Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

UNASSIGNED: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).
UNASSIGNED: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.
UNASSIGNED: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.
UNASSIGNED: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.
UNASSIGNED: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.

Neurological disorders, including spinal cord injury, peripheral nerve injury, traumatic brain injury, and neurodegenerative diseases, pose significant challenges in terms of diagnosis, treatment, and understanding the underlying pathophysiological processes. Label-free multiphoton microscopy techniques, such as coherent Raman scattering, two-photon excited autofluorescence, and second and third harmonic generation microscopy, have emerged as powerful tools for visualizing nervous tissue with high resolution and without the need for exogenous labels. Coherent Raman scattering processes as well as third harmonic generation enable label-free visualization of myelin sheaths, while their combination with two-photon excited autofluorescence and second harmonic generation allows for a more comprehensive tissue visualization. They have shown promise in assessing the efficacy of therapeutic interventions and may have future applications in clinical diagnostics. In addition to multiphoton microscopy, vibrational spectroscopy methods such as infrared and Raman spectroscopy offer insights into the molecular signatures of injured nervous tissues and hold potential as diagnostic markers. This review summarizes the application of these label-free optical techniques in preclinical models and illustrates their potential in the diagnosis and treatment of neurological disorders with a special focus on injury, degeneration, and regeneration. Furthermore, it addresses current advancements and challenges for bridging the gap between research findings and their practical applications in a clinical setting.

Photodynamic therapy (PDT) relies on a series of photophysical and photochemical reactions leading to cell death. While effective for various cancers, PDT has been less successful in treating pigmented melanoma due to high light absorption by melanin. Here, this limitation is addressed by 2-photon excitation of the photosensitizer (2p-PDT) using ~100 fs pulses of near-infrared laser light. A critical role of melanin in enabling rather than hindering 2p-PDT is elucidated using pigmented and non-pigmented murine melanoma clonal cell lines in vitro. The photocytotoxicities were compared between a clinical photosensitizer (Visudyne) and a porphyrin dimer (Oxdime) with ~600-fold higher σ2p value. Unexpectedly, while the 1p-PDT responses are similar in both cell lines, 2p activation is much more effective in killing pigmented than non-pigmented cells, suggesting a dominant role of melanin 2p-PDT. The potential for clinical translational is demonstrated in a conjunctival melanoma model in vivo, where complete eradication of small tumors was achieved. This work elucidates the melanin contribution in multi-photon PDT enabling significant advancement of light-based treatments that have previously been considered unsuitable in pigmented tumors.

UNASSIGNED: Intravital cellular calcium imaging has emerged as a powerful tool to investigate how different types of neurons interact at the microcircuit level to produce seizure activity, with newfound potential to understand epilepsy. Although many methods exist to measure seizure-related activity in traditional electrophysiology, few yet exist for calcium imaging.
UNASSIGNED: To demonstrate an automated algorithmic framework to detect seizure-related events using calcium imaging-including the detection of pre-ictal spike events, propagation of the seizure wavefront, and terminal spreading waves for both population-level activity and that of individual cells.
UNASSIGNED: We developed an algorithm for precise recruitment detection of population and individual cells during seizure-associated events, which broadly leverages averaged population activity and high-magnitude slope features to detect single-cell pre-ictal spike and seizure recruitment. We applied this method to data recorded using awake in vivo two-photon calcium imaging during pentylenetetrazol-induced seizures in mice.
UNASSIGNED: We demonstrate that our detected recruitment times are concordant with visually identified labels provided by an expert reviewer and are sufficiently accurate to model the spatiotemporal progression of seizure-associated traveling waves.
UNASSIGNED: Our algorithm enables accurate cell recruitment detection and will serve as a useful tool for researchers investigating seizure dynamics using calcium imaging.

Rats are used in neuroscience research because of their physiological similarities with humans and accessibility as model organisms, trainability, and behavioral repertoire. In particular, rats perform a wide range of sophisticated social, cognitive, motor, and learning behaviors within the contexts of both naturalistic and laboratory environments. Further progress in neuroscience can be facilitated by using advanced imaging methods to measure the complex neural and physiological processes during behavior in rats. However, compared with the mouse, the rat nervous system offers a set of challenges, such as larger brain size, decreased neuron density, and difficulty with head restraint. Here, we review recent advances in in vivo imaging techniques in rats with a special focus on open-source solutions for calcium imaging. Finally, we provide suggestions for both users and developers of in vivo imaging systems for rats.

UNASSIGNED: Genetic cellular calcium imaging has emerged as a powerful tool to investigate how different types of neurons interact at the microcircuit level to produce seizure activity, with newfound potential to understand epilepsy. Although many methods exist to measure seizure-related activity in traditional electrophysiology, few yet exist for calcium imaging.
UNASSIGNED: To demonstrate an automated algorithmic framework to detect seizure-related events using calcium imaging - including the detection of pre-ictal spike events, propagation of the seizure wavefront, and terminal spreading waves for both population-level activity and that of individual cells.
UNASSIGNED: We developed an algorithm for precise recruitment detection of population and individual cells during seizure-associated events, which broadly leverages averaged population activity and high-magnitude slope features to detect single-cell pre-ictal spike and seizure recruitment. We applied this method to data recorded using awake in vivo two-photon calcium imaging during pentylenetetrazol induced seizures in mice.
UNASSIGNED: We demonstrate that our detected recruitment times are concordant with visually identified labels provided by an expert reviewer and are sufficiently accurate to model the spatiotemporal progression of seizure-associated traveling waves.
UNASSIGNED: Our algorithm enables accurate cell recruitment detection and will serve as a useful tool for researchers investigating seizure dynamics using calcium imaging.

Multiphoton microscopy is a powerful imaging tool for biomedical applications. A variety of techniques and respective benefits exist for multiphoton microscopy, but an enhanced resolution is especially desired. Additionally multiphoton microscopy requires ultrafast pulses for excitation, so optimization of the pulse duration at the sample is critical for strong signals.
We aim to perform enhanced resolution imaging that is robust to scattering using a structured illumination technique while also providing a rapid and easily repeatable means to optimize group delay dispersion (GDD) compensation through to the sample.
Spatial frequency modulation imaging (SPIFI) is used in two domains: the spatial domain (SD) and the wavelength domain (WD). The WD-SPIFI system is an in-line tool enabling GDD optimization that considers all material through to the sample. The SD-SPIFI system follows and enables enhanced resolution imaging.
The WD-SPIFI dispersion optimization performance is confirmed with independent pulse characterization, enabling rapid optimization of pulses for imaging with the SD-SPIFI system. The SD-SPIFI system demonstrates enhanced resolution imaging without the use of photon counting enabled by signal to noise improvements due to the WD-SPIFI system.
Implementing SPIFI in-line in two domains enables full-path dispersion compensation optimization through to the sample for enhanced resolution multiphoton microscopy.

Independent control over the Young\'s modulus and topography of a hydrogel cell culture substrate is necessary to characterize how attributes of its adherent surface affect cellular responses. Arbitrary, real-time manipulation of these parameters at the micron scale would further provide cellular biologists and bioengineers with the tools to study and control numerous highly dynamic behaviors including cellular adhesion, motility, metastasis, and differentiation. Although physical, chemical, thermal, and light-based strategies have been developed to influence Young\'s modulus and topography of hydrogel substrates, independent control of these physical attributes has remained elusive, spatial resolution is often limited, and features commonly must be pre-patterned. We recently reported a strategy in which biomaterials having specified three-dimensional (3D) morphologies are micro-3D printed in a two-step process: laser-scanning bioprinting of a protein-based hydrogel, followed by biocompatible hydrogel re-scanning to create microscale imprinted features at user-defined times. In this approach, a pulsed near-infrared laser beam is focused within the printed hydrogel to promote matrix contraction through multiphoton crosslinking, where scanning the laser focus projects a user-defined topographical pattern on the surface without subjecting the hydrogel-solution interface to damaging light intensities. Here, we extend this strategy, demonstrating the ability to decouple dynamic topographical changes from changes in hydrogel Young\'s modulus at the substrate surface by increasing the isolation distance between the surface and re-scanning planes. Using atomic force microscopy, we show that robust topographic changes can be imposed without altering the Young\'s modulus measured at the substrate surface by scanning at a depth of greater than ~6 μm. Transmission electron microscopy of hydrogel thin sections reveals changes to hydrogel porosity and density distribution within scanned regions, and that such changes to the hydrogel matrix are highly localized to regions of laser exposure. These results represent valuable new capabilities for deconvolving the effects of substrate dynamic physical attributes on the behavior of adherent cells.

With recent advances in cancer therapeutics, there is a great need for improved imaging methods for characterizing cancer onset and progression in a quantitative and actionable way. Collagen, the most abundant extracellular matrix protein in the tumor microenvironment (and the body in general), plays a multifaceted role, both hindering and promoting cancer invasion and progression. Collagen deposition can defend the tumor with immunosuppressive effects, while aligned collagen fiber structures can enable tumor cell migration, aiding invasion and metastasis. Given the complex role of collagen fiber organization and topology, imaging has been a tool of choice to characterize these changes on multiple spatial scales, from the organ and tumor scale to cellular and subcellular level. Macroscale density already aids in the detection and diagnosis of solid cancers, but progress is being made to integrate finer microscale features into the process. Here we review imaging modalities ranging from optical methods of second harmonic generation (SHG), polarized light microscopy (PLM), and optical coherence tomography (OCT) to the medical imaging approaches of ultrasound and magnetic resonance imaging (MRI). These methods have enabled scientists and clinicians to better understand the impact collagen structure has on the tumor environment, at both the bulk scale (density) and microscale (fibrillar structure) levels. We focus on imaging methods with the potential to both examine the collagen structure in as natural a state as possible and still be clinically amenable, with an emphasis on label-free strategies, exploiting intrinsic optical properties of collagen fibers.