Two-photon Fluorescence Microscopy Research Articles

FAST: Fast, free, consistent, and unsupervised oligodendrocyte segmentation and tracking system.

To develop reparative therapies for neurological disorders like multiple sclerosis (MS), we need to better understand the physiology of loss and replacement of oligodendrocytes, the cells that make myelin and are the target of damage in MS. In vivo two-photon fluorescence microscopy allows direct visualization of oligodendrocytes in the intact brain of transgenic mouse models, promising a deeper understanding of the longitudinal dynamics of replacing oligodendrocytes after damage. However, the task of tracking the fate of individual oligodendrocytes requires extensive effort for manual annotation and is especially challenging in three-dimensional images. While several models exist for annotating cells in two-dimensional images, few models exist to annotate cells in three-dimensional images and even fewer are designed for tracking cells in longitudinal imaging. Notably, existing options often come with a substantial financial investment, being predominantly commercial or confined to proprietary software. Furthermore, the complexity of processes and myelin formed by individual oligodendrocytes can result in the failure of algorithms that are specifically designed for tracking cell bodies alone. Here, we propose a fast, free, consistent, and unsupervised beta-mixture oligodendrocyte segmentation system (FAST) that is written in open-source software, and can segment and track oligodendrocytes in three-dimensional images over time with minimal human input. We showed that the FAST model can segment and track oligodendrocytes similarly to a blinded human observer. Although FAST was developed to apply to our studies on oligodendrocytes, we anticipate that it can be modified to study four-dimensional in vivo data of any brain cell with associated complex processes.Significance Statement We have developed "FAST: Fast, free, consistent, and unsupervised oligodendrocyte segmentation and tracking system" to solve our challenge of quantification of four-dimensional data acquired from longitudinal in vivo imaging. Although it was developed for oligodendrocytes, we will make the code entirely open source and user-friendly, and expect that it will be useful for segmentation for any cell body from a complex cell amenable to longitudinal in vivo imaging.

Advancements in organic fluorescent materials: unveiling the potential of peripheral group modification in dithienyl-diketopyrrolopyrrole derivatives for one- and two-photon bioimaging.

The quest for novel organic fluorescent materials capable of two-photon absorption (2PA) has intensified in recent years due to their promising applications in biological imaging. Two-photon fluorescence microscopy (2PFM) offers high spatial-temporal resolution, reduced photodamage, and deeper tissue penetration compared to conventional techniques. However, the development of bright two-photon molecular markers remains a challenge, necessitating compounds with high fluorescence quantum yield and 2PA cross-section (σ2PA). Strategies such as increasing π-conjugation have shown promise but are hindered by synthesis complexities and limited biocompatibility. Alternatively, incorporating electron-donating (ED) or electron-withdrawing (EW) peripheral groups in a main structure has emerged as a viable approach, leading to significant enhancements in σ2PA. This study highlights the advantages and challenges of these strategies, emphasizing the importance of exploring new organic compounds and evaluating the efficacy of peripheral groups for advanced two-photon bioimaging applications.

Exploring Imaging Applications of a Red-Emitting π-Acceptor (π-A) Pyrene-Benzothiazolium Dye.

Bright biocompatible fluorescent imaging dyes with red to near-infrared (NIR) emissions are ideal candidates for fluorescence microscopy applications. Pyrene-benzothiazolium hemicyanine dyes are a new class of lysosome-specific probes reported on recently. In this work, we conduct a detailed implementation study for a pyrene-benzothiazolium derivative, BTP, to explore its potential imaging applications in fluorescence microscopy. The optical properties of BTP are studied in intracellular environments through advanced fluorescence microscopy techniques, with BTP exhibiting a noticeable shift toward blue (λem ≈ 590 nm) emissions in cellular lysosomes. The averaged photon arrival time (AAT)-based studies exhibit two different emissive populations of photons, indicating the probe's dynamic equilibrium between two distinctively different lysosomal microenvironments. Here, BTP is successfully utilized for time-lapse fluorescence microscopy imaging in real-time as a 'wash-free' imaging dye with no observed background interference. BTP exhibits an excellent ability to highlight microorganisms (i.e., bacteria) such as Bacillus megaterium through fluorescence microscopy. BTP is found to be a promising candidate for two-photon fluorescence microscopy imaging. The two-photon excitability of BTP in COS-7 cells is studied, with the probe exhibiting an excitation maximum at λTP ≈ 905 nm.

Adaptive optical correction for in vivo two-photon fluorescence microscopy with neural fields.

Adaptive optics (AO) restore ideal imaging performance in complex samples by measuring and correcting optical aberrations, but often require custom-built microscopes with carefully aligned wavefront sensing/shaping devices and can be susceptible to sample motion. Here we describe NeAT, a computational framework using neural fields for AO two-photon fluorescence microscopy. NeAT estimates wavefront aberration and recovers sample structure from a 3D image stack without requiring external datasets for training. Incorporating motion correction in learning and correcting conjugation errors commonly found in commercial microscopes, NeAT is designed for deployment in biological laboratories for in vivo imaging. We validate NeAT's performance using a custom-built microscope with a wavefront sensor under varying signal-to-noise ratios, aberration, and motion conditions. With a commercial microscope, we demonstrate real-time aberration correction for in vivo morphological and functional imaging in the living mouse brain, with NeAT improving signal and accuracy of glutamate and calcium imaging of synapses and neurons.

Light on abnormal red blood cell subpopulations: Label-free optics-based approach for studying in vitro rigidified blood cells

RBCs deformability plays a crucial role in maintaining proper blood flow and oxygen delivery throughout the body. Conventional ektacytometry fails to differentiate between variations in deformability of RBC subpopulations as the averaging measurement process obscures these differences. In this study, we introduced an approach that integrates label-free optics-based techniques (flow cytometry, phase-contrast, and two-photon excitation fluorescent microscopy) with ektacytometry to evaluate subpopulations that exhibit decreased RBCs deformability upon an in vitro oxidation using 0.5 mM TBHP, as a low-level oxidative agent.We found that flow cytometry can easily detect rigidified and oxidized subpopulations based on FSC/SSC light distribution, as well as RBCs fluorescence intensity and peak area likely originating from hemoglobin photo and/or degradation products. Two-photon excitation fluorescence microscopy proved altered morphology and spatial location of fluorescence intensity signal near the membrane of oxidized RBCs, when compared to control RBCs, indicating a link with the reduced deformability. The proposed label-free optics-based methodology, which combines established techniques with more sophisticated microscopy, emerges as a promising tool for detecting mechano-biological changes in different RBC subpopulations induced by oxidative stress. The findings suggest potential applications in clinical practice for monitoring pathological conditions influenced by physical or environmental stress and as a quality control measure for stored RBCs.

Simultaneous dual-colour labelling of mitochondria and lysosomes: An indolium-based approach

Fluorescence is a valuable tool for understanding mitochondria-lysosome interactions, which is central for unveiling their roles in health and disease. Herein, we describe novel indolium-based molecules for labelling mitochondria and lysosomes simultaneously with organelle-specific fluorescence properties. These probes readily enable clear distinction of mitochondria and lysosomes by combining confocal and two-photon fluorescence microscopy. Our results show that this unique behaviour relies on aggregation. The functioning of these probes is not compromised by mitophagy, allowing the visualisation of these organelles in separate emission channels by using a single fluorescent marker under all scenarios. Their enhanced experimental simplicity compared to using multiple dyes makes these novel compounds well-suited for automated screening applications within living cells.

TPF stitching imaging of rubber tree leaves

This paper presents a two-photon fluorescence (TPF) microscopy platform based on a femtosecond oscillator. The system images rubber tree leaf samples by exciting and detecting TPF signals generated by the tissue using broadband femtosecond pulses. The imaging system utilizes a detection window of 565–615 nm to capture TPF signals from flavin adenine dinucleotide (FAD) in the rubber tree leaf tissue. Additionally, multiple TPF images were acquired through sample movement and Z-axis scanning, followed by image stitching to achieve large-area comprehensive visualization of the rubber tree leaf samples. This study provides detailed observations and analyses of various biological structures within the rubber tree leaf samples, contributing significantly to the understanding of plant growth, physiological functions, and environmental adaptability.

Application Exploration of the Photon Chip Technology in Real time Processing of Biomedical Images

In the biomedical field, photonic chip technology is gradually becoming the key to improving the speed and accuracy of medical imaging. The development of this technology has given us new hope in early disease diagnosis and cell monitoring. Recent research has not only sorted out the hotspots and future trends of biomedical photonics, but also paid special attention to the application of photon technology in these fields. Specifically, photonic chips have demonstrated their unique capabilities in multi-color two-photon excitation fluorescence microscopy imaging, two-photon fluorescence lifetime imaging, and two-photon fiber endoscopic imaging. These applications not only improve the clarity of imaging, but also accelerate processing speed, which is crucial for rapid diagnosis and treatment. At the end of the article, the author summarized the contributions of photonic chip technology in the field of biomedical imaging and provided prospects for future development directions.

Acne-related UVA-induced facial fluorescence: An exploratory study from physiological properties to tissue structure information

UVA-induced facial fluorescence (UVAF) is recognized as an objective measurement technique to quantify the severity of acne. However, notable inconsistencies in quantitative outcomes have been observed in various studies, possibly due to the fact that different colors of fluorescence represent different pathophysiological implications. This study investigated the pathophysiological importance of UVAF color differences and improved its reliability in assessing acne severity. MIDOO Smart Skin Imager was used to capture UVAF and analyze the correlation between fluorescence colors and acne lesions. Techniques such as two-photon excited fluorescence microscopy, scanning electron microscopy, western blot, and high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) were used to examine the biochemical composition and structure of comedonal plugs and follicular casts associated with different fluorescence colors. We found that green fluorescence correlates with non-inflammatory acne lesions (comedones), while orange-red fluorescence shows no correlation with either type of lesion. Green fluorescence is associated with higher levels of keratin, indicating keratinization, while orange-red fluorescence is associated with porphyrin from S. epidermidis. UVAF color differences - orange-red are from porphyrins and green from keratin. This distinction helps to understand the structural and physiological bases of facial fluorescence, with potential implications for clinical evaluations of acne.

Photobleaching analysis of fluorescent proteins in two-photon microscopy at high repetition rates

Severe photobleaching in two-photon excitation fluorescence microscopy (TPM) has limited its potential for long-term and quantitative imaging of live cells and tissues. One solution is to excite fluorophores with a high-repetition rate which reduces the peak intensity of irradiance. However, there is a lack of knowledge about the general utility of this strategy for fluorescent proteins. Here, using simple photobleaching assay, we studied the photobleaching of EGFP and tdTomato at 80 MHz and 640 MHz repetition rates. Our analysis shows that the impact of high repetition rate excitation on reducing photobleaching in TPM is highly dependent on the excitation wavelength and fluorophores. Our results are useful for selecting optimal parameters to minimize photobleaching and photodamage in TPM.

Ultrasensitive Dual Fluorophore-Conjugated Carbon Dots for Intracellular pH Sensing in 3D Tumor Models.

The dysregulation of pH has been linked to the onset of chronic conditions, such as cancer and neurological diseases. Consequently, the development of a highly sensitive tool for intracellular pH sensing is imperative to investigate the interplay between pH and the biochemical changes accompanying disease pathogenesis. Here, we present the development of a ratiometric fluorescent nanoprobe, NpRhoDot, designed for precisely measuring pH levels. We demonstrate its efficacy in sensitively reporting intracellular pH in monolayer A549 lung cancer cells, primary fibroblast cells, and 3D tumor spheroids derived from the DLD-1 colorectal adenocarcinoma cell line. NpRhoDot leverages a novel design, where stable carbon dots are functionalized with a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine moiety, NpRho1. This design confers NpRhoDot with the high pH sensitivity characteristics of organic fluorescent probes, along with excellent photostability up to 1 h and biocompatibility of carbon dots. Through one-photon and two-photon fluorescence microscopy, we validate the reliability of NpRhoDot for biosensing intracellular pH in monolayer and three-dimensional tumor models from pH 4 to 7.

Convolutional neural network transformer (CNNT) for fluorescence microscopy image denoising with improved generalization and fast adaptation

Deep neural networks can improve the quality of fluorescence microscopy images. Previous methods, based on Convolutional Neural Networks (CNNs), require time-consuming training of individual models for each experiment, impairing their applicability and generalization. In this study, we propose a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), that outperforms CNN based networks for image denoising. We train a general CNNT based backbone model from pairwise high-low Signal-to-Noise Ratio (SNR) image volumes, gathered from a single type of fluorescence microscope, an instant Structured Illumination Microscope. Fast adaptation to new microscopes is achieved by fine-tuning the backbone on only 5–10 image volume pairs per new experiment. Results show that the CNNT backbone and fine-tuning scheme significantly reduces training time and improves image quality, outperforming models trained using only CNNs such as 3D-RCAN and Noise2Fast. We show three examples of efficacy of this approach in wide-field, two-photon, and confocal fluorescence microscopy.

Variational calculus approach to Zernike polynomials with application to FCS

Zernike polynomials are a sequence of orthogonal polynomials that play a crucial role in optics and, in particular, modeling microscopy systems. Introduced by Frits Zernike in 1934, they are particularly useful in expressing wavefront aberrations and, thus, imperfections of imaging systems. However, their origin and properties are rarely discussed and proven. Here, we present a novel approach to Zernike polynomials using variational calculus and apply them to describe aberrations in fluorescence microscopy. In particular, we model the impact of various optical aberrations on the performance of one-photon and two-photon excitation fluorescence microscopy.

Localization of protoporphyrin IX during glioma-resection surgery via paired stimulated Raman histology and fluorescence microscopy.

The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas.

High-speed two-photon microscopy with adaptive line-excitation.

We present a two-photon fluorescence microscope designed for high-speed imaging of neural activity at cellular resolution. Our microscope uses an adaptive sampling scheme with line illumination. Instead of building images pixel by pixel via scanning a diffraction-limited spot across the sample, our scheme only illuminates the regions of interest (i.e., neuronal cell bodies) and samples a large area of them in a single measurement. Such a scheme significantly increases the imaging speed and reduces the overall laser power on the brain tissue. Using this approach, we performed high-speed imaging of the neuronal activity in mouse cortex in vivo. Our method provides a sampling strategy in laser-scanning two-photon microscopy and will be powerful for high-throughput imaging of neural activity.

Three-Dimensional Imaging of Bioinspired Lipidic Mesophases Using Multicolored Light-Emitting Carbon Nanodots.

Recent progress in the design of carbon nanostructures exhibiting strong multiphoton-excited emission opens new pathways to explore the self-organization of lipids found in living organisms. Phospholipid-based lyotropic myelin figures (MFs) are promising materials as simplified models of biomembranes due to their structural resemblance to a multilamellar sheath insulating the axon. This study demonstrates the possibility of selective labeling of MFs by strongly emitting multicolor phloroglucinol-derived carbon nanodots (PG CNDs). Such dopants are efficiently excited by visible and near-infrared light; therefore, one- and two-photon fluorescence microscopies are incorporated to gain 3D insights into the MFs. Combining nondestructive fluorescence microscopy and spectroscopy techniques along with polarized light microscopy gives details on the stability and morphology of lipidic mesophases. Our findings suggest that PG CNDs can be a viable and simple alternative to conventional fluorescent lipid stains to image biologically relevant phospholipid-based structures.

Improved image contrast in nonlinear light-sheet fluorescence microscopy using i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE Pulse compression

Nonlinear microscopy has become an invaluable tool for biological imaging, offering high-resolution visualization of biological specimens. In this manuscript, we present the application of a spectral phase measurement technique, i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE, to compress broad-bandwidth supercontinuum pulses for two-photon excitation fluorescence light-sheet fluorescence microscopy. The results demonstrated a significant improvement in the two-photon excitation response achieved. We also showed that the implementation of i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE allowed for enhanced image contrasts when compared to conventional compression techniques, with i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE producing an image contrast improvement over conventional methods by over 50%.

An antagonist-based two-photon fluorogenic probe for imaging metabotropic glutamate receptor 5 in neuronal cells

The expression of metabotropic glutamate receptor 5 (mGluR5) is subject to developmental regulation and undergoes significant changes in neuropsychiatric disorders and diseases. Visualizing mGluR5 by fluorescence imaging is a highly desired innovative technology for biomedical applications. Nevertheless, there are substantial problems with the chemical probes that are presently accessible. In this study, we have successfully developed a two-photon fluorogenic probe, mGlu-5-TP, based on the structure of mGluR5 antagonist 6-methyl-2-(phenylethynyl)pyridine (MPEP). Due to this antagonist-based probe selectively recognizes mGluR5, high expression of mGluR5 on living SH-SY5Y human neuroblastoma cells has been detected during intracellular inflammation triggered by lipopolysaccharides (LPS). Of particular significance, the probe can be employed along with two-photon fluorescence microscopy to enable real-time visualization of the mGluR5 in Aβ fiber-treated neuronal cells, thereby establishing a connection to the progression of Alzheimer's disease (AD). These results revealed that the probe can be a valuable imaging tool for studying mGluR5-related diseases in the nervous system.

293 The Impact of Tumor Mutational Status on Protoporphyrin IX Accumulation in Tumor-Associated Macrophages in Glioblastoma Patients

INTRODUCTION: Immunotherapies provide promising new treatment options for lung cancer and melanoma, however success in glioblastoma (GBM) patients has been much rarer and uneven. Identifying candidates with GBM that are most likely to benefit from immunotherapy remains a challenge. The abundance of tumor-associated macrophages (TAMs) correlates response to immunotherapy in GBM patients. Here we leverage a new imaging technique for quantifying TAMs via tandem stimulated Raman histology (SRH) and two-photon excitation fluorescence microscopy (TPEF) to assess the impact of genetic classification on the abundance of TAMs. METHODS: We conducted tandem SRH and TPEF on specimens from 79 patients undergoing high-grade glioma resection with 5-ALA. We trained a MaskR-CNN deep neural network to identify TAMs and compute their density in each specimen. Corresponding clinical, demographic, genomic and methylomic data were collected for each patient. Independent sample t-tests were used to identify significant correlations between genetic markers, treatment, and TAM density. RESULTS: We analyzed 3,121,524 300 x 300 micron fields of view, from 151 fresh specimens, enabling segmentation of 611,789 TAMs. IDH-mutant tumors had significantly lower TAM density than IDH-wildtype tumors (p < 0.01). The absence of MGMT promoter hypermethylation was associated with significantly higher TAM density (p = 0.03). Tumors with EGFR amplification averaged significantly lower TAM density than EGFR wildtype specimens (p < 0.01). Recurrent tumors possessed 31.5% lower TAM density than primary resections. CONCLUSIONS: Tandem SRH and TPEF can estimate variations in TAM density in human GBMs. We found TAM density to be associated with IDH status, MGMT promoter methylation, and EGFR amplification. This imaging technique creates a new avenue for forecasting response to immunotherapy in GBM patients.

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)