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

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

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

LUMINOS-102: Lerapolturev with and without α-PD- 1 in unresectable α-PD- 1 refractory melanoma

Lerapolturev (lera, formerly PVSRIPO) is a novel poliovirus based intratumoral immunotherapy that infects both cancer cells and antigen-presenting cells (APCs) via CD155, the poliovirus receptor. Lera has direct anticancer effects while also generating type I/III interferon-dominated inflammation and anti-tumor T-cell priming and activation via infection of local APCs. LUMINOS-102 (NCT04577807) is a multi-center, open-label, two-arm randomized Phase 2 study investigating the efficacy and safety of lera ± α-PD- 1 in patients with unresectable melanoma who failed prior α-PD- 1 therapy. Cross-over to the α-PD- 1 arm is permitted after progression, PR for ≥6 mo or 6 mo on treatment with SD. The maximum initial lera dose was 6x108 TCID50 /visit every 3 or 4 weeks (Q3/4 W). As of March 2022, the maximum lera dose was increased to 1.6 x 109 TCID50/visit, every week (QW) for 7 weeks (induction), followed by Q3/4 W dosing (maintenance). As of 20-Jun- 2022, 21 participants (10 male, 11 female, median 64 yrs) received lera (n = 14 at initial dose, Q3/4 W; n = 4 at increased dose, Q3/4 W; n = 3 at increased dose, QW) ± αPD-1. Five patients are currently on treatment. With the initial regimen, no objective responses and a CBR of 7% were observed. However, with the higher dose regimen, 1 complete response and a CBR of 71% (5/7) has been observed. Two of 4 participants with stable disease have evidence of response (1 with resolution of uninjected lung metastasis, 1 with decreased PET signal in injected and uninjected lesions receiving combination therapy). The only treatment related AE in \u3e1 pt was fatigue (19%, all grade 1 or 2). No dose-limiting toxicities or treatment-related SAEs were reported. Multiplex-IF analysis of on-treatment tumor biopsies will be presented. Lera ± αPD-1 is well tolerated, with early signs of efficacy at the higher dose level. Enrollment and randomization are ongoing

Low tumour cell proliferation at the invasive margin is associated with a poor prognosis in Dukes' stage B colorectal cancers

The conflicting results about the prognostic impact of tumour cell proliferation in colorectal cancer might be explained by the heterogeneity observed within these tumours. We have investigated whether a systematic spatial heterogeneity exists between different compartments, and whether the presence of such a systematic heterogeneity has any impact on survival. Fifty-six Dukes' stage B colorectal cancers were carefully morphometrically quantified with respect to the immunohistochemical expression of the proliferative marker Ki-67 at both the luminal border and the invasive margin. The proliferative activity was significantly higher at the luminal border compared with the invasive margin (P < 0.001), although the two compartments were also significantly correlated with each other. Tumours with low proliferation at the invasive margin had a significantly poorer prognosis both in univariate (P = 0.014) and in multivariate survival analyses (P = 0.042). We conclude that Dukes' B colorectal cancers exhibit a systematic spatial heterogeneity with respect to proliferation, and tumours with low proliferation at the invasive margin had a poor prognosis. The present data independently confirm recent results from the authors, and provide new insights into the understanding of tumour cell proliferation in colorectal cancer. © 1999 Cancer Research Campaig

Ensheathing cells utilize dynamic tiling of neuronal somas in development and injury as early as neuronal differentiation

Abstract Background Glial cell ensheathment of specific components of neuronal circuits is essential for nervous system function. Although ensheathment of axonal segments of differentiated neurons has been investigated, ensheathment of neuronal cell somas, especially during early development when neurons are extending processes and progenitor populations are expanding, is still largely unknown. Methods To address this, we used time-lapse imaging in zebrafish during the initial formation of the dorsal root ganglia (DRG). Results Our results show that DRG neurons are ensheathed throughout their entire lifespan by a progenitor population. These ensheathing cells dynamically remodel during development to ensure axons can extend away from the neuronal cell soma into the CNS and out to the skin. As a population, ensheathing cells tile each DRG neuron to ensure neurons are tightly encased. In development and in experimental cell ablation paradigms, the oval shape of DRG neurons dynamically changes during partial unensheathment. During longer extended unensheathment neuronal soma shifting is observed. We further show the intimate relationship of these ensheathing cells with the neurons leads to immediate and choreographed responses to distal axonal damage to the neuron. Conclusion We propose that the ensheathing cells dynamically contribute to the shape and position of neurons in the DRG by their remodeling activity during development and are primed to dynamically respond to injury of the neuron