DeepNoise: Signal and Noise Disentanglement Based on Classifying Fluorescent Microscopy Images via Deep Learning

The high-content image-based assay is commonly leveraged for identifying the phenotypic impact of genetic perturbations in biology field.However,a persistent issue remains unsolved during experiments:the interferential technical noises caused by systematic errors(e.g.,temperature,reagent concentration,and well location)are always mixed up with the real biological signals,leading to misinterpretation of any conclusion drawn.Here,we reported a mean teacher-based deep learning model(Deep Noise)that can disentangle biological signals from the experimental noises.Specifically,we aimed to classify the phenotypic impact of 1108 different genetic perturbations screened from 125,510 fluorescent microscopy images,which were totally unrecognizable by the human eye.We validated our model by participating in the Recursion Cellular Image Classification Challenge,and Deep Noise achieved an extremely high classification score(accuracy:99.596%),ranking the 2nd place among 866 participating groups.This promising result indicates the successful separation of biological and technical factors,which might help decrease the cost of treatment development and expedite the drug discovery process.The source code of Deep Noise is available at https://github.com/Scu-sen/Recursion-Cellular-Image-Classification-Challenge.

From static to dynamic:live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy

Ischemic stroke is one of the most common causes of mortality and disability worldwide.However,treatment efficacy and the progress of research remain unsatisfactory.As the critical support system and essential components in neurovascular units,glial cells and blood vessels(including the bloodbrain barrier)together maintain an optimal microenvironment for neuronal function.They provide nutrients,regulate neuronal excitability,and prevent harmful substances from entering brain tissue.The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis,supporting neuronal function,and reacting to injuries.However,most studies have focused on postmortem animals,which inevitably lack critical information about the dynamic changes that occur after ischemic stroke.Therefore,a high-precision technique for research in living animals is urgently needed.Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions.Twophoton fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure,information on multicellular component interactions,and provide images of structure and function in the cranial window.This technique shifts the existing research paradigm from static to dynamic,from flat to stereoscopic,and from single-cell function to multicellular intercommunication,thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain.In this review,we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy,highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain’s support systems.We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

Lipid droplets imaging with three-photon microscopy

Lipid droplets(LDs)participate in many physiological processes,the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity.It is crucial to monitor the distribution of LDs at high spatial resolution and large depth.Herein,we carried three-photon imaging of LDs in fat liver.Owing to the large three-photon absorption cross-section of the luminogen named NAP-CF_(3)(1:67×10^(-79) cm^(6) s^(2)),three-photon fluorescence fat liver imaging reached the largest depth of 80μm.Fat liver diagnosis was successfully carried out with excellent performance,providing great potential for LDs-associated pathologies research.

Myelin histology:a key tool in nervous system research

The myelin sheath is a lipoprotein-rich,multilayered structure capable of increasing conduction velocity in central and peripheral myelinated nerve fibers.Due to the complex structure and composition of myelin,various histological techniques have been developed over the centuries to evaluate myelin under normal,pathological or experimental conditions.Today,methods to assess myelin integrity or content are key tools in both clinical diagnosis and neuroscience research.In this review,we provide an updated summary of the composition and structure of the myelin sheath and discuss some histological procedures,from tissue fixation and processing techniques to the most used and practical myelin histological staining methods.Considering the lipoprotein nature of myelin,the main features and technical details of the different available methods that can be used to evaluate the lipid or protein components of myelin are described,as well as the precise ultrastructural techniques.

Observation of Insulin Exocytosis by a Pancreatic β Cell Line with Total Internal Reflection Fluorescence Microscopy

INSULIN secretion was traditionally measured with biochemical and immunological methods such as enzyme linked immunosorbant assay and radioimmunoassay. However, these methods can only tell the amount of insulin secreted; they give no information about the secretion process or mechanism of exocytosis. In recent years, an imaging technique known as total internal reflection fluorescence （TIRF） microscopy has been employed to study insulin secretion.

Super-resolution fluorescence polarization microscopy

Fluorescence polarization is related to the dipole orientation of chromophores,making fuores-cence polarization microscopy possible to_reveal structures and functions of tagged cellularorganelles and biological macromolecules.Several recent super resolution techniques have beenapplied to fluorescence polarization microscopy,achieving dipole measurement at nanoscale.In this review,we summarize both difraction limited and super resolution fluorescence polari-zation microscopy techniques,as well as their applications in biological imaging.

Two-photon excitation fuorescence lifetime imaging microscopy:A promising diagnostic tool for digestive tract tumors

Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.

Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy

AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Modulated illumination localization microscopy-enabled sub-10 nm resolution

Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.

Monitoring microenvironment of Hep G2 cell apoptosis using two-photon fluorescence lifetime imaging microscopy

Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.

Fluorescence life-time imaging microscopy(FLIM)monitors tumor cell death triggered by photothermal therapy with MoS_(2) nanosheets

Recently,photothermal therapy(PTT)has been proved to have great potential in tumor therapy.In the last several years,MoS_(2),as one novel member of nanomaterials,has been applied into PTT due to its excellent photothermal conversion efficacy.In this work,we applied fuorescence lifetime imaging microscopy(FLIM)techniques into monitoring the PPT-triggered cell death under MoS_(2) nanosheet treatment.Two types of MoS_(2) nanosheets(single layer nanosheets and few layer nanosheets)were obtained,both of which exhibited presentable photothermal conversion fficacy,leading to high cell death rates of 4T1 cells(mouse breast cancer cells)under PTT.Next,live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123,which were then continuously observed with FLIM technique.FLIM data showed that the fuorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS_(2) nanosheets significantly increased during PTT treatment.By contrast,the fuorescence lifetime of the same dye in control cells(without nanomaterials)remained constant after laser irradiation.These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells,which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.

U-Net-based deep learning for tracking and quantitative analysis of intracellular vesicles in time-lapse microscopy images

Fluorescence microscopy has become an essential tool for biologists,to visualize the dynamics of intracellular structures with specific labeling.Quantitatively measuring the dynamics of moving objects inside the cell is pivotal for understanding of the underlying regulatory mechanism.Protein-containing vesicles are involved in various biological processes such as material transportation,organelle interaction,and hormonal regulation,whose dynamic characteristics are signi¯cant to disease diagnosis and drug screening.Although some algorithms have been developed for vesicle tracking,most of them have limited performance when dealing with images with low resolution,poor signal-to-noise ratio(SNR)and complicated motion.Here,we proposed a novel deep learning-based method for intracellular vesicle tracking.We trained the U-Net for vesicle localization and motion classification,with demonstrates great performance in both simulated datasets and real biological samples.By combination with fan-shaped tracker(FsT)we have previously developed,this hybrid new algorithm significantly improved the performance of particle tracking with the function of subsequently automated vesicle motion classification.Furthermore,its performance was further demonstrated in analyzing with vesicle dynamics in different temperature,which achieved reasonable outcomes.Thus,we anticipate that this novel method would have vast applications in analyzing the vesicle dynamics in living cells.

Preliminary Study of the CAS-LIBB Single-Particle Microbeam Ⅱ Endstation: Ⅰ. Proposed Multi-Dimensional Quantitative Fluorescence Microscopy

Single-particle microbeam as a powerful tool can open a research field to find answers to many enigmas in radiobiology. A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering （LIBB）, Chinese Academy of Sciences （CAS）, China. However there has been less research activities in this field concerning the original process of the interaction between low-energy ions and complicated organisms. To address this challenge, an in situ multi-dimensional quantitative fluorescence microscopy system combined with the CAS-LIBB single-particle microbeam II endstation is proposed. In this article, the rationale, logistics and development of many aspects of the proposed system are discussed.

Fluorescence emission difference microscopy based on polarization modulation

In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.

Three-dimensional tracking of GLUT4 vesicles in TIRF microscopy

TIRF microscopy has provided a means to view mobile granules within 100 nm in size in two dimensions.However quantitative analysis of the position and motion of those granules requires an appropriate tracking method.In this paper,we present a new tracking algorithm combined with the unique features of TIRF.Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time.Mobile granules were then segmented from a time-lapse image stack by an adaptive background subtraction method.Kalman filter was introduced to estimate and track the granules that allowed reducing searching range and hence greater reliability in tracking process.After the tracked granules were located in x-y plane,the z-position was indirectly inferred from the changes in their intensities.In the experiments the algorithm was applied in tracking GLUT4 vesicles in living adipose cells.The results indicate that the algorithm has achieved robust estimation and tracking of the vesicles in three dimensions.

Advancing biological fluorescence microscopy with deep learning:a bibliometric perspective

Background:Fluorescence microscopy has increasingly promising applications in life science.This bibliometrics-based review focuses on deep learning assisted fluorescence microscopy imaging techniques.Methods:Papers on this topic retrieved by Core Collection on Web of Science between 2017 and July 2022 were used for the analysis.In addition to presenting the representative papers that have received the most attention,the process of development of the topic,the structure of authors and institutions,the selection of journals,and the keywords are analyzed in detail in this review.Results:The analysis found that this topic gained immediate popularity among scholars from its emergence in 2017,gaining explosive growth within three years.This phenomenon is because deep learning techniques that have been well established in other fields can be migrated to the analysis of fluorescence micrographs.From 2020 onwards,this topic tapers off but has attracted a few stable research groups to tackle the remaining challenges.Although this topic has been very popular,it has not attracted scientists from all over the world.The USA,China,Germany,and the UK are the key players in this topic.Keyword analysis and clustering are applied to understand the different focuses on this topic.Conclusion:Based on the bibliometric analysis,the current state of this topic to date and future perspectives are summarized at the end.

Unleashing the potential:super-resolution microscopy as the key to advanced mitochondrial research

Investigating the fine structure of mitochondria and their dynamic interactions with other organelles is crucial for unraveling the mechanisms underlying mitochondrial-related diseases.The development of super-resolution techniques has provided powerful visualization tools for mitochondrial research,which is significant for investigating mitochondrial cristae structure,the localization of mitochondrial-related protein complex,and the interactions between mitochondria and other organelles.In this perspective,we introduce several advanced super-resolution techniques and their applications in mitochondrial research,and discuss the potential roles these techniques may play in future studies of mitochondria.

EFFECT OF BUNGARUS MULTICINCTUS CRUDE VENOM AND ITS COMPONENTS ON TUMOR CELLS

Objective: To confirm whetherBungarus multicinctus crude venom induces the apoptosis of K562 tumor cells and to find out the components inducing apoptosis of K562 cells from the crude venom. Methods: the crude venom separated and purified by cation exchange chromatography, and the effect of venoms on K562 was studied by MTT method and flow cytometry. Results: The crude venom began to kill K562 cells at than 8×l03ng/ml (the survival rate was 82.5%) concentration and the effect was more significant in 24 h when administrating 8×l05ng/ml (the survival rate was 29.4%) crude venom. Apoptotic bodies were observed in the K562 tumor cells by fluorescent microscopy after administration of 5 μg/ml cycloheximide (CHX) or the peak VI solution at about 8×105 ng/ml. The same results were detected by the flow cytometry. A sub-Gi peak appeared after administration of CHX or the sixth peak solution. Conclusion: The authors found that the venom can kill K562 tumor cells in time- and dose-dependent manner. However, the killing effect of the venom is not apoptosis. What’s more, the peak VI solution, a component of the crude venom can induce the apoptosis of K562 tumor cells.

Single DNA Condensation Induced by Hexammine Cobalt with Molecular Combing

We investigated the interaction between DNA and hexammine cobalt III [Co（NH3）6]3＋ by a simple molecular combing method and dynamic light scattering. The average extension of A- DNA-YOYO-1 complex is found to be 20.9μm, about 30% longer than the contour length of the DNA in TE buffer （10 mmol/L Tris, 1 mmol/L EDTA, pH=8.0）, due to bis-intercalation of YOYO-1. A multivalent cation, hexammine cobalt, is used for DNA condensation. We find that the length of DNA-[Co（NH3）6]3＋ complexes decrease from 20.9 μm to 5.9μm as the concentration of the [Co（NH3）6]3＋ vary from 0 to 3 μmol/L. This observation provides a direct visualization of single DNA condensation induced by hexammine cobalt. The results from the molecular combing studies are supported by dynamic light scattering investigation, where the average hydrodynamic radius of the DNA complex decreases from 203.8 nm to 39.26 nm under the same conditions. It shows that the molecular combing method is feasible for quantitative conformation characterization of single bio-macromolecules.

Hydrocarbon charge history of the Paleogene reservoir in the northern Dongpu Depression,Bohai Bay Basin,China

The hydrocarbon charge history of the Paleogene in the northern Dongpu Depression was analyzed in detail based on a comprehensive analysis of the generation and expulsion history of the major hydrocarbon source rocks, fluorescence microscopic features and fluid inclusion petrography. There were two main stages of hydrocarbon generation and expulsion of oil from the major hydrocarbon source rocks. The first stage was the main hydrocarbon expulsion stage. The fluorescence microscopic features also indicated two stages of hydrocarbon accumulation. Carbonaceous bitumen, asphaltene bitumen and colloidal bitumen reflected an early hydrocarbon charge, whereas the oil bitumen reflected a second hydrocarbon charge. Hydrocarbon inclusions also indicate two distinct charges according to the diagenetic evolution sequence, inclusion petrography features combined with the homogenization temperature and reservoir burial history analysis. According to these comprehensive analysis results, the hydrocarbon charge history of the Paleogene reservoir in the northern Dongpu Depression was divided into two phases. The first phase was from the late Dongying depositional period of the Oligocene to the early uplift stages of the late Paleogene. The second phase was from the late Minghuazhen period of the Pliocene to the Quaternary. Reservoirs formed during the first period were widely distributed covering the entire area. In contrast,reservoirs formed during the second period were mainly distributed near the hydrocarbon generation sags. Vertically, it was characterized by a single phase in the upper layers and two phases in the lower layers of the Paleogene.