Accuracy of Ex-vivo Fluorescence Confocal Microscopy in Margin Assessment of Solid Tumors: A Systematic Review.

Fluorescence confocal microscopy (FCM) is a novel technology that enables rapid high-resolution digital imaging of non-formalin-fixed tissue specimens and offers real-time positive surgical margin identification. In this systematic review, we evaluated the accuracy metrics of ex vivo FCM for intraoperative margin assessment of different tumor types. A systematic search of MEDLINE via PubMed, Embase, Cochrane Central Register of Controlled Trials, Web of Science, and Scopus was performed for relevant papers (PROSPERO ID: CRD42022372558). We included 14 studies evaluating four types of microscopes in six different tumor types, including breast, prostate, central nervous system, kidney, bladder, and conjunctival tumors. Using the Quality Assessment of Diagnostic Accuracy Studies tool, we identified a high risk of bias in patient selection (21%) and index test (36%) of the included studies. Overall, we found that FCM has good accuracy metrics in all tumor types, with high sensitivity and specificity (>80%) and almost perfect concordance (>90%) against final pathology results. Despite these promising findings, the quality of the available evidence and bias concerns highlight the need for adequately designed studies to further define the role of ex vivo FCM in replacing the frozen section as the tool of choice for intraoperative margin assessment.

Fluorescence lifetime imaging microscopy as an instrument for human sperm assessment.

Mammalian spermatozoa are highly energized cells in which most of the proteins and activated signaling cascades are involved in the metabolic pathways. Flavin adenine dinucleotide (FAD) has one of the most important roles in the correct functional activity of spermatozoa since it acts as a cofactor for flavoenzymes, critical for proper metabolism and predominantly located in mitochondria. Non-invasive, vital and non-traumatic examination of sperm FAD level and microenvironment could be performed by fluorescence lifetime imaging microscopy (FLIM). In this study, we assessed the metabolic status of spermatozoa from healthy donors and found that FLIM could be used to segregate and separate the male germ cells according to the type of metabolic activity which corresponds with spermatozoa motility measured in standard spermogram tests.

i-scope: a compact automated fluorescence microscope for cell counting applications in low resource settings.

Fluorescence microscopy has widespread applications across biological sciences. It has been routinely used for cell counting, which provides a preliminary diagnostic test for many infectious diseases. Conventional fluorescence microscopes are bulky, expensive, time-intensive and laborious. They often require trained operators to acquire and analyze data. We report a compact automated digital fluorescence microscopy system,i-scope, for cell counting applications. Thei-scopeemploys a total internal reflection fluorescence (TIRF) mode of sample illumination, along with a brightfield mode. It has a magnification of 30X, an optical resolution of ∼0.2µm/pixel and offers sample scanning over 20 mm × 20 mm. A custom-written program enables automated image acquisition and analysis, thereby enhancing ease of operation. It has a compact form-factor and has been developed into a standalone system with a processing unit, screen, and other accessories to offer a portable and economic point-of-care diagnostic solution in low-resource settings. We analysed the performance of the i-scopefor milk somatic cell enumeration and benchmarked it against that of a conventional fluorescence microscope.

Quality assessment in light microscopy for routine use through simple tools and robust metrics.

Although there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results.

Rapid tool for cell nanoarchitecture integrity assessment.

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.

Assessment of Mitochondrial Ca2+ Uptake.

Mitochondrial Ca2+ uptake regulates mitochondrial function and contributes to cell signaling. Accordingly, quantifying mitochondrial Ca2+ signals and elaborating the mechanisms that accomplish mitochondrial Ca2+ uptake are essential to gain our understanding of cell biology. Here, we describe the benefits and drawbacks of various established old and new techniques to assess dynamic changes of mitochondrial Ca2+ concentration ([Ca2+]mito) in a wide range of applications.

Immunofluorescence microscopy-based assessment of cytosolic DNA accumulation in mammalian cells.

Here, we describe an immunofluorescence (IF) microscopy-based approach to quantify cytosolic double-stranded DNA molecules in cultured eukaryotic cells upon the selective and specific permeabilization of plasma membranes. This technique is compatible with widefield microscopy coupled with automated image analysis for mid- to high-throughput applications and high-resolution confocal microscopy for subcellular assessments and co-localization studies. In addition to enabling single-cell and subcellular resolution, this approach circumvents most constraints associated with alternative approaches based on subcellular fractionation. For complete use and execution of this protocol, please refer to Yamazaki et al. (2020).

Assessment of Cytotoxicity of α-Synuclein in Budding Yeast Using a Spot Growth Assay and Fluorescent Microscopy.

The budding yeast Saccharomyces cerevisiae is a model organism amenable both to genetic analysis and cell biology. Due to these advantages, yeast has provided platforms to examine the properties of pathogenic proteins involved in human diseases. The methods used to examine the cytotoxicity and intracellular localization of α-Synuclein, a human neuronal protein implicated in Parkinson's disease, using yeast have been described herein. These methods are readily accessible to researchers or graduate students unfamiliar with experiments using yeast and applicable to larger scale analyses, such as high-throughput genetic and chemical screenings.

A cross-nearest neighbor/Monte Carlo algorithm for single-molecule localization microscopy defines interactions between p53, Mdm2, and MEG3.

The functions of long noncoding (lnc)RNAs, such as MEG3, are defined by their interactions with other RNAs and proteins. These interactions, in turn, are shaped by their subcellular localization and temporal context. Therefore, it is important to be able to analyze the relationships of lncRNAs while preserving cellular architecture. The ability of MEG3 to suppress cell proliferation led to its recognition as a tumor suppressor. MEG3 has been proposed to activate p53 by disrupting the interaction of p53 with mouse double minute 2 homolog (Mdm2). To test this mechanism in the native cellular context, we employed two-color direct stochastic optical reconstruction microscopy, a single-molecule localization microscopy technique, to detect and quantify the localizations of p53, Mdm2, and MEG3 in U2OS cells. We developed a new cross-nearest neighbor/Monte Carlo algorithm to quantify the association of these molecules. Proof of concept for our method was obtained by examining the association between FKBP1A and mTOR, MEG3 and p53, and Mdm2 and p53. In contrast to previous models, our data support a model in which MEG3 modulates p53 independently of the interaction with Mdm2.

Rapid, user-friendly, and inexpensive detection of azidothymidine.

Strict adherence to highly active antiretroviral therapy (HAART) is very important to improve the quality of life for HIV-positive patients to reduce new infections and determine treatment success. Azidothymidine (AZT) is an antiretroviral drug commonly used in HAART treatment. In this research, an "add, mix, and measure" assay was developed to detect AZT within minutes. Three different probes designed to release fluorophores when samples containing AZT are added were synthesized and characterized. The limit of detection to AZT in simulated urine samples was determined to be 4 µM in 5 min for one of the probes. This simple and rapid point-of-care test could potentially be used by clinicians and health care workers to monitor the presence of AZT in low resource settings.

High-speed super-resolution imaging of rotationally symmetric structures using SPEED microscopy and 2D-to-3D transformation.

Various super-resolution imaging techniques have been developed to break the diffraction-limited resolution of light microscopy. However, it still remains challenging to obtain three-dimensional (3D) super-resolution information of structures and dynamic processes in live cells at high speed. We recently developed high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy and its two-dimensional (2D)-to-3D transformation algorithm to provide an effective approach to achieving 3D sub-diffraction-limit information in subcellular structures and organelles that have rotational symmetry. In contrast to most other 3D super-resolution microscopy or 3D particle-tracking microscopy approaches, SPEED microscopy does not depend on complex optical components and can be implemented onto a standard inverted epifluorescence microscope. SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside sub-micrometer biological channels or cavities at high spatiotemporal resolution. After data collection, post-localization 2D-to-3D transformation is applied to obtain 3D super-resolution structural and dynamic information. The complete protocol, including cell culture and sample preparation (6-7 d), SPEED imaging (4-5 h), data analysis and validation through simulation (5-13 h), takes ~9 d to complete.

Sources of widefield fluorescence from the brain.

Widefield fluorescence microscopy is used to monitor the spiking of populations of neurons in the brain. Widefield fluorescence can originate from indicator molecules at all depths in cortex and the relative contributions from somata, dendrites, and axons are often unknown. Here, I simulate widefield illumination and fluorescence collection and determine the main sources of fluorescence for several GCaMP mouse lines. Scattering strongly affects illumination and collection. One consequence is that illumination intensity is greatest ~300-400 µm below the pia, not at the brain surface. Another is that fluorescence from a source deep in cortex may extend across a diameter of 3-4 mm at the brain surface, severely limiting lateral resolution. In many mouse lines, the volume of tissue contributing to fluorescence extends through the full depth of cortex and fluorescence at most surface locations is a weighted average across multiple cortical columns and often more than one cortical area.

A rapid response near-infrared ratiometric fluorescent probe for the real-time tracking of peroxynitrite for pathological diagnosis and therapeutic assessment in a rheumatoid arthritis model.

Peroxynitrite (ONOO-) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO-in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO-. The recognition unit diene in the probes could be specifically cleaved by ONOO- with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO- in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO- levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.

Single-cell radioluminescence microscopy with two-fold higher sensitivity using dual scintillator configuration.

Radioluminescence microscopy (RLM) is an imaging technique that allows quantitative analysis of clinical radiolabeled drugs and probes in single cells. However, the modality suffers from slow data acquisition (15-30 minutes), thus critically affecting experiments with short-lived radioactive drugs. To overcome this issue, we suggest an approach that significantly accelerates data collection. Instead of using a single scintillator to image the decay of radioactive molecules, we sandwiched the radiolabeled cells between two scintillators. As proof of concept, we imaged cells labeled with [18F]FDG, a radioactive glucose popularly used in oncology to image tumors. Results show that the double scintillator configuration increases the microscope sensitivity by two-fold, thus reducing the image acquisition time by half to achieve the same result as the single scintillator approach. The experimental results were also compared with Geant4 Monte Carlo simulation to confirm the two-fold increase in sensitivity with only minor degradation in spatial resolution. Overall, these findings suggest that the double scintillator configuration can be used to perform time-sensitive studies such as cell pharmacokinetics or cell uptake of short-lived radiotracers.

Quantitative Assessment of Tendon Hierarchical Structure by Combined Second Harmonic Generation and Immunofluorescence Microscopy.

Histological evaluation of healing tendons is primarily focused on monitoring restoration of longitudinal collagen alignment, although the elastic property of energy-storing flexor tendons is largely attributed to interfascicular sliding facilitated by the interfascicular matrix (IFM). The objectives of this study were to explore the utility of second harmonic generation (SHG) imaging to objectively assess cross-sectional tendon fascicle architecture, to combine SHG microscopy with elastin immunofluorescence to assess the ultrastructure of collagen and elastin in longitudinal and transverse sections, and lastly, to quantify changes in IFM elastin and fascicle collagen alignment of normal and collagenase-injured flexor tendons. Paraffin-embedded transverse and longitudinal histological sections (10-µm thickness) derived from normal and collagenase-injured (6- and 16-week time-points) equine superficial digital flexor tendons were de-paraffinized, treated with Tris EDTA at 80°C for epitope retrieval, and incubated with mouse monoclonal anti-elastin antibody (1:100 dilution) overnight. Anti-mouse IgG Alexa Flour 546 secondary antibody was applied, and sections were mounted with ProLong Gold reagent with 4',6-diamidino-2-phenylindole (DAPI). Nuclei (DAPI) and elastin (Alexa Fluor 546) signals were captured by using standard confocal imaging with 405 and 543 nm excitation wavelengths, respectively. The SHG signal was captured by using a tunable Ti:Sapphire laser tuned to 950 nm to visualize type I collagen. Quantitative measurements of fascicle cross-sectional area (CSA), IFM thickness in transverse SHG-DAPI merged z-stacks, fascicle/IFM elastin area fraction (%), and elastin-collagen alignment in longitudinal SHG-elastin merged z-stacks were conducted by using ImageJ software. Using this methodology, fascicle CSA, IFM thickness, and IFM elastin area fraction (%) at 6 weeks (∼2.25-fold; ∼2.8-fold; 60% decrease; p < 0.001) and 16 weeks (∼2-fold; ∼1.5-fold; 70% decrease; p < 0.001) after collagenase injection, respectively, were found to be significantly different from normal tendon. IFM elastin and fascicle collagen alignment characterized via fast Fourier transform (FFT) frequency plots at 16 weeks demonstrated that collagen re-alignment was more advanced than that of elastin. The integration of SHG-derived quantitative measurements in transverse and longitudinal tendon sections supports comprehensive assessment of tendon structure. Our findings demonstrate the importance of including IFM and non-collagenous proteins in tendon histological evaluations, tasks that can be effectively carried out by using SHG and immunofluorescence microscopy. Impact statement This work demonstrated that second harmonic generation microscopy in conjunction with elastin immunofluorescence provided a comprehensive assessment of multiscale structural re-organization in healing tendon than when restricted to longitudinal collagen fiber alignment alone. Utilizing this approach for tendon histomorphometry is ideal not only to improve our understanding of hierarchical structural changes that occur after tendon injury and during remodeling but also to monitor the efficacy of therapeutic approaches.

Imaging unlabeled proteins on DNA with super-resolution.

Fluorescence microscopy is invaluable to a range of biomolecular analysis approaches. The required labeling of proteins of interest, however, can be challenging and potentially perturb biomolecular functionality as well as cause imaging artefacts and photo bleaching issues. Here, we introduce inverse (super-resolution) imaging of unlabeled proteins bound to DNA. In this new method, we use DNA-binding fluorophores that transiently label bare DNA but not protein-bound DNA. In addition to demonstrating diffraction-limited inverse imaging, we show that inverse Binding-Activated Localization Microscopy or 'iBALM' can resolve biomolecular features smaller than the diffraction limit. The current detection limit is estimated to lie at features between 5 and 15 nm in size. Although the current image-acquisition times preclude super-resolving fast dynamics, we show that diffraction-limited inverse imaging can reveal molecular mobility at ∼0.2 s temporal resolution and that the method works both with DNA-intercalating and non-intercalating dyes. Our experiments show that such inverse imaging approaches are valuable additions to the single-molecule toolkit that relieve potential limitations posed by labeling.

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

A rapid-response and ratiometric fluorescent probe for nitric oxide: From the mitochondria to the nucleus in live cells.

Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (<1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.

A fluorescent hydrazone exchange probe of pyridoxal phosphate for the assessment of vitamin B6 status.

Abnormal vitamin B6 status, marked by deficient intracellular concentrations of pyridoxal phosphate (PLP), is classified as a direct biomarker based on its biomedical significance. However, there exist no direct methods for measuring vitamin B6 status in intact cells. Here we describe the development of a fluorogenic probe, RAB6, which shows remarkable selectivity for PLP among the B6 vitamers and other cellular aldehydes.