Multichannel Fluorescence Research Articles

Photosystems functioning, the content of chemical elements, flavonoids and brassinosteroids in leaves of chimera Cornus controversa

he formation of chimera is a well-known phenomenon in variegated ornamental and wild plants, but the biochemical peculiarities of this phenomenon are not well studied. The purpose of this work was to conduct a comparative study of the photosynthetic apparatus functioning, the content of macro-, microelements, flavonoids and brassinosteroids in the variegated and green leaves of the periclinal chimera Cornus controversa plant grown in the collection of M. M. Gryshko National Botanical Garden of the NAS of Ukraine. Sampling was made during the period from June to October 2022. The content of photosynthetic pigments and brassinosteroids was measured spectrophotometrically, autofluorescence of leaf tissues was studied using an inverted microscope with a multi-channel fluorescence imaging system, Normalized difference vegetation index (NDVI) – using GreenSeeker devices and fluorimeter, the level of macro- and microelements – using plasma emission spectrometer. Phenolic secondary metabolites were separated by HPTLC. To assess the protective capacity of flavonoids, a bioassay on the germination of radish seeds Raphanus in the presence of AlCl3 was used. It was shown that green leaves of C. controversa chimera, compared to variegated ones, had a twice as high NDVI index, maintained photosynthetic function longer in autumn, and had higher content of macronutrients (Mg, Ca and S) but lower concentration of toxic metals (Al, Cu, Fe, Zn, Ti). Instead, variegated leaves accumulated more brassinosteroids and phenolic metabolites, which were found not only to reduce the toxic effect of aluminum but also to have a growth-stimulating effect on the roots of radish seedlings. Such specialization of green and variegated leaves may contribute to better plant chimeras adaptation to environmental pollution. Keywords: brassinosteroids, Cornus controversa, macroelements, microelements, NDVI index, periclinal chimera, phenolic metabolites, photosynthetic pigments, tissue autofluorescence

B-026 Transforming PCR protocols in pandemic response with automated non-contact liquid dispensing

Abstract Background During the COVID-19 pandemic, effective Polymerase Chain Reaction (PCR) protocols were crucial for detecting SARS-CoV-2 in clinical samples, playing a key role in rapid infection control and easing the strain on healthcare services. However, with the critical need for fast and accurate pathogen surveillance, the manual execution of PCR workflows presented numerous challenges, including limited throughput, risk of human error and variability, and risk to human health. Several automated liquid handling systems from a range of vendors were considered to prepare extraction plates for this process. Ultimately, RFL decided on dragonfly® discovery, a non-contact positive displacement dispenser. Methods The analytical sensitivity for the UltraDx SARS-CoV-2 N1/N2/RP assay was evaluated by the RFL development team using RNA extracted from a panel of virus inputs from a Qnostics Analytical Q Panel, Product Number: SCV2AQP. Samples were extracted using a modified RNA extraction procedure for Thermo MagMax reagents and consumables with dragonfly discovery. dragonfly discovery was utilized to distribute a combination of inactivation buffer and beads into a 96-well kingfisher flex plate. This process was accomplished using a peristaltic pump and a 6-way Auto Feed Reservoir (AFR) to ensure a constant buffer flow. Simultaneously, the AFR ensured that the beads were kept in constant suspension, resulting in precise dispensing across all wells. The EPCR assay, UltraDx SARS-CoV-2 N1/N2/RP assay (LGC, Biosearch Technologies), utilized two CDC-characterized amplicon primer sets alongside two additional probes using fluorescence-based (FAM dye) detection. The input RNA extracted from clinical samples was combined with RT-PCR reaction mixes, before the reaction arrays were amplified. The reactions were then measured by a single scan on a multi-channel fluorescence reader. Results The UltraDx SARS-CoV-2 N1/N2/RP assay (when performed using the manufacturer method and in the absence of dragonfly discovery) has an LoD of 80 copies per reaction for the N1 assay, and 20 copies per reaction for the N2 assay, across 20 replicates for each copy number tested. Using the same/equivalent reagents and control materials, inclusion of dragonfly discovery showed an LoD of 70 copies/mL (0.9 copies/reaction) sensitivity when compared to the LoD suggested by the manufacture. Conclusions Integrating dragonfly discovery into the UltraDx SARS-CoV-2 N1/N2/RP assay led to an 80-fold reduction in the limit of detection for the N1 assay compared to the reagent manufacturer's recommendation. Some of this improvement can be attributed to dragonfly discovery's non-contact positive displacement dispensing, coupled with the AFR pump mechanism that ensures a continuous suspension of magnetic beads, enhancing accuracy and precision. dragonfly discovery's versatility supports a robust business-as-usual (BAU) process and is easily scalable for pandemic-level testing. Its user-friendly software interface requires zero computer programming knowledge, providing end-users with the flexibility to run multiple protocols on the same instrument, making it ideal for BAU assay processing. The instrument played a pivotal role in the UltraDx SARS-CoV-2 N1/N2/RP assay conducted by UKHSA during the pandemic, and SPT Labtech will continue to support UKHSA in their mission to protect communities from the impact of health threats.

Red/near-infrared (NIR) difluoroboron β-diketonate derivatives with reversible mechanochromism for cellular imaging

Near-infrared (NIR) fluorophores have promoted the development of materials for bioimaging, but traditional NIR dyes usually suffer from aggregation-caused quenching (ACQ), impeding their applications. Herein, we propose two difluoroboron β-diketonate complexes TBO and TBS, consisting a donor–acceptor (D-A) structure with triphenylamine (TPA) moiety as an electron donors and difluoroboron as well as furan or thiophene building block as an electron acceptor. The theoretical calculation and optical data shows that both of them have intramolecular charge transfer (ICT) characteristics. Such ICT characteristics endow them with both solvatochromism and dual-state emission (DSE) properties. In the solvent CH2Cl2, the emission wavelength of TBO ranges from 550 nm to 750 nm, with a low fluorescence quantum yield (Φ = 7.0 %). However, in the less polar solvent hexane, the emission wavelength blue-shifts, with an increased Φ reaching up to 18 %. Moreover, TBO and TBS exhibit mechanochromic characteristics and rare multi-channel fluorescence emission phenomena at solid-state. Their solid-state samples can emit fluorescence in four spectral bands with maximum emission wavelengths at 300 nm, 400 nm, 600 nm, and 770 nm under excitation at 240 nm. These unique optical properties are expected to be utilized for detecting polarity of system and deformation. Moreover, according to the results of cell imaging and flow cytometry, TBO molecular were easily internalized into Hela cells and distributed in the cytoplasm with strong red fluorescence. Therefore, this research inspires more insight into development of NIR luminogens for biomedical imaging.

Psudo: Exploring Multi-Channel Biomedical Image Data with Spatially and Perceptually Optimized Pseudocoloring.

Over the past century, multichannel fluorescence imaging has been pivotal in myriad scientific breakthroughs by enabling the spatial visualization of proteins within a biological sample. With the shift to digital methods and visualization software, experts can now flexibly pseudocolor and combine image channels, each corresponding to a different protein, to explore their spatial relationships. We thus propose psudo, an interactive system that allows users to create optimal color palettes for multichannel spatial data. In psudo, a novel optimization method generates palettes that maximize the perceptual differences between channels while mitigating confusing color blending in overlapping channels. We integrate this method into a system that allows users to explore multi-channel image data and compare and evaluate color palettes for their data. An interactive lensing approach provides on-demand feedback on channel overlap and a color confusion metric while giving context to the underlying channel values. Color palettes can be applied globally or, using the lens, to local regions of interest. We evaluate our palette optimization approach using three graphical perception tasks in a crowdsourced user study with 150 participants, showing that users are more accurate at discerning and comparing the underlying data using our approach. Additionally, we showcase psudo in a case study exploring the complex immune responses in cancer tissue data with a biologist.

Dynamic modulation of multicolor upconversion luminescence of Er3+ via excitation pulse width.

Lanthanide-doped upconversion (UC) luminescent materials display multicolor emissions, making them ideal for a variety of applications, such as multi-channel biological imaging, fluorescence encryption, anti-counterfeiting, and 3D display. Manipulating the UC emissions of the luminescent materials with a fixed composition is crucial for their applications. Herein, we propose a facile strategy to achieve pulse-width-dependent multicolor UC emissions in NaYF4:Yb/Er/Tm nanocrystals. Upon excitation with a 980nm continuous-wave laser diode, Er3+ ions in NaYF4:20%Yb,15%Er,1%Tm nanocrystals exhibited UC emissions with a red-to-green (R/G) ratio of 11.3. Nevertheless, by employing a 980nm pulse laser with pulse widths from 0.1 to 10ms, the UC R/G ratio can be easily adjusted from 0.9 to 11.3, resulting in continuous and remarkable color transformation from green, yellow, orange, to red. By virtue of the dynamic luminescence color variation of these NaYF4:20%Yb,15%Er,1%Tm nanocrystals, we demonstrated their potential applications in the areas of anti-counterfeiting and information encryption. These findings provide deep insights into the excited-state dynamics and energy transfer of Er3+ in NaYF4:Yb/Er/Tm nanocrystals upon 980nm pulse excitation, which may pave the way for designing multicolor UC materials toward versatile applications.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Ultrastructural localization of Porphyromonas gingivalis gingipains in the substantia nigra of Parkinson’s disease brains

Gingipains are protease virulence factors produced by Porphyromonas gingivalis, a Gram-negative bacterium best known for its role in chronic periodontitis. Gingipains were recently identified in the middle temporal gyrus of postmortem Alzheimer’s disease (AD) brains, where gingipain load correlated with AD diagnosis and tau and ubiquitin pathology. Since AD and Parkinson’s disease (PD) share some overlapping pathologic features, including nigral pathology and Lewy bodies, the current study explored whether gingipains are present in the substantia nigra pars compacta of PD brains. In immunohistochemical techniques and multi-channel fluorescence studies, gingipain antigens were abundant in dopaminergic neurons in the substantia nigra of both PD and neurologically normal control brains. 3-dimensional reconstructions of Lewy body containing neurons revealed that gingipains associated with the periphery of alpha-synuclein aggregates but were occasionally observed inside aggregates. In vitro proteomic analysis demonstrated that recombinant alpha-synuclein is cleaved by lysine-gingipain, generating multiple alpha-synuclein fragments including the non-amyloid component fragments. Immunogold electron microscopy with co-labeling of gingipains and alpha-synuclein confirmed the occasional colocalization of gingipains with phosphorylated (pSER129) alpha-synuclein. In dopaminergic neurons, gingipains localized to the perinuclear cytoplasm, neuromelanin, mitochondria, and nucleus. These data suggest that gingipains localize in dopaminergic neurons in the substantia nigra and interact with alpha-synuclein.

Cell Painting-based bioactivity prediction boosts high-throughput screening hit-rates and compound diversity

Identifying active compounds for a target is a time- and resource-intensive task in early drug discovery. Accurate bioactivity prediction using morphological profiles could streamline the process, enabling smaller, more focused compound screens. We investigate the potential of deep learning on unrefined single-concentration activity readouts and Cell Painting data, to predict compound activity across 140 diverse assays. We observe an average ROC-AUC of 0.744 ± 0.108 with 62% of assays achieving ≥0.7, 30% ≥0.8, and 7% ≥0.9. In many cases, the high prediction performance can be achieved using only brightfield images instead of multichannel fluorescence images. A comprehensive analysis shows that Cell Painting-based bioactivity prediction is robust across assay types, technologies, and target classes, with cell-based assays and kinase targets being particularly well-suited for prediction. Experimental validation confirms the enrichment of active compounds. Our findings indicate that models trained on Cell Painting data, combined with a small set of single-concentration data points, can reliably predict the activity of a compound library across diverse targets and assays while maintaining high hit rates and scaffold diversity. This approach has the potential to reduce the size of screening campaigns, saving time and resources, and enabling primary screening with more complex assays.

Abstract 6444: Assessing cytotoxic T cell responses to immune checkpoint inhibitors in murine and human tumor samples using metabolic imaging

Abstract Background Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment. Unfortunately, existing FDA approved biomarkers do not accurately predict which patients will respond. The Elephas Cybrid™ platform aims to improve ICI response prediction by imaging a patient’s biopsy treated with ICI ex vivo. Multiphoton microscopy (MPM) is a powerful imaging technique to interrogate the biological changes in live tumor fragments (LTFs) following ICI treatment. MPM allows rapid visualization of biological processes in 3D living tissues in a non-destructive fashion allowing for longitudinal assessment of a sample. We and others have used label-free metabolic imaging to demonstrate that changes in T cell metabolism and activation status are correlated. Using MPM imaging we successfully demonstrated T cell activation and T cell mediated cytotoxicity in response to ICI in LTFs and tumor biopsies. Methods Tumors were cut into LTFs, sorted, and cultured using the Cybrid platform. LTF structure and metabolic status were assessed using intrinsically fluorescent NAD(P)H and FAD. Anti-CD8 nanobodies and cleaved caspase 3/7 dye were used to characterize T cell cytotoxicity. Multi-channel fluorescence intensity and lifetime were obtained over 48 hours using MPM. Results Murine CT26 LTFs generated with the Cybrid platform were treated with ICI and cytotoxicity was assessed in the intact LTFs by monitoring caspase 3/7 activity using MPM over 48 hours. We found that ICI treatment resulted in a significant increase in caspase 3/7 activity compared to control treated LTFs. By blocking the interaction of T cells with tumor cells through antibody blockade, we demonstrated that the cytotoxicity observed was T cell mediated. As additional confirmation, we measured changes in cytotoxicity in the ICI resistant LLC1 tumor model. As expected, we did not observe an increase in cytotoxicity in LLC1 LTFs treated with ICI. MPM allows for the tracking of labeled T cells and characterization of their metabolism as a means of assessing changes in their activation state. We used a CD8 nanobody to label T cells in CT26 LTFs and then characterized the shift in metabolism following ICI treatment. Finally, we applied the Cybrid platform to human biopsies treated with ICI and characterized the cytotoxicity and shifts in T cell metabolism in living tissue. These data show that the Cybrid platform can measure changes in T cell metabolism and T cell cytotoxicity in response to ICI in 3D living tissue. Conclusions The methods presented herein will advance the field of cancer diagnostics by progressing from fixed tissue imaging and genetic characterization to the assessment of clinically relevant ICI-treated biopsies. Understanding the response of biopsies to immunotherapies will allow clinicians to make more informed treatment decisions and improve the lives of those with cancer. Citation Format: Jonathan Ouellette, Janey Degnan, Anna Kellner, Evan Flietner, Lindsay Doebert, Zachary Swider, Eric Wait, Victoria Pope, Laura Hrycyniak, Sean Caenepeel, Kevin Eliceiri, T S. Ramasubramanian, Michael J. Korrer. Assessing cytotoxic T cell responses to immune checkpoint inhibitors in murine and human tumor samples using metabolic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6444.

Astrocytes modulate cerebral blood flow and neuronal response to cocaine in prefrontal cortex.

Cocaine affects both cerebral blood vessels and neuronal activity in brain. Cocaine can also disrupt astrocytes, which modulate neurovascular coupling-a process that regulates cerebral hemodynamics in response to neuronal activation. However, separating neuronal and astrocytic effects from cocaine's direct vasoactive effects has been challenging, partially due to limitations of neuroimaging techniques able to differentiate vascular from neuronal and glial effects at high temporal and spatial resolutions. Here, we used a newly-developed multi-channel fluorescence and optical coherence Doppler microscope (fl-ODM) that allows for simultaneous measurements of neuronal and astrocytic activities (reflected by the intracellular calcium changes in neurons Ca2+N and astrocytes Ca2+A, respectively) alongside their vascular interactions in vivo to address this challenge. Using green and red genetically-encoded Ca2+ indicators differentially expressed in astrocytes and neurons, fl-ODM enabled concomitant imaging of large-scale astrocytic and neuronal Ca2+ fluorescence and 3D cerebral blood flow velocity (CBFv) in vascular networks in the mouse cortex. We assessed cocaine's effects in the prefrontal cortex (PFC) and found that the CBFv changes triggered by cocaine were temporally correlated with astrocytic Ca2+A activity. Chemogenetic inhibition of astrocytes during the baseline state resulted in blood vessel dilation and CBFv increases but did not affect neuronal activity, suggesting modulation of spontaneous blood vessel's vascular tone by astrocytes. Chemogenetic inhibition of astrocytes during a cocaine challenge prevented its vasoconstricting effects alongside the CBFv decreases, but it also attenuated the neuronal Ca2+N increases triggered by cocaine. These results document a role of astrocytes both in regulating vascular tone and consequently blood flow, at baseline and for modulating the vasoconstricting and neuronal activation responses to cocaine in the PFC. Strategies to inhibit astrocytic activity could offer promise for ameliorating vascular and neuronal toxicity from cocaine misuse.

Amphiphilic Polyurethane with Cluster-Induced Emission for Multichannel Bioimaging in Living Cell Systems.

The development of single-component materials with low cytotoxicity and multichannel fluorescence imaging capability is a research hotspot. In the present work, highly electron-deficient pyrazine monomers were covalently connected into a polyurethane backbone using addition polymerization with terminal poly(ethylene glycol) monomethyl ether units containing a high density of electron pairs. Thereby, an amphiphilic polyurethane-pyrazine (PUP) derivative has been synthesized. The polymer displays cluster-induced emission through compact inter- and/or intramolecular noncovalent interactions and extensive through-space electron coupling and delocalization. Molecular rigidity facilitates red-shifted emission. Based on hydrophilic/hydrophobic interactions and excitation dependence emission at low concentrations, PUP has been self-assembled into fluorescent nanoparticles (PUP NPs) without additional surfactant. PUP NPs have been used for cellular multicolor imaging to provide a variety of switchable colors on demand. This work provides a simple molecular design for environmentally sustainable, luminescent materials with excellent photophysical properties, biocompatibility, low cytotoxicity, and color modulation.

Self-confocal NIR-II fluorescence microscopy for multifunctional in vivo imaging

Fluorescence imaging in the second near-infrared window (NIR-II, 900–1880[Formula: see text]nm) with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution. However, the traditional NIR-II fluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal efficiency, as well as difficultly to adjust. Two types of upgraded NIR-II fluorescence confocal microscopes, sharing the same pinhole by excitation and emission focus, leading to higher confocal efficiency, are built in this work. One type is fiber-pinhole-based confocal microscope applicable to CW laser excitation. It is constructed for fluorescence intensity imaging with large depth, high stabilization and low cost, which could replace multiphoton fluorescence microscopy in some applications (e.g., cerebrovascular and hepatocellular imaging). The other type is air-pinhole-based confocal microscope applicable to femtosecond (fs) laser excitation. It can be employed not only for NIR-II fluorescence intensity imaging, but also for multi-channel fluorescence lifetime imaging to recognize different structures with similar fluorescence spectrum. Moreover, it can be facilely combined with multiphoton fluorescence microscopy. A single fs pulsed laser is utilized to achieve up-conversion (visible multiphoton fluorescence) and down-conversion (NIR-II one-photon fluorescence) excitation simultaneously, extending imaging spectral channels, and thus facilitates multi-structure and multi-functional observation.

A Multichannel Fluorescence Isothermal Amplification Device with Integrated Internet of Medical Things for Rapid Sensing of Pathogens through Deep Learning.

The landscape of diagnostic assessments has experienced a paradigm shift driven by the advent of isothermal amplification techniques on point-of-care testing (POCT). The development of compact, portable isothermal amplification devices further emphasizes their transformative influence on diagnostic approaches. However, in prioritizing portability, these devices may exhibit limitations in functionality, rendering them less effective in addressing urgent public health emergencies during sudden pathogen outbreaks. In this paper, an efficient isothermal fluorescence amplification device has been fabricated for the rapid detection of pathogens during public health crises. The device features multichannel capability for simultaneous detection of various targets, integrates with the Internet of Medical Things (IoMT) for remote control and data uploading, and includes a deep learning-based batch processing system for rapid (9.4 ms) and accurate discrimination of pathogen type with excellent accuracy. The device has been successfully employed to simultaneously detect Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) with limits of detection (LODs) of 18 CFU/mL (SA) and 20 CFU/mL (MRSA) within 35 min by multiplex RPA assay and CRISPR/Cas12a-mediated nucleic acid detection assay.

Delivery of Small-Molecule Drugs and Protein Drugs by Injectable Acid-Responsive Self-Assembled COF Hydrogels for Combinatorial Lung Cancer Treatment.

Covalent organic frameworks (COFs) have revealed enormous application prospects for cancer therapeutics recently, but their assembly systems face considerable challenges, such as the codelivery of hydrophobic and hydrophilic protein drugs with different physicochemical properties for in vivo delivery and release, as well as endosomal/lysosomal escape of protein drugs. To address these issues, we leveraged the high specific surface area, lipotropism, and structural tunability of boronate ester-linked COFs (COF-1) for the construction of advanced drug delivery systems. We first encapsulated the small-molecule drug doxorubicin (DOX) into a lipophilic COF (COF-1@DOX) and immobilized the functional protein drug ribonuclease A (RNase A) on the surface of the COF (RNase A-COF-1@DOX). We then created a novel composite delivery system (RNase A-COF-1@DOX gel) by cross-linking an albumin-oxygenated hydrogel (gel) network into the pores of COFs, allowing targeted codelivery of protein and small-molecule drugs in vivo. Using in-living body and multichannel fluorescence imaging, we analyzed the in vivo codelivery of protein and small-molecule drugs in a Lewis lung carcinoma (LLC) model. Finally, we applied the RNase A-COF-1@DOX gel to treat lung cancer in mice. This study paves an avenue for constructing COF-based drug delivery systems for lung cancer treatment and holds the potential to be extended to other types of cancer for more effective and targeted therapeutic treatments.

Near-Infrared-II Fluorophores for In Vivo Multichannel Biosensing

The pathological process involves a range of intrinsic biochemical markers. The detection of multiple biological parameters is imperative for providing precise diagnostic information on diseases. In vivo multichannel fluorescence biosensing facilitates the acquisition of biochemical information at different levels, such as tissue, cellular, and molecular, with rapid feedback, high sensitivity, and high spatiotemporal resolution. Notably, fluorescence imaging in the near-infrared-II (NIR-II) window (950–1700 nm) promises deeper optical penetration depth and diminished interferential autofluorescence compared with imaging in the visible (400–700 nm) and near-infrared-I (NIR-I, 700–950 nm) regions, making it a promising option for in vivo multichannel biosensing toward clinical practice. Furthermore, the use of advanced NIR-II fluorophores supports the development of biosensing with spectra-domain, lifetime-domain, and fluorescence-lifetime modes. This review summarizes the versatile designs and functions of NIR-II fluorophores for in vivo multichannel biosensing in various scenarios, including biological process monitoring, cellular tracking, and pathological analysis. Additionally, the review briefly discusses desirable traits required for the clinical translation of NIR-II fluorophores such as safety, long-wavelength emission, and clear components.

Simultaneous Spectral Temporal Modelling for a Time-Resolved Fluorescence Emission Spectrum

Innovations in complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) technology has featured in the development of next-generation instruments for point-based time-resolved fluorescence spectroscopy (TRFS). These instruments provide hundreds of spectral channels, allowing the collection of fluorescence intensity and fluorescence lifetime information over a broad spectral range at a high spectral and temporal resolution. We present Multichannel Fluorescence Lifetime Estimation, MuFLE, an efficient computational approach to exploit the unique multi-channel spectroscopy data with an emphasis on simultaneous estimation of the emission spectra, and the respective spectral fluorescence lifetimes. In addition, we show that this approach can estimate the individual spectral characteristics of fluorophores from a mixed sample.

Clinical assessment of a low-cost, hand-held, smartphone-attached intraoral imaging probe for 5-aminolevulinic acid photodynamic therapy monitoring and guidance.

India has one of the highest rates of oral squamous cell carcinoma (OSCC) in the world, with an incidence of 15 per 100,000 and more than 70,000 deaths per year. The problem is exacerbated by a lack of medical infrastructure and routine screening, especially in rural areas. New technologies for oral cancer detection and timely treatment at the point of care are urgently needed. Our study aimed to use a hand-held smartphone-coupled intraoral imaging device, previously investigated for autofluorescence (auto-FL) diagnostics adapted here for treatment guidance and monitoring photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence (FL). A total of 12 patients with 14 buccal mucosal lesions having moderately/well-differentiated micro-invasive OSCC lesions ( diameter and depth) were systemically (in oral solution) administered three doses of ALA (total ). Lesion site PpIX and auto-FL were imaged using the multichannel FL and polarized white-light oral cancer imaging probe before/after ALA administration and after light delivery (fractionated, total of 635nm red LED light). The handheld device was conducive for access to lesion site images in the oral cavity. Segmentation of ratiometric images in which PpIX FL is mapped relative to auto-FL enabled improved demarcation of lesion boundaries relative to PpIX alone. A relative FL (-value) threshold of 1.4 was found to segment lesion site PpIX production among the patients with mild to severe dysplasia malignancy. The segmented lesion size is well correlated with ultrasound findings. Lesions for which -value was at the time of treatment were associated with successful outcomes. These results indicate the utility of a low-cost, handheld intraoral imaging probe for image-guided PDT and treatment monitoring while also laying the groundwork for an integrated approach, combining cancer screening and treatment with the same hardware.

Optimizing the Cell Painting assay for image-based profiling.

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Customizable Dual-Fluorescent Nanoparticles for Tracing and Quantifying of Cell Transport.

Nanotechnology-based drug delivery systems (nano-DDS) have been developed to be a promising strategy to improve the efficacy, safety, physicochemical and pharmacokinetic/pharmacodynamics properties of drugs. It is very necessary to elucidate the delivery process in vivo or in cells for the rational design and accurate preparation of nano-DDS. The aim of this study was to construct a nano-DDS to visualize and quantify the intracellular behavior of the loaded cargo and carrier in such a system. A carboxyl-terminal end of poly(lactic-co-glycolic acid) polymer was fluorescently labeled with rhodamine B by conjugation of ethylenediamine. Dual-fluorescent nanoparticles (DFPs) were prepared from this fluorescently labeled polymer to encapsulate a fluorescent cargo, coumarin 6. The carrier and cargo of DFPs were monitored by confocal fluorescence microscopy during cellular uptake. Furthermore, the transcellular transportation of DFPs was evaluated quantitatively by measuring the fluorescence intensity. The obtained fluorescent polymer showed stable and quantifiable characteristics. DFPs could be customized in terms of coumarin 6 content (97.7±1.0%), size (367.3±1.7 nm) and dual-emission fluorescence (green cargo and red carrier). DFPs did not significantly affect cell viability, the integrity of cell monolayers and the microscopic morphology at concentrations below 0.7 mg/mL within 3 h of co-incubation with Caco-2 cells. Multichannel fluorescence monitoring revealed that the fluorescence intensity of the carrier and cargo increased with time, but not synchronously. By calculating the residual, intracellular, and transport amounts of DFPs, the material balance between the total amount of cellular transport and the dose administered was obtained. Based on the advantages of dual fluorescent labeling, the differential behavior of cell trafficking can be visualized and quantitatively analyzed for the cargo and carrier of DFPs. These results provide insights into the cellular transport process of holistic nanoparticles and complement our understanding of the biological behaviors of nano-DDS.