Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera

Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.Comment: Overview of FLIM techniques, StreakFLIM instrument, FRET application

Electrophonic sounds from the re-entry of the Molniya 1-67 satellite over Australia: Confirmation of the electromagnetic link.

Accepted versio

Southern Hemisphere automated supernova search

The Perth Astronomy Research Group has developed an automated supernova search program, using the 61 cm Perth–Lowell reflecting telescope at Perth Observatory in Western Australia, equipped with a CCD camera. The system is currently capable of observing about 15 objects per hour, using 3 min exposures, and has a detection threshold of 18th–19th magnitude. The entire system has been constructed using low‐cost IBM‐compatible computers. Two original discoveries (SN 1993K, SN 1994R) have so far been made during automated search runs. This paper describes the hardware and software used for the supernova search program, and shows some preliminary results from the search system

A Concept for an STJ-based Spectrograph

We describe a multi-order spectrograph concept suitable for 8m-class telescopes, using the intrinsic spectral resolution of Superconducting Tunneling Junction detectors to sort the spectral orders. The spectrograph works at low orders, 1-5 or 1-6, and provides spectral coverage with a resolving power of R~8000 from the atmospheric cutoff at 320 nm to the long wavelength end of the infrared H or K band at 1800 nm or 2400 nm. We calculate that the spectrograph would provide substantial throughput and wavelength coverage, together with high time resolution and sufficient dynamic range. The concept uses currently available technology, or technologies with short development horizons, restricting the spatial sampling to two linear arrays; however an upgrade path to provide more spatial sampling is identified. All of the other challenging aspects of the concept - the cryogenics, thermal baffling and magnetic field biasing - are identified as being feasible.Comment: Accepted in Monthly Notices of the Royal Astronomical Society, 12 pages with 10 figure

Multi-session electrical neuromodulation effects on craving, relapse and cognitive functions in cocaine use disorder: A randomized, sham-controlled tDCS study

Background: The use of transcranial Direct Current Stimulation (tDCS) has previously shown promising results for reducing craving in cocaine use disorder. In this study we further explored the potential of tDCS as add-on intervention in the treatment of cocaine use disorder. Methods: In a randomized, placebo-controlled, between subject study, we applied tDCS bilaterally with the anodal electrode targeting the right dorsolateral prefrontal cortex (DLPFC; https://clinicaltrials.gov/ct2/show/ NCT03025321). Patients with cocaine use disorder were allocated to ten sessions of either active tDCS (n = 29) or sham (n = 30) on five consecutive days. Inhibitory control and risky decision-making were measured via a Go-NoGo task and a two-choice gambling task, respectively, each at baseline, one day after all tDCS sessions and after three months. Relapse at follow-up and craving were also assessed. Results: There was no significant effect of active tDCS on the number of cocaine use days and craving. Relapse was frequent among patients who had received either active or sham tDCS (48.0 % and 69.2 %, respectively), despite an overall decrease in craving during the first two weeks of treatment. No effects were found on cognitive functions. An exploratory analysis for crack cocaine use only revealed that relapse rates were significantly reduced after active tDCS (n = 17) as compared to sham (n = 19). Conclusions: No beneficial effects of tDCS on number of cocaine use days, craving and cognitive functions were found in the present study, but somewhat promising results were obtained regarding relapse rates among crackcocaine users specifically. Further research is required to determine the efficacy of tDCS as a complementary treatment in cocaine use disorde

Fast fluorescence microscopy for imaging the dynamics of embryonic development

Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field

Multi-session electrical neuromodulation effects on craving, relapse and cognitive functions in cocaine use disorder: A randomized, sham-controlled tDCS study

Background: The use of transcranial Direct Current Stimulation (tDCS) has previously shown promising results for reducing craving in cocaine use disorder. In this study we further explored the potential of tDCS as add-on intervention in the treatment of cocaine use disorder. Methods: In a randomized, placebo-controlled, between subject study, we applied tDCS bilaterally with the anodal electrode targeting the right dorsolateral prefrontal cortex (DLPFC; https://clinicaltrials.gov/ct2/show/NCT03025321). Patients with cocaine use disorder were allocated to ten sessions of either active tDCS (n = 29) or sham (n = 30) on five consecutive days. Inhibitory control and risky decision-making were measured via a Go-NoGo task and a two-choice gambling task, respectively, each at baseline, one day after all tDCS sessions and after three months. Relapse at follow-up and craving were also assessed. Results: There was no significant effect of active tDCS on the number of cocaine use days and craving. Relapse was frequent among patients who had received either active or sham tDCS (48.0 % and 69.2 %, respectively), despite an overall decrease in craving during the first two weeks of treatment. No effects were found on cognitive functions. An exploratory analysis for crack cocaine use only revealed that relapse rates were significantly reduced after active tDCS (n = 17) as compared to sham (n = 19). Conclusions: No beneficial effects of tDCS on number of cocaine use days, craving and cognitive functions were found in the present study, but somewhat promising results were obtained regarding relapse rates among crack-cocaine users specifically. Further research is required to determine the efficacy of tDCS as a complementary treatment in cocaine use disorder

W2S: Microscopy Data with Joint Denoising and Super-Resolution for Widefield to SIM Mapping

In fluorescence microscopy live-cell imaging, there is a critical trade-off between the signal-to-noise ratio and spatial resolution on one side, and the integrity of the biological sample on the other side. To obtain clean high-resolution (HR) images, one can either use microscopy techniques, such as structured-illumination microscopy (SIM), or apply denoising and super-resolution (SR) algorithms. However, the former option requires multiple shots that can damage the samples, and although efficient deep learning based algorithms exist for the latter option, no benchmark exists to evaluate these algorithms on the joint denoising and SR (JDSR) tasks. To study JDSR on microscopy data, we propose such a novel JDSR dataset, Widefield2SIM (W2S), acquired using a conventional fluorescence widefield and SIM imaging. W2S includes 144,000 real fluorescence microscopy images, resulting in a total of 360 sets of images. A set is comprised of noisy low-resolution (LR) widefield images with different noise levels, a noise-free LR image, and a corresponding high-quality HR SIM image. W2S allows us to benchmark the combinations of 6 denoising methods and 6 SR methods. We show that state-of-the-art SR networks perform very poorly on noisy inputs. Our evaluation also reveals that applying the best denoiser in terms of reconstruction error followed by the best SR method does not necessarily yield the best final result. Both quantitative and qualitative results show that SR networks are sensitive to noise and the sequential application of denoising and SR algorithms is sub-optimal. Lastly, we demonstrate that SR networks retrained end-to-end for JDSR outperform any combination of state-of-the-art deep denoising and SR networksComment: ECCVW 2020. Project page: \<https://github.com/ivrl/w2s

Development of a Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM) system using a Streak Camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging

Semi-Automated Reconstruction of Neural Processes from Large Numbers of Fluorescence Images

We introduce a method for large scale reconstruction of complex bundles of neural processes from fluorescent image stacks. We imaged yellow fluorescent protein labeled axons that innervated a whole muscle, as well as dendrites in cerebral cortex, in transgenic mice, at the diffraction limit with a confocal microscope. Each image stack was digitally re-sampled along an orientation such that the majority of axons appeared in cross-section. A region growing algorithm was implemented in the open-source Reconstruct software and applied to the semi-automatic tracing of individual axons in three dimensions. The progression of region growing is constrained by user-specified criteria based on pixel values and object sizes, and the user has full control over the segmentation process. A full montage of reconstructed axons was assembled from the ∼200 individually reconstructed stacks. Average reconstruction speed is ∼0.5 mm per hour. We found an error rate in the automatic tracing mode of ∼1 error per 250 um of axonal length. We demonstrated the capacity of the program by reconstructing the connectome of motor axons in a small mouse muscle