Generating intravital super-resolution movies with conventional microscopy reveals actin dynamics that construct pioneer axons

Super-resolution microscopy is broadening our in-depth understanding of cellular structure. However, super-resolution approaches are limited, for numerous reasons, from utilization in longer-term intravital imaging. We devised a combinatorial imaging technique that combines deconvolution with stepwise optical saturation microscopy (DeSOS) to circumvent this issue and image cells in their native physiological environment. Other than a traditional confocal or two-photon microscope, this approach requires no additional hardware. Here, we provide an open-access application to obtain DeSOS images from conventional microscope images obtained at low excitation powers. We show that DeSOS can be used in time-lapse imaging to generate super-resolution movies in zebrafish. DeSOS was also validated in live mice. These movies uncover that actin structures dynamically remodel to produce a single pioneer axon in a 'top-down' scaffolding event. Further, we identify an F-actin population - stable base clusters - that orchestrate that scaffolding event. We then identify that activation of Rac1 in pioneer axons destabilizes stable base clusters and disrupts pioneer axon formation. The ease of acquisition and processing with this approach provides a universal technique for biologists to answer questions in living animals

High-speed, long-term, 4D in vivo lifetime imaging in intact and injured zebrafish and mouse brains by instant FLIM

Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in the emission decay lifetime. With fluorescence lifetime imaging microscopy (FLIM), physiological parameters such as pH, refractive index, ion concentration, dissolved gas concentration, and fluorescence resonance energy transfer (FRET) can be measured. Despite these benefits, existing FLIM techniques are typically slow, noisy, and hard to implement due to expensive instrumentation and complex post-processing. To overcome these limitations, we present instant FLIM, a method that allows real-time acquisition and display of two-photon intensity, lifetime, and phasor imaging data. Using analog signal processing, we demonstrate in vivo four-dimensional (4D) FLIM movies by imaging mouse and zebrafish glial cell response to injury over 12 hours through intact skulls. Instant FLIM can be implemented as an upgrade to an existing multiphoton microscope using cost-effective off-the-shelf components, requires no data post-processing, and is demonstrated to be compatible with FD-FLIM super-resolution techniques

Discovery of a Transiting Adolescent Sub-Neptune Exoplanet with K2

The role of stellar age in the measured properties and occurrence rates of exoplanets is not well understood. This is in part due to a paucity of known young planets and the uncertainties in age-dating for most exoplanet host stars. Exoplanets with well-constrained ages, particularly those which are young, are useful as benchmarks for studies aiming to constrain the evolutionary timescales relevant for planets. Such timescales may concern orbital migration, gravitational contraction, or atmospheric photo-evaporation, among other mechanisms. Here we report the discovery of an adolescent transiting sub-Neptune from K2 photometry of the low-mass star K2-284. From multiple age indicators we estimate the age of the star to be 120 Myr, with a 68% confidence interval of 100-760 Myr. The size of K2-284 b ($R_P$ = 2.8 $\pm$ 0.1 $R_\oplus$) combined with its youth make it an intriguing case study for photo-evaporation models, which predict enhanced atmospheric mass loss during early evolutionary stages.Comment: Accepted to AJ, 36 pages, 17 figures, 5 table

Automatic segmentation of intravital fluorescence microscopy images by K-means clustering of FLIM phasors

Fluorescence lifetime imaging microscopy (FLIM) provides additional contrast for fluorophores with overlapping emission spectra. The phasor approach to FLIM greatly reduces the complexity of FLIM analysis and enables a useful image segmentation technique by selecting adjacent phasor points and labeling their corresponding pixels with different colors. This phasor labeling process, however, is empirical and could lead to biased results. In this Letter, we present a novel and unbiased approach to automate the phasor labeling process using an unsupervised machine learning technique, i.e., K-means clustering. In addition, we provide an open-source, user-friendly program that enables users to easily employ the proposed approach. We demonstrate successful image segmentation on 2D and 3D FLIM images of fixed cells and living animals acquired with two different FLIM systems. Finally, we evaluate how different parameters affect the segmentation result and provide a guideline for users to achieve optimal performance

Convolutional Neural Network Denoising in Fluorescence Lifetime Imaging Microscopy (FLIM)

Fluorescence lifetime imaging microscopy (FLIM) systems are limited by their slow processing speed, low signal-to-noise ratio (SNR), and expensive and challenging hardware setups. In this work, we demonstrate applying a denoising convolutional network to improve FLIM SNR. The network will be integrated with an instant FLIM system with fast data acquisition based on analog signal processing, high SNR using high-efficiency pulse-modulation, and cost-effective implementation utilizing off-the-shelf radio-frequency components. Our instant FLIM system simultaneously provides the intensity, lifetime, and phasor plots \textit{in vivo} and \textit{ex vivo}. By integrating image denoising using the trained deep learning model on the FLIM data, provide accurate FLIM phasor measurements are obtained. The enhanced phasor is then passed through the K-means clustering segmentation method, an unbiased and unsupervised machine learning technique to separate different fluorophores accurately. Our experimental \textit{in vivo} mouse kidney results indicate that introducing the deep learning image denoising model before the segmentation effectively removes the noise in the phasor compared to existing methods and provides clearer segments. Hence, the proposed deep learning-based workflow provides fast and accurate automatic segmentation of fluorescence images using instant FLIM. The denoising operation is effective for the segmentation if the FLIM measurements are noisy. The clustering can effectively enhance the detection of biological structures of interest in biomedical imaging applications.Comment: SPIE Proceedings Volume 11648, Multiphoton Microscopy in the Biomedical Sciences XXI; 116481C (2021

SPIRITS 15c and SPIRITS 14buu: Two Obscured Supernovae in the Nearby Star-Forming Galaxy IC 2163

SPIRITS---SPitzer InfraRed Intensive Transients Survey---is an ongoing survey of nearby galaxies searching for infrared (IR) transients with Spitzer/IRAC. We present the discovery and follow-up observations of one of our most luminous ($M_{[4.5]} = -17.1\pm0.4$ mag, Vega) and red ($[3.6] - [4.5] = 3.0 \pm 0.2$ mag) transients, SPIRITS 15c. The transient was detected in a dusty spiral arm of IC 2163 ($D\approx35.5$ Mpc). Pre-discovery ground-based imaging revealed an associated, shorter-duration transient in the optical and near-IR (NIR). NIR spectroscopy showed a broad ($\approx 8400$ km s$^{-1}$), double-peaked emission line of He I at $1.083 \mu$m, indicating an explosive origin. The NIR spectrum of SPIRITS 15c is similar to that of the Type IIb SN 2011dh at a phase of $\approx 200$ days. Assuming $A_V = 2.2$ mag of extinction in SPIRITS 15c provides a good match between their optical light curves. The IR light curves and the extreme $[3.6]-[4.5]$ color cannot be explained using only a standard extinction law. Another luminous ($M_{4.5} = -16.1\pm0.4$ mag) event, SPIRITS 14buu, was serendipitously discovered in the same galaxy. The source displays an optical plateau lasting $\gtrsim 80$ days, and we suggest a scenario similar to the low-luminosity Type IIP SN 2005cs obscured by $A_V \approx 1.5$ mag. Other classes of IR-luminous transients can likely be ruled out in both cases. If both events are indeed SNe, this may suggest $\gtrsim 18\%$ of nearby core-collapse SNe are missed by currently operating optical surveys.Comment: 19 pages, 7 Figures, 4 Table

SPIRITS 16tn in NGC 3556: A heavily obscured and low-luminosity supernova at 8.8 Mpc

We present the discovery by the SPitzer InfraRed Intensive Transients Survey (SPIRITS) of a likely supernova (SN) in NGC 3556 at only 8.8 Mpc, which, despite its proximity, was not detected by optical searches. A luminous infrared (IR) transient at $M_{[4.5]} = -16.7$ mag (Vega), SPIRITS 16tn is coincident with a dust lane in the inclined, star-forming disk of the host. Using IR, optical, and radio observations, we attempt to determine the nature of this event. We estimate $A_V \approx$ 8 - 9 mag of extinction, placing it among the three most highly obscured IR-discovered SNe to date. The [4.5] light curve declined at a rate of 0.013 mag day$^{-1}$, and the $[3.6] - [4.5]$ color grew redder from 0.7 to $\gtrsim$ 1.0 mag by 184.7 days post discovery. Optical/IR spectroscopy shows a red continuum, but no clearly discernible features, preventing a definitive spectroscopic classification. Deep radio observations constrain the radio luminosity of SPIRITS 16tn to $L_{\nu} \lesssim 10^{24}$ erg s$^{-1}$ Hz$^{-1}$ between 3 - 15 GHz, excluding many varieties of radio core-collapse SNe. A type Ia SN is ruled out by the observed red IR color, and lack of features normally attributed to Fe-peak elements in the optical and IR spectra. SPIRITS 16tn was fainter at [4.5] than typical stripped-envelope SNe by $\approx$ 1 mag. Comparison of the spectral energy distribution to SNe II suggests SPIRITS 16tn was both highly obscured, and intrinsically dim, possibly akin to the low-luminosity SN 2005cs. We infer the presence of an IR dust echo powered by a peak luminosity of the transient of $5 \times 10^{40}$ erg s$^{-1} < L_{\mathrm{peak}} < 4\times10^{43}$ erg s$^{-1}$, consistent with the observed range for SNe II. This discovery illustrates the power of IR surveys to overcome the compounding effects of visible extinction and optically sub-luminous events in completing the inventory of nearby SNe.Comment: 25 pages, 10 figures, submitted to Ap