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Through the use of bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single-cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, noninvasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single-cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio during the night compared to the day. Furthermore, we demonstrate that single-cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that, in proliferating stem cells, the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity.

We have developed a two-photon fluorescence microscope capable of imaging up to 4mm in turbid media with micron resolution. The key feature of this instrument is the innovative detector, capable of collecting emission photons from a wider surface area of the sample than detectors in traditional two-photon microscopes. This detection scheme is extremely efficient in the collection of emitted photons scattered by turbid media which allows eight fold increase in the imaging depth when compared with conventional two-photon microscopes. Furthermore, this system also has in-depth fluorescence lifetime imaging microscopy (FLIM) imaging capability which increases image contrast. The detection scheme captures emission light in a transmission configuration, making it extremely efficient for the detection of second harmonic generation (SHG) signals, which is generally forward propagating. Here we present imaging experiments of tissue phantoms and in vivo and ex vivo biological tissue performed with this microscope.

We describe a novel two-photon fluorescence microscopy system capable of producing high-quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes.

We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons. © 2013 SPIE.

We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons. © 2013 SPIE.

Abstract.We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, while maintaining high resolution imaging at millimeter depth. In Lgr5-GFP mice, we have been successful in imaging Lgr5-eGFP positive stem cells, present in SI and colon crypt bases.

Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).

Recently we described a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. This technique was applied to in vivo imaging of murine small intestine and colon. Individuals with Inflammatory Bowel Disease (IBD), commonly presenting as Crohn's disease or Ulcerative Colitis, are at increased risk for developing colorectal cancer. We have developed a Giα2 gene knock out mouse IBD model that develops colitis and colon cancer. The challenge is to study the disease in the whole animal, while maintaining high resolution imaging at millimeter depth. In the Giα2-/- mice, we have been successful in imaging Lgr5-GFP positive stem cell reporters that are found in crypts of niche structures, as well as deeper structures, in the small intestine and colon at depths greater than 1mm. In parallel with these in vivo deep tissue imaging experiments, we have also pursued autofluorescence FLIM imaging of the colon and small intestine-at more shallow depths (roughly 160μm)- on commercial two photon microscopes with excellent structural correlation (in overlapping tissue regions) between the different technologies. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).

The depth of two-photon fluorescence imaging in turbid media can be significantly enhanced by the use of the here described fluorescence detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector we were able to perform imaging of turbid samples, simulating brain tissue, at depths up to 3 mm, where the two-photon induced fluorescence signal is too weak to be detected by means used in conventional two-photon microscopy.

Bertrand's paradox (Bertrand 1889 "Calcul des Probabilites" (Paris: Gauthier-Villars)) can be considered as a cautionary memento, to practitioners and students of probability calculus alike, of the possible ambiguous meaning of the term "at random" when the sample space of events is continuous. It deals with the existence of different possible answers to the following question: what is the probability that a chord, drawn at random in a circle of radius "R", is longer than the side R[square root]3 of an inscribed equilateral triangle? Physics can help to remove the ambiguity by identifying an actual experiment, whose outcome is obviously unique and prescribes the physical variables to which the term "random" can be correctly applied. In this paper, after briefly describing Bertrand's paradox, we associate it with an experiment, which is basically a variation of the famous Buffon's needle experiment (Buffon 1733 "Histoire de l'Acad. Roy. des. Sciences" pp 43-5; Buffon 1777 "Histoire naturelle, generale et particuliere" Supplement 4 46) for estimating the value of [pi]. Its outcome is compared with the analytic predictions of probability calculus, that is with the probability distribution of variables whose uniform distribution can be considered a sound implementation of complete randomness. (Contains 6 figures.)

Background: There are only two countries in the world (the United States and New Zealand) that allow the pharmaceutical branch to advertise prescription medication directly to consumers. There is pressure on governments to allow direct-to-consumer advertising (DTCA) for prescription drugs elsewhere too. One argument the industry uses frequently is the claim that exposure to DCTA, through various methods and occasions, is supposed to improve customers' knowledge of a disease and treatment. This argument has been part of the health care community's wider discussion of whether DTCA of prescription drugs benefits the population's general interest or is only an attempt to increase the sales of the pharmaceutical branch. Belief in true learning by DTCA is rooted in concepts of empowered consumers and their autonomous and empowered decision-making. Objective: In this study, we tested the hypotheses that contact with DTCA increases recipients' literacy/knowledge, especially regarding the side effects of treatment (hypothesis 1), and empowerment (hypothesis 2). We further hypothesized that DTCA exposure would not increase depression knowledge (ie, about treatments, symptoms, and prevalence) (hypothesis 3). Methods: A snowball sample of 180 participants was randomly split into three experimental groups receiving (1) a traditional information sheet, (2) a DTCA video clip for an antidepressant prescription drug, or (3) both. The video was original material from the United States translated into Italian for the experiment. Dependent variables were measures of depression knowledge (regarding treatments, symptoms and prevalence, and antidepressant side effects), depression literacy, and empowerment. Results: None of the experimental groups differed significantly from the others in the empowerment measure (hypothesis 2 not confirmed). Partial confirmation of hypothesis 1 was obtained. Lower values on the depression literacy scale were obtained when participants had been given the video compared to the sheet condition. However, the general depression knowledge and its subscale on side effects reached higher scores when participants were exposed to the DTCA, alone or in combination with the information sheet. Finally, participants showed lower scores on knowledge about treatment and symptoms or prevalence after watching the video compared to the sheet condition (hypothesis 3 confirmed). Symptoms and prevalence knowledge increased only when the video was presented in combination with the sheet. Conclusions: There is no evidence for an increase in empowerment following DTCA exposure. An increase in knowledge of the side effects of the medication was observed in the group exposed to the DTCA video. This was the only result that confirmed the hypothesis of the beneficial effect of DTCA videos on knowledge. Written information proved to be the most suitable way to convey knowledge on treatments and symptoms prevalence. Our findings support the necessity of studying health literacy and patient empowerment together and the consequences of such an increase in knowledge in terms of help-seeking behavior. [ABSTRACT FROM AUTHOR]