE imaging of hands (or even the whole body[17][18]) might be a useful technique not only for basic research in human physiology and medical applications.

    Since it is known that spontaneous UPE is correlated with the oxidative state of the tissue (oxidative stress)[25][26], it would be very interesting for future studies to quantify the oxidative stress level at different parts of the hands and capture subsequently UPE images of them, enabling to analyze the correlation of both phenomena. Since there is also a correlation with local blood flow and tissue oxygenation with UPE[27], it would be of interest for future studies to analyze the correlation of the local microcirculatory state of hand tissue with UPE intensity.

    Limitationslink

    There are three main limitations to this study. First, only 2 subjects and only a quite small number (n = 10) of UPE images were analysed in the present study. A larger number would have made it easier to statistically investigate the dependence of the spatio-temporal characteristics of the UPE hand image patterns with several factors like the time of measurement (time of day, date) or individual subject. Second, the parallel measurement of additional local and systemic physiological parameters (like cortisol concentration, thermal image of the hand, microcirculation, melanin concentration, body temperature, heart rate, partial pressure of CO2 in the blood, blood biomarkers of oxidative stress as well as O2, CO2 and CO concentration of the air in the darkroom in combination with temperature, humidity and pressure in the darkroom) would have given additional insights into the origin of the spatio-temporal characteristics of the UPE hand image patterns. It has been shown already that the temperature of the hand is positively correlated with UPE intensity[21]. Third, a complete removal of cosmic ray artifacts in the UPE images was not conducted since there are no standard algorithms available to perform this task and an algorithm specifically dedicated to this task is currently still under development.

    Methodslink

    Camera and lens

    An iXon 888EM+ (back-illuminated) EMCCD camera (Andor Technology Ltd, Belfast, Northern Ireland) with the following specifications was used: CCD201 (e2v technologies Ltd., Essex, UK), pixel format 1024×1024, pixel size 13 µm2, 16-bits at 1 MHz readout rate, system readout noise < 1e- at 16-bit 1 MHz readout rate, peak quantum efficiency (QE) 92.5% at 575 nm, and a dark current of <<1 electrons/pixel/s. The spectral sensitivity of the CCD is in the range of about 200–1000 nm.

    The detector was cooled to -100°C with thermoelectric cooling including extra cooling by a water-cooled chiller (OasisTM 160LT, Solid State Cooling Systems, NY, USA) which was set to 15°C to avoid condensation in the camera. When using water cooling, the internal fan of the camera can be turned off and exceptional cooling performance is maintained indefinitely while absolutely no errors will be introduced due to vibrations of the internal fan.

    A Xenon 0.95/25 mm C-mount lens (Jos. Schneider Optische Werke GmbH, Bad Kreuznach, Germany) was used. It covers a 1” format sensor with a 16 mm image circle. The high aperture (f/0.95) provides good image quality even under low light conditions. Xenon lenses are designed to cover the visible spectrum (400–700 nm) whereby the overall spectral sensitivity of the camera and lens system was reduced in the UV and the IR region. A lens with a slightly smaller image circle than the diagonal of the EMCCD detector (18.8 mm) was chosen as a trade-off for higher aperture since there are not many lenses with high aperture available on the market for a C-mount of 25 mm.

    The acquisition parameters for UPE imaging of two human hands were empirically determined by imaging a representative set of subjects with different acquisition settings. There is a trade-off between imaging time and resolution (as well as signal-to-noise ratio) when choosing acquisition parameters. As a long imaging time ensures a high signal-to-noise ratio and a good resolution, it is demanding for the subject that needs to sit motionless in a completely darkroom for the respective time. For our UPE images the following settings were found to be optimal and were used: 1800 s (= 30 min) exposure time, 4×4 binning, 1.7 µs vertical shift speed, 1 MHz pixel readout rate, ‘normal’ clock amplitude, Electron Multiplying ‘on’, EM gain level= 300, pre-amplifier gain= 5.2. Pilot tests revealed that shorter exposure times than 30 min did not give enough signal intensity for some subjects, resulting in a poor signal-to-noise ratio. This could not be satisfactorily compensated by larger binning (e.g. 8×8) because cosmic ray effects and other noise on the detector would, in case of larger binning, affect a larger area of the image at the same time. The readout rate was the slowest possible to introduce less noise; a fast readout was not required for this experiment. For the two-hand imaging setup, the camera was placed at a distance of 65 cm from the hands to achieve an adequate field of view encompassing both left and right hand.

    Measurement room

    A darkroom for applications up to whole body UPE imaging was utilized. It was designed to be absolutely dark for reliable and high-quality UPE imaging of different kinds of biosystems. The darkroom itself was positioned in a temperature controlled room. It was ensured that the darkroom was free from any phosphorescent objects (walls, surfaces, etc.) that would interfere with the UPE imaging. A light lock with 2 doors was installed for entering the room without introducing stray light. The inner walls of the darkroom were covered with a black graphite paint (RAL 9005) having no phosphorescence. Electricity cables were introduced into the room through a sophisticated light-tight access. The cables were zigzagged from the bottom to the top of a double wall with alternating light blocking beams such that no light was able to come through the hole in the wall. In the inner wall, a fan was installed to introduce fresh air in the room.

    For UPE imaging of two hands simultaneously, the hands were situated on a wooden plate with 2 wooden stubs designating the points between middle and ring finger of left and right hand. The plate and stubs were painted with black graphite. To avoid possible light reflections from one hand to the other, a wooden plate or black cardboard was introduced between the hands.

    The light-tightness of the room was checked utilizing 2 procedures: (i) As conventionally done, a photomultiplier tube (PMT) was placed in the darkroom at various locations and with different orientations to estimate the signal as compared to the dark count of the PMT. This method provided first insights into light leakages. (ii) Since a PMT may have different spectral sensitivity than the EMCCD camera, a second procedure was introduced. A (dark adapted) white ball that reflects light was imaged with the EMCCD camera itself to see if a part of the ball was illuminated by stray light. Light leakage into the room relevant to the field of view could thus be located using the orientation of light on the ball. The same acquisition parameters of the camera were used for this purpose. The room was said to be completely dark when the image had similar intensity to an image with the camera shutter closed. For the UPE images of the hands shown in this manuscript this was ensured, i.e. the room was completely dark.

    Experimental protocol

    2 subjects participated in this experiment. The study was conducted in accordance with the principles of the Declaration of Helsinki. Subject #1 was a female (age: 32 years) and subject #2 a male (age: 44 years). All subjects adequately understood the study procedure and gave their oral informed consent.

    On 5 different days, UPE hand images were taken twice (one in the morning and one in the afternoon), on 4 days with subject #1 (16th, 19th, 30th and August 31st, 2011) and on one day (August 27th, 2011) with subject #2. The time difference between the UPE images taken in the morning and the afternoon was 4.2 ± 2.5 h.

    Right and left hand were imaged simultaneously with an exposure duration of 30 min. The dorsum side of the hand was imaged. Before starting the imaging, the subject was out of bright daylight for 2 h and the hands were dark-adapted for 30 min using hand gloves and once inside the darkroom, a dark-adapted black cloak was used to cover clothing to ensure that no delayed luminescence but only spontaneous UPE was imaged.

    Image processing and statistical analysis

    The UPE images were processed with Matlab (version 2013b; Natick, MA, USA). Noise from cosmic rays was removed by thresholding the images at 2000 total photon counts. Adaptive noise-removal filtering was applied to each image using a pixel-wise adaptive low-pass Wiener filter with a 5×5 pixel kernel to estimate the local image mean and standard deviation. Mean values of regions of interest (ROI) from the dorsum side of each hand (right and left) were determined by a 150×150 pixel ROI. These ROI values were used for a correlation analysis with respect to the time of day the measurements were taken. A second-order polynomial function was fitted to the data and the goodness of the fit was determined as well as the statistical significance of the correlation (t-test).

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    According to Medical Research Involving Human Subjects Act, this study did not require a medical ethics review. All subjects adequately understood the study procedure and gave their oral informed consent. The subjects measured were persons that were executing the experiments and writing the paper, i.e. no external subjects.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

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