Purpose: To describe a case of persistent visual distortion following clinical resolution of intracameral cefuroxime-induced cystoid macular edema (CME). Methods: A single case was evaluated. Results: A 68-year-old woman developed persistent visual distortion after cataract surgery with intracameral cefuroxime administration. Although optical coherence tomography (OCT) revealed only a small ellipsoid zone defect indicating near-complete recovery, adaptive optics scanning laser ophthalmoscopy (AOSLO) uncovered a pronounced fissure-like lesion coursing through the macular photoreceptor mosaic. This discrepancy demonstrated localized photoreceptor disruption that was not apparent on OCT. Conclusions: Intracameral cefuroxime-induced CME may result in lasting photoreceptor damage despite apparent anatomic recovery on OCT. This case underscores the limitations of current clinical imaging and highlights the potential of AOSLO to detect photoreceptor damage underlying persistent blurred vision after resolution of CME.

Assessing cell viability is important in many fields of research. Current optical methods to assess cell viability typically involve fluorescent dyes, which are often less reliable and have poor permeability in primary tissues. Dynamic optical coherence microscopy (dOCM) is an emerging tool that provides label-free contrast reflecting changes in cellular metabolism. In this work, we compare the live contrast obtained from dOCM to viability dyes, and for the first time to our knowledge, demonstrate that dOCM can distinguish live cells from dead cells in murine syngeneic tumors. We further demonstrate a strong correlation between dOCM live contrast and optical redox ratio by metabolic imaging in primary mouse liver tissue. The dOCM technique opens a new avenue to apply label-free imaging to assess the effects of immuno-oncology agents, targeted therapies, chemotherapy, and cell therapies using live tumor tissues.

Supplementary Methods, Figure Legend from Optical Detection of Buccal Epithelial Nanoarchitectural Alterations in Patients Harboring Lung Cancer: Implications for Screening

Single photon avalanche diode (SPAD) array sensors can increase the imaging speed for fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser scanning to widefield geometries. While a SPAD camera in epi-fluorescence geometry enables widefield FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus features masks weak cell autofluorescence and biases lifetime measurements. Here, we address this problem by integrating the SPAD camera in a light sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling fast label-free FLIM of single-cell NAD(P)H autofluorescence. The feasibility of this NAD(P)H light sheet FLIM system was confirmed with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times, and in vivo NAD(P)H light sheet FLIM was demonstrated with live neutrophil imaging in a zebrafish tail wound, also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light sheet geometries, indicating a 30X to 6X frame rate advantage for the light sheet compared to the laser scanning geometry. This light sheet system provides faster frame rates for 3D NAD(P)H FLIM for live cell imaging applications such as monitoring single cell metabolism and immune cell migration throughout an entire living organism.

SPAD array sensors support higher-throughput fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser-scanning to wide-field geometries. While a SPAD camera in epi-fluorescence geometry enables wide-field FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus culture medium masks cell autofluorescence and biases lifetime measurements. Here, we address this problem in a proof-of-concept implementation by integrating the SPAD camera in a light-sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling label-free wide-field FLIM of single-cell NAD(P)H autofluorescence.

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.

An optical platform is presented for examining intrinsic contrast detection strategies when imaging retinal structure using ex vivo tissue. A custom microscope was developed that scans intact tissue and collects scattered light distribution at every image pixel, allowing digital masks to be applied after image collection. With this novel approach at measuring the spatial distribution of multiply scattered light, known and novel methods of detecting intrinsic cellular contrast can be explored, compared, and optimized for retinal structures of interest.

Changes in the multi-level physical structure of biological features going from cellular to tissue level composition is a key factor in many major diseases. However, we are only beginning to understand the role of these structural changes because there are few dedicated multiscale imaging platforms with sensitivity at both the cellular and macrostructural spatial scale. A single platform reduces bias and complications from multiple sample preparation methods and can ease image registration. In order to address these needs, we have developed a multiscale imaging system using a range of imaging modalities sensitive to tissue composition: Ultrasound, Second Harmonic Generation Microscopy, Multiphoton Microscopy, Optical Coherence Tomography, and Enhanced Backscattering. This paper details the system design, the calibration for each modality, and a demonstration experiment imaging a rabbit eye.

A liquid crystal variable retarder (LCVR) enables fast, automated control of retardance that can be used as a variable waveplate in polarimetric instruments. However, precise control of the polarization state requires calibration of the LCVR. A manufacturer calibration curve is typically supplied for a single specific wavelength and temperature, but for applications under different conditions, additional calibration is needed. Calibration is typically performed with crossed polarizers to generate an intensity curve that is converted to retardance, but this method is prone to noise when retardance is close to zero. Here, we demonstrate a simple common-path Sagnac interferometer to measure retardance and provide open source software for automated generation of calibration curves for retardance as a function of wavelength and voltage. We also provide a curve fitting method and closed-form functional representation that outputs the voltage needed to achieve a desired retardance given a specified wavelength.

ABSTRACT Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.