Picosecond Fluorescence Spectroscopy By Time-Correlated Single-Photon Counting

Photophysics of ethidium bromide complexed to ct-DNA: a maximum entropy study

Excitation energy transfer pathways and mechanisms in the antenna systems of oxygenic photosynthetic organisms were discussed from the viewpoint of methodology of analyses. Time-resolved fluorescence spectroscopy in the picosecond and femtosecond time range and its analytical method were applied to intact cells of cyanobacteria and one species of cryptophyceae, furthermore, a unique phycobiliproteins of cyanobacteria, APC-B. Finally, the most abundant antenna complex of higher plants, that is, light-harvesting complex II (LHC II) was described in detail on the molecular arrangement of chlorophyll molecules and energy transfer pathways among those molecules.

This report with recommendations is the result of an expert panel meeting on PAT applications in food industry that was organized by the M3C Section of the European Society of Biochemical Engineering Science (ESBES) at the 10th ESBES Symposium. The aim of the panel was to provide an update on the present status of the subject and to identify critical needs and issues for wider applications of PAT in food industry. A brief description of the current state-of-the-art and industrial uptake of the methodology is provided in this report. It concludes with a number of recommendations to facilitate further developments and a wider application of PAT in food industry. Process Analytical Technologies (PAT) [1] (European Medicines Agency EMA-FDA pilot program for parallel assessment of Quality by Design applications; Document EMA/172347/2011) have been extensively discussed in literature, particularly with respect to (bio)pharmaceutical process modelling, monitoring and control [2]. Table 1 provides an historic overview of the PAT development in the context of food applications. Although successful applications within food industry are increasingly being reported [3, 4], the session on PAT in food industries at the ESBES-IFIBiop 2014 in Lille highlighted significant challenges and opportunities for further development in this area. This position paper briefly reviews the current state-of-the-art, industrial needs and opportunities as well as scientific challenges to be addressed in order to extend the use of PAT methodology in the food industries. Currently, quality and safety control are still based mainly on discontinuous analysis with traditional analytical methods in the lab or, at best, at-line measurements. This is no longer sufficient to fulfill the needs of the food industry. Due to higher safety and quality standards and demands and high throughput of production facilities, the number of samples to be analyzed is increasing. Rapid analysis methods and PAT are required to address these needs along the complete production chain leading to a better understanding and control of raw materials, intermediate products in the production process as well as the final products to be packaged and delivered. The goal is to achieve real time analysis in order: • to avoid usage of any out-of-specification raw material and to detect adulteration, substitution, tampering and counterfeiting leading to non-authentic products; • to be able to intervene and stop/change processes in order to secure the target quality; • to assess the final quality to avoid out-of-specification products being packaged and shipped, thus leading to undesirable customer dissatisfaction and costs associated with the resolution of complaints. Over a hundred years ago (on May 22nd, 1913) the first patent on a PAT device ("Pfeiffenanalysator" for measuring the ratio of H2 and N2 gas for ammonia synthesis) was granted to Paul Gmelin from Badische Anilin- und Soda-Fabrik, BASF (Patentschrift Nr. 281157, Klasse 42/. Gruppe 4). Since then, PAT found broad application in chemical industry, which is dominated by highly automated, continuous processes. Today in chemical processes, such as the synthesis of isocyanates, e.g. hexamethylendiisocyanate (HDI), typically 60-130 PAT measurements are collected. In contrast, in sectors like (bio)pharmaceutical or food industry, the application of PAT is significantly less frequent today, especially, in terms of on-line analytics, where the measurement takes place in or close to the production step (or unit operation). One reason for this is the reduced degree of automation of the processes in these industries, which are dominated by unit operations, single production steps consecutively executed after each other, resulting in production batches. Analytics of a production batch takes place mostly off-line in analytical laboratories during hold-up times of the process intermediate between the different unit operations. Regulatory requirements represent an equally important driver for extensive online analytics. With the 2004 FDA's Quality by Design initiative, regulatory agencies demand systematic risk based process development and understanding of the processing space. This also allows for flexible process adjustments within the explored space for producing the desired product quality. In order to achieve the required understanding, more extensive analytical data is required, ideally obtained in-time on raw materials, process intermediates and ideally also on product quality attributes. In food industry, where typically high product titers are obtained, product analytics is sometimes possible by in-line measurements, such as spectroscopy (section 4). In biopharmaceutical processes, product titers are usually very low, the products are proteins with high molecular weight, and quality attributes are challenging to analyse. In these situations, online sampling with automated sample preparation and analytics may open up a solution [5]. But, identifying and implementing analytical methods, which are fast enough for delivering results on product quality attributes (e.g. protein glycosylation) in time, during the unit operation for allowing process modulation, remains a challenge. The overall typical goals in industry are high safety, high and stable product quality, high yield, low consumption of resources (materials, energy, room, time, and people), reduced influence of variability in raw materials as well as an increased shelf life of products which holds also in food processes. However, compared to the chemical and biopharmaceutical industries, food industry has to deal with certain characteristics, which renders PAT a formidable challenge in this context. The characteristic features in food industry are: • Raw materials are not pure substances: they are complex combinations of pure substances with varying compositions • Raw materials are soft, variable size, fragile and slippery • Physical properties of raw materials depend heavily on temperature, pressure, moisture and harvesting, and storage conditions • Raw materials undergo usually a phase transformation during processing and hence change their physical properties during processing • Micro processes (physical, (bio)-chemical, microbiological) are frequently not known • Highly perishable products. Here the challenge is to produce good quality, which maintains for a long time (shelf life) • High demand for hygiene These characteristics lead to a challenge for the application of sensors in the food industry. For important quality and process variables, such as sensory assessment, micro flora or spoilage, reliable and robust sensors are not yet available. Moreover, the sensors which are available are typically used only in isolated applications and frequently provide insufficient reliability. They are often not integrated in a common data management infrastructure. The materials of construction do not always consider suitability of contact with food and the solutions developed within advanced research projects frequently demand high care and maintenance. One reason why PAT is not as common as in other areas is that in food industry knowledge from various subject areas is necessary (such as physics, chemistry, biology, mathematics, informatics, engineering, nutritional science). For unit operations mathematical models are available in principle, but frequently they are too complicated to adapt. One of the biggest challenges in food industries is the dynamic nature of the processes. Changes in geometry, porosity, microstructure, solubility as well as mass and energy transfer coefficients must be addressed. Mostly gradients of temperature and moisture have to be considered as well as changes in kinetic parameters during process run. End product qualities like color, smell and 'brokens' also strongly influence the process. Therefore, food processes can be considered to be significantly more complex then chemical and bio-pharmaceutical processes. To achieve more stable processes PAT applications are necessary to check the quality of the raw and processed materials and their relationship to each other. If the quality is changing, then control actions resulting in online parameter changes are required in order to maintain constant product quality. Measurement systems are required, which guarantee that the process is in accordance with recipe and formulation. Furthermore, disturbances must be compensated for using control actions to reduce process variability and to conform to food regulations. As Glassey [6] argues, PAT methodology can aid in product design and testing as well as in ensuring full compliance with the HACCP and ISO 22000:2005 requirements during processing. As demonstrated below, PAT methodologies have the potential to aid the identification of critical control points and their critical limits, their effective monitoring and control, but also effective communication with suppliers and customers. Documentation which requires measurements of important variables is especially important so that traceability can be guaranteed. Here PAT has to deliver the corresponding measurement systems. Clearly a high demand for PAT in food industry is evident as is the need for further developments in the science and technology that help address the specific challenges posed by the characteristics of the food industry highlighted above. The complexity of raw materials that are typically soft and easily damageable is arguably one of the major scientific and technological challenges in food industry. During storage and processing the quality of such raw materials can decline due to oxidation processes, pressure and temperature effects. In addition, the visual impression of the final product (i.e. its appearance) is much more important than in other industries due to the fact that this will influence the purchase decision of the consumer. During the whole processing from raw material to the storage of the final product, hygiene is of utmost importance. Therefore non-invasive sensor systems are required in these applications. Measurement systems based on optical principles are particularly suitable from this perspective. Such measurement systems, including near infrared (NIR), Raman and fluorescence spectroscopy as well as computer-assisted image-based systems, potentially have a number of advantages in food process supervision and automation. Spectroscopic methods and imaging devices are well suited for PAT purposes because they are fast, non-destructive, provide multiple chemical information, allowing remote in-process analysis via fiber optics or instruments mounted directly on-line. Fluorescence spectroscopy is the most sensitive spectroscopic technique. Recently, many applications have been developed using fluorescence techniques [3, 7, 8]. Raw materials, the supervision of processing as well as product quality and the contamination of the equipment can be monitored by fluorescence. For example, Everard et al. [3] presented a method for detection of fecal contamination on spinach leaves. They coupled three hyperspectral imaging (HSI) configurations with two multivariate image analysis techniques and compared fluorescence imaging in the visible region with ultra violet and violet excitation sources, and reflectance imaging in the visible to near-infrared regions. They showed, that both fluorescence configurations had 100% detection rates for fecal contamination up to 1:10 dilution level and violet HSI had 99% and 87% detection rates for 1:20 and 1:30 levels, respectively. Everard et al. emphasized that on-line detection of fecal contamination on leaves has the potential to reduce the cases of food borne illnesses and their associated costs. A similar approach is presented by Lee et al. [7] where bovine faeces on Romaine lettuce and baby spinach leaves were investigated. They pointed out that two-band ratios using bands at 665.6 nm and 680.0 nm for lettuce and at 660.8 nm and 680.0 nm for spinach effectively differentiated all contamination spots applied. Grote et al. [8] described a fluorescence measurement technique to monitor a sourdough fermentation process. For the prediction of pH value and acidity during rye sourdough fermentations they applied partial least squares regression and principal component regression models for prediction and compared them with an evaluation where principal component analysis was combined with artificial neural networks. Depending on process operation and evaluation technique the average percentage root mean square errors of prediction for pH values were between 2.5 and 5.1%. For the prediction of the acidity level, the best results were between 6.0 and 8.1%. Liu et al. [9] used the Hoffman reaction to convert acrylamide to a compound which shows strong fluorescence emission at 480 nm. They showed good correlation of acrylamide in the range of 0.015 μg/mL to 20 μg/mL. Using this technique the food security will be increased. A fluorescence imaging device to detect deli residues on deli slicers were used by Beck et al. [10] processing four cheeses and four processed meats. The authors suggested that a fluorescence imaging device can be applied for routine use even in delicatessens. Fig. 1 clearly shows that the application of fluorescence for the monitoring of food processes increased from less than 10 before 2003 to more than 60 a year since 2013. This figure includes all papers from a search containing the key words "Food" and "Fluorescence". Although most of these papers describe laboratory applications rather than industrial PAT applications, they indicate potential future applications in this technology in food industry. Number of published papers obtained from the Scopus database searching for the words "Food" and "Image analysis" or "Raman" or "Near infrared spectroscopy" or "Fluorescence spectroscopy". Raman spectroscopy can only be applied if no fluorescence occurs in the corresponding excitation range. He et al. [11] applied the surface enhanced Raman scattering spectroscopy to detect banned food additives, such as Sudan I dye and Rhodamine B in food, Malachite green residues in aquaculture fish. They concluded, that Raman spectroscopy and chemometric evaluation techniques can be used to identify banned food additives to ensure food safety. Ilaslan et al. [12] presented a method based on Raman spectroscopy to provide a rapid method for evaluating the quantitative analysis of glucose, fructose, and sucrose in soft drinks. Wang et al. [13] applied a Raman spectrometer as a process analyzer to monitor the wine fermentation. They demonstrated that sugar, ethanol and glycerol can be measured on-line with high correlation (higher than 0.98) to the HPLC reference measurements. Nache et al. [14] investigated Raman spectra from pork meat to monitor the early postmortem lactate accumulation and pH decline. They suggested that the locally weighted regression applied to the standard normal variate (SNV) normalized Raman spectra provide one of the most accurate and robust models with a cross-validated coefficient of determination (r2cv) of 0.97 for pH and lactate, a cross-validated root mean square error (RMSECV) of 4.5 mmol/kg for the lactate prediction and 0.06 pH-units for the pH prediction. These results demonstrate significant potential of combining chemometrics and Raman spectroscopy for on-line meat quality control applications. Fig. 1 shows the number of papers recorded in Scopus (search words "Food" and "Raman"). Compared to fluorescence, the number of papers describing Raman spectroscopy is twice as high in recent years with a significant rate of increase, demonstrating the increasing interest in this method for food process monitoring. Computer vision systems enable one of the main aspects of consumer preference – the appearance of a product – to be inherently considered. Therefore, computer vision systems for the supervision of food processing also gained importance over the years. An overview of several examples is given by Sun [15]. Especially the supervision of food drying processes is discussed by Aghbashlo at al. [16]. They pointed out that there is a large unexploited potential in the image data captured during various food processing operations. More informative feature extraction algorithms or novel pattern recognition procedures have to be developed. Paquet-Durand et al. [4] described a system for the supervision of the baking process. They demonstrated, by using the Viola–Jones-algorithm as well as neural networks, that the pastry can be identified and the volume increase as well as color development can be monitored. To detect defective apples Zhang et al. [17] used a computer vision system, which was combined with an automatic lightness correction system. For 160 samples they showed a 95% overall detection accuracy. For the evaluation they used a weighted relevance vector machine classifier. Fig. 1 shows the number of papers over time from Scopus searching for "Food" and "Image analysis". Here the applications start earlier compared to the spectroscopic methods, the number of applications per year is higher than in the case of spectroscopic applications; except in the year 2014, where more reports of Raman applications were published. Moreover imaging systems using visible and NIR region are available to perform quality checks based on the spectroscopic information derived from each point in the image. One example is the on-line analysis of the widely varying fat distribution in salmon for sorting purposes [18]. Near infrared (NIR) is a well-established method for rapid analysis of food raw materials and products [18], either on-/in-line, at-line or off-line. In the lab or at-line multiple components such as fat, protein, moisture and many more, can be analyzed without any sample preparation in all kinds of liquid, solid and semi-solid samples. Raw materials can be verified for identity and further characterized regarding composition already in the goods reception. The composition, freshness and adulteration of edible oils [19], e.g. olive oil [20], can be analyzed rapidly before a truck is unloaded. Many other sample types like meat, grains, flour, dairy products [21] and others can be analyzed. Even mixtures like vitamin premixes can be analyzed to make sure that the correct material was delivered and can be used in production. There are several approaches and technologies for PAT available, ranging from simple filter based devices over dispersive diode array spectrometers to Fourier-Transform (FT)-NIR instruments. Samples can be analyzed in-line by fiber optic probes or by contactless systems (Fig. 2). Smaller instruments can be directly attached to pipes, chutes or in other installations. A) NIR reflection probe in a fluid bed dryer. (B) Probe head for contactless measurement of grain and other solids. Applications cover a broad range from a simple monitoring of moisture content of a product on a conveyor belt to more complex control situations. In dairy industry there are demands to control milk powder production by monitoring the feed of the spray tower and the powder after fluid bed drying. Other important production steps to control are the standardization of milk, cream, whey and concentrates [22]. In frying processes with big volumes of oil an in-line monitoring of the oil quality with regards to acid value, anisidine value and the content of polar and polymerized components [23] are of particular interest. Another example of high volume processes where monitoring of protein, moisture and ash is important is the milling of grain to flour and the production of cereals of any kind. Finally there are more and more fermentation processes controlled by in-line NIR to optimize the conditions and follow the feeding and consumption cycles during biomass buildup [24]. In 2013 roughly 120 papers were published, however the recent increase is not as steep as in the case of Raman spectroscopy (Fig. 1). The economic benefits due to PAT can be attributed to higher product quality and yield as well as decreased product variation. This will reduce the overall production costs significantly and increase competitiveness. Furthermore, the knowledge and understanding of the process will increase. However, the level of automation in industrial food processes is significantly lower compared to the chemical and pharmaceutical processes. This is partly due to the complexity of food processes. On the other hand, the lack of reliable sensor systems to determine important process parameters and variables contributed to this in the past. Sensors represent a fundamentalpart of all automation systems, although they only represent a part of the requirements for wider application of PAT in food industry. The areas of further development and recommendations enabling more extensive use of the PAT within this industry also include: • Further industrial case studies and wider dissemination of the positive impact of reliable sensor technology in raw material and product quality as well as process monitoring. • Operational and maintenance requirements of sensor technologies will remain a significant aspect of future sensor technology uptake in this industry. • Data management and analysis software will play a significant role in extending the PAT application within the food industry. Platform solutions, preferably supporting wireless data transfer, at competitive pricing levels are required. • Cost benefit analysis studies are particularly important given the business drivers of this industry. • The impact of PAT is likely to be more pronounced in the new processes, although it is important to continue to encourage the application of PAT approaches within established food processes to enhance the process economics and compliance with the standards ensuring safe food supply chains. • PAT can significantly contribute to the stringent traceability and documentation requirements, although issues of compatibility and data standardization will impact upon data management systems employed by the companies within the supply chain. The authors declare no financial or commercial conflict of interest.

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH₂ provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

A unique tissue phantom is reported here that mimics the optical and acoustical properties of biological tissue and enables testing and validation of a dual-modality clinical diagnostic system combining time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasound backscatter microscopy (UBM). The phantom consisted of contrast agents including silicon dioxide particles with a range of diameters from 0.5 to 10 μm acting as optical and acoustical scatterers, and FITC-conjugated dextran mimicking the endogenous fluorophore in tissue. The agents were encapsulated in a polymer bead attached to the end of an optical fiber with a 200 μm diameter using a UV-induced polymerization technique. A set of beads with fibers were then implanted into a gel-based matrix with controlled patterns including a design with lateral distribution and a design with successively changing depth. The configuration presented here allowed the validation of the hybrid fluorescence spectroscopic and ultrasonic system by detecting the lateral and depth distribution of the contrast agents, as well as for coregistration of the ultrasonic image with spectroscopic data. In addition, the depth of the beads in the gel matrix was changed to explore the effect of different concentration ratio of the mixture on the fluorescence signal emitted.

This thesis was aimed at investigating the physical-chemical properties and the behaviour in physiological environment of two classes of bioceramics: calcium silicate-based dental cements and alumina-based femoral heads for hip joint prostheses. The material characterization was performed using spectroscopic techniques such as that allow to obtain information on the molecular structure of the species and phases present in the analyzed samples. Raman, infrared and fluorescence spectroscopy was principally used. Calcium silicate cements, such as MTA (Mineral Trioxide Aggregate), are hydraulic materials that can set in presence of water: this characteristic makes them suitable for oral surgery and in particular as root-end filling materials. With the aim to improve the properties of commercial MTA cements, several MTA-based experimental formulations have been tested with regard to bioactivity (i.e. apatite forming ability) upon ageing in simulated body fluids. The formation of a bone-like apatite layer may support the integration in bone tissue and represents an essential requirement for osteoconduction and osteoinduction. The spectroscopic studies demonstrated that the experimental materials under study had a good bioactivity and were able to remineralize demineralized dentin. . Bioceramics thanks to their excellent mechanical properties and chemical resistance, are widely used as alternative to polymer (UHMWPE) and metal alloys (Cr-Co) for hip-joint prostesis. In order to investigate the in vivo wear mechanisms of three different generations of commercial bioceramics femoral heads (Biolox®, Biolox® forte, and Biolox® delta), fluorescence and Raman spectroscopy were used to investigate the surface properties and residual stresses of retrieved implants. Spectroscopic results suggested different wear mechanisms in the three sets of retrievals. Since Biolox® delta is a relatively recent material, the Raman results on its retrievals has been reported for the first time allowing to validate the in vitro ageing protocols proposed in the literature to simulate the effects of the in vivo wear.

Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.

In order to provide an alternative to day-old chick culling in the layer hatcheries, a noninvasive method for egg sexing is required at an early stage of incubation before onset of embryo sensitivity. Fluorescence and Raman spectroscopy of blood offers the potential for precise and contactless in ovo sex determination of the domestic chicken (Gallus gallus f. dom.) eggs already during the fourth incubation day. However, such kind of optical spectroscopy requires a window in the egg shell, is thus invasive to the embryo and leads to decreased hatching rates. Here, we show that near infrared Raman and fluorescence spectroscopy can be performed on perfused extraembryonic vessels while leaving the inner egg shell membrane intact. Sparing the shell membrane makes the measurement minimally invasive, so that the sexing procedure does not affect hatching rates. We analyze the effect of the membrane above the vessels on fluorescence signal intensity and on Raman spectrum of blood, and propose a correction method to compensate for it. After compensation, we attain a correct sexing rate above 90% by applying supervised classification of spectra. Therefore, this approach offers the best premises towards practical deployment in the hatcheries.

Fast Cherenkov and scintillation detectors based on microchannel-plate photomultiplier tubes and silicon photomultipliers are being developed for application in the multi-purpose detector and baryonic matter at the Nuclotron experiments with the heavy ion beams at the Joint Institute for Nuclear Research. The aim of the detectors is the fast triggering of nucleus–nucleus collisions with high efficiency and to generate the start signal with picosecond time resolution for time-of-flight measurements. The detectors provide a time resolution better than 50 ps and can operate in the strong magnetic field of the experimental facilities.

We report on work to develop a system with about 100 picoseconds (ps) time resolution for time of flight positron emission tomography [TOF-PET]. The chosen photo detectors for the study were Silicon Photomultipliers (SiPM's). This study was based on extensive experience in studying timing properties of SiPM's. The readout of these devices used the commercial high speed digitizer DRS4. We applied different algorithms to get the best time resolution of 155 ps Guassian (sigma) for a LYSO crystal coupled to a SiPM. We consider the work as a first step in building a prototype TOF-PET module. The field of positron-emission-tomography (PET) has been rapidly developing. But there are significant limitations in how well current PET scanners can reconstruct images, related to how fast data can be acquired, how much volume they can image, and the spatial and temporal resolution of the generated photons. Typical modern scanners now include multiple rings of detectors, which can image a large volume of the patient. In this type of scanner, one can treat each ring as a separate detector and require coincidences only within the ring, or treat the entire region viewed by the scanner as a single 3 dimensional volume. This 3d technique has significantly better sensitivity since more photon pair trajectories are accepted. However, the scattering of photons within the volume of the patient, and the effect of random coincidences limits the technique. The advent of sub-nanosecond timing resolution detectors means that there is potentially much better rejection of scattered photon events and random coincidence events in the 3D technique. In addition, if the timing is good enough, then the origin of photons pairs can be determined better, resulting in improved spatial resolution - so called 'Time-of-Flight' PET, or TOF-PET. Currently a lot of activity has occurred in applications of SiPMs for TOF-PET. This is due to the devices very good time resolution, low profile, lack of high voltage needed, and their non-sensitivity to magnetic fields. While investigations into this technique have begun elsewhere, we feel that the extensive SiPM characterization and data acquisition expertise of Fermilab, and the historical in-depth research of PET imaging at University of Chicago will combine to make significant strides in this field. We also benefit by a working relationship with the SiPM producer STMicroelectronics (STM).

The Mars 2020 mission will analyze samples in situ and identify any that could have preserved biosignatures in ancient habitable environments for later return to Earth. Highest priority targeted samples include aqueously formed sedimentary lithologies. On Earth, such lithologies can contain fossil biosignatures as aromatic carbon (kerogen). In this study, we analyzed nonextracted kerogen in a diverse suite of natural, complex samples using colocated UV excitation (266 nm) time-gated (UV-TG) Raman and laser-induced fluorescence spectroscopies. We interrogated kerogen and its host matrix in samples to (1) explore the capabilities of UV-TG Raman and fluorescence spectroscopies for detecting kerogen in high-priority targets in the search for possible biosignatures on Mars; (2) assess the effectiveness of time gating and UV laser wavelength in reducing fluorescence in Raman spectra; and (3) identify sample-specific issues that could challenge rover-based identifications of kerogen using UV-TG Raman spectroscopy. We found that ungated UV Raman spectroscopy is suited to identify diagnostic kerogen Raman bands without interfering fluorescence and that UV fluorescence spectroscopy is suited to identify kerogen. These results highlight the value of combining colocated Raman and fluorescence spectroscopies, similar to those obtainable by SHERLOC on Mars 2020, to strengthen the confidence of kerogen detection as a potential biosignature in complex natural samples. Key Words: Raman spectroscopy-Laser-induced fluorescence spectroscopy-Mars Sample Return-Mars 2020 mission-Kerogen-Biosignatures. Astrobiology 18, 431-453.

Time-of-flight resolution of scintillating counters with Burle 85001 microchannel plate photomultipliers in comparison with Hamamatsu R2083

TORCH is a novel time-of-flight detector, designed to provide charged particle identification of pions, kaons and protons in the momentum range 2–20 GeV/c over a 9.5 m flight path. A detector module, comprising a 10 mm thick quartz plate, provides a source of Cherenkov photons which propagate via total internal reflection to one end of the plate. Here, the photons are focused onto an array of custom-designed Micro-Channel Plate Photo-Multiplier Tubes (MCP-PMTs) which measure their positions and arrival times. The target time resolution per photon is 70 ps which, for 30 detected photons per charged particle, results in a 10–15 ps time-of-flight resolution. A 1.25 m length TORCH prototype module employing two MCP-PMTs has been developed, and tested at the CERN PS using a charged hadron beam of 8 GeV/c momentum. The construction of the module, the properties of the MCP-PMTs and the readout electronics are described. Measurements of the collected photon yields and single-photon time resolutions have been performed as a function of particle entry points on the plate and compared to expectations. These studies show that the performance of the TORCH prototype approaches the design goals for the full-scale detector.

A new setup has been made at Fermilab for detector timing measurements at picosecond level. The core timing resolution of the amplifiers, discriminators and TAC/ADC combination is approximately 2 picosecond. We have made a study of a single photoelectron time resolution (SPTR) measured for signals coming from silicon photomultipliers (SiPm) made by different manufacturers. The obtained SPTR is of the order of 180 picosecond with SiPms illuminated by red (635 nm) PiLas laser light. IRST SiPms show better SPTR when illuminated by the blue laser light (405 nm). Most of the data were taken with 1 Volt of the overvoltage. The SiPms time resolution is inversely proportional to the square root of the number of photoelectrons. A time-of-flight (TOF) system with few tens of picosecond time resolution, based SiPms with quartz Cherenkov radiators looks practically achievable. A simple model is proposed to explain the difference in SPTR of the IRST SiPms when illuminating by the blue and red light. The explanation of the origin of the tail in the MPPC SiPm's single photoelectron time spectra is presented. Finally, requirements for the SiPms temperature and bias voltage stability to maintain few picosecond time resolution are discussed.

Time-correlated single photon counting (TCSPC) is a widely used, sensitive, precise, robust and mature technique to measure photon arrival times in applications such as fluorescence spectroscopy and microscopy, light detection and ranging (lidar) and optical tomography. Wide-field TCSPC detection techniques, where the position and the arrival time of the photons are recorded simultaneously, have seen several advances in the last few years, from the microsecond to the picosecond time scale. Here, we summarise some of our recent work in this field with emphasis on microsecond resolution phosphorescence lifetime imaging (PLIM) and nanosecond fluorescence lifetime imaging (FLIM) microscopy.