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.

MULTIPHOTON MICROSCOPIC IMAGING OF MOUSE INTESTINAL MUCOSA BASED ON TWO-PHOTON EXCITED FLUORESCENCE AND SECOND HARMONIC GENERATION

Multiphoton microscopy(MPM),based on two-photon excited fuorescence and second harmonic generation,enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents.In this paper,we used MPM to image the microstructures of the mucosa in fresh,unfixed,and unstained intestinal tissue of mouse.The morphology and distribution of the main components in mucosa layer such as columnar cells,goblet cells,intestinal glands,and a little collagen fibers were clearly observed in MPM images,and then compared with standard H&:E images from paired specimens.Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer.

Multi-branched carbazole derivatives for two-photon absorption and two-photon excited fluorescence

Carbazole-core multi-branched chromophores 9-ethyl- 3, 6-bis （ 2- { 4- [ 5- （4-tert-butyl-phenyl） - [ 1, 3, 4 ] oxadiazol-2-yl ] - phenyl }-vinyl） -carbazole（3） and 9-ethyl-3-（ 2- {4-[ 5-（4-tert-butyl- phenyl） -[ 1, 3, 4 ] oxadiazol-2-yl ] -phenyl }-vinyl ） -carbazole （ 2 ） are synthesized through Wittig reaction and characterized by nuclear magnetic resonance（NMR）and infrared（IR）. The two- photon absorption properties of chromophores are investigated. These chromophores exhibit large two-photon absorption crosssections and strong blue two-photon excited fluorescence. The cooperative enhancement of two-photon absorption（TPA） in the multi-branched structures is observed. This enhancement is partly attributed to the electronic coupling between the branches. The electronic push-pull structures in the arm and their cooperative effects help the extended charge transfer for TPA.

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.

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.

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.

Two-photon Excited Fluorescence of Bithiophene Derivatives

Two new bithiophene derivatives named as 5, 5-bis(p-N,N-dimethylaminostyryl)-2, 2 -bithiophene (BMSBT), and 5, 5-bis(p-N,N-diethylaminostyryl)-2, 2-bithiophene (BESBT) have been synthesized. Both compounds can emit strong single-photon excited fluorescence (SPEF) and two-photon excited fluorescence (TPEF) with the emission peaks around ~560 nm and with the lifetime of ~1ns.

Crystal Structure, One-and Two-photon Excited Fluorescence and Bioimaging of a D-π-A Structural Triphenylamine Derivative

An electron donor-π-bridge-electron acceptor(D-π-A) optical functional organic compound comprising a triphenylamine moiety as the electron donor and pyridine moiety as the electron acceptor was synthesized. The structure of the compound was solved by single-crystal X-ray diffraction analysis. It crystallizes in monoclinic, space group P21, with a = 9.753(5), b = 8.815(5), c = 25.554(5) ?, β = 96.315(5)°, V = 2184(2) ?~3, Z = 2, D_c = 1.136 g/m^3, F(000) = 792, Μr = 746.92, μ = 0.069 mm^(-1), the final R = 0.0658 and wR = 0.1730 for 6790 observed reflections with I > 2(I). Study of nonlinear optical properties shows that the compound exhibits excellent two-photon excited fluorescence with the two-photon absorption cross-section value of 116 GM. The structure-property relationship was researched in detail through X-ray crystallography and quantum chemical calculation. Result of living cell imaging experiment shows its potential in fluorescence microscopy bioimaging.

Synthesis, Structure of a Symmetrically Substituted Stilbene with Strong Two-photon Absorption and Up-converted Blue Fluorescence

Efficient Ti-catalyzed reductive coupling methodology was first employed to synthesize the symmetrical bis-donor stilbene, trans-4, 4'-bis[diphenyl amino] stilbene (BDPAS). X-ray diffraction analyses reveal that this new crystal belongs to the triclinic crystal system of centro-symmetric P-1 space group. The DBPAS solution, with the linear transmission at wavelength of greater than or equal to 450 nm, possesses large two-photon absorption cross section as high as 39.4x10(-48) cm(4).s/photon resulting in strong two-photon induced blue fluorescence of 460 nm, pumped by 740 nm laser irradiation.

Improved Detection of Sleeping Sickness Cases by LED Fluorescence Microscopy: Evidence from a Prospective Multi-Centric Study in the Democratic Republic of the Congo

Background: Confirmatory diagnosis of Trypanosoma brucei gambiense human African trypanosomiasis (HAT) is based on demonstration of parasites by microscopy. However, the sensitivity of routine microscopy methods is very low, and many cases are missed and left untreated. A clinical study was conducted in the Democratic Republic of the Congo to evaluate the accuracy of improved microscopy methods in diagnosis of HAT. These included examination by fluorescence microscopy (FM) of acridine orange (AO) stained smears of whole blood and smears made following a new procedure for concentrating trypanosomes by selective lysis of red blood cells (RBC). Methodology/Principal Findings: Venous blood was collected from 213 HAT cases, 101 HAT suspects and 95 controls and used to determine the accuracy of four microscopy methods: bright field microscopy of Giemsa-stained thick blood smears, FM of AO-stained thick blood smears, FM of AO-stained thick blood smears prepared after RBC lysis and concentration, and FM of AO-stained thin blood smears prepared after RBC lysis and concentration. The sensitivity of FM using thick blood smears stained with AO was 3 times higher than bright field microscopy using Giemsa-stained thick blood smears [19.7% (95% CI: 14.9% - 25.6%) versus 6.1% (95% CI: 3.6% - 10.2%)]. When the RBC lysis and concentration procedure was included, sensitivity of the test was further enhanced to 23.0% (95% CI: 17.9% - 29.1%) with thick blood smears and 34.3% (95% CI: 28.2% - 40.9%) with thin blood smears. Specificity of all four microscopy methods was 100% (95% CI: 96.1% - 100.0%). However, the miniature anion exchange chromatography technique (mAECT) and capillary tube centrifugation (CTC) method remained more sensitive. Conclusions: These new methods have practical advantages, including shorter staining time, ease of demonstration of parasites, and the possibility of archiving slides. They could, therefore, be alternative methods to improve case detection where concentration procedures such as mAECT or CTC are not performed.

3D fluorescence emission difference microscopy based on spatial light modulator

We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2
vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid,doughnut and z-axis hollow excitation spot,respectively.Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors.Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED.Also,the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads.The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.

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.

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.

New Thiophene Derivatives with Two-photon Excited Fluorescence

Two new compounds involving a thiophene moiety named as 2,5-bis[4-(N,N- diphenyl- amino)styryl]thiophene (BPST) and 2,5-bis[4-(N,N-diethylamino)styryl]thiophene (BEST) have been synthesized. The two-photon absorption cross section of BPST was measured as large as 256 × 10-50 cm4·s/photon, when it was excited by 800 nm femtosecond laser.

Transparent Electrode Materials for Simultaneous Amperometric Detection of Exocytosis and Fluorescence Microscopy

We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12 - 17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.

Single & Two-photon Excited Fluorescence of Two New Compounds with 2-Benzothiazolyl as Electron Acceptor

Two new D--A type compounds, where electron-donor D is tertiary amino group, electron-acceptor A is 2-benzothiazolyl and ?is two conjugated styryl units, have been synthesized. They are named as trans, trans-2-{4-[4-(N, N-diethylamino)styryl]styryl}-1, 3-benzothiazole and trans, trans-2-{4-[4-(N, N-diphenylamino)styryl]styryl}-1, 3-benzothiazole. Both compounds show strong two-photon excited fluorescence in yellow-orange region when excited by a femtosecond laser at 800 nm.

Field Evaluation of LED Fluorescence Microscopy for Demonstration of <i>Trypanosoma brucei rhodesiense</i>in Patient Blood

Diagnosis of Trypanosoma brucei rhodesiense human African trypanosomiasis requires demonstration of parasites in body fluids by microscopy. The microscopy methods that are routinely used are difficult to deploy in resource-limited settings due to practical challenges, including lengthy and tedious procedures, and the need for specific equipment to centrifuge samples in glass capillary tubes. We report here on a study that was conducted in a rural region of eastern Uganda to evaluate new methods that take advantage of a field-deployable LED fluorescence microscope. Examination of acridine orange-stained blood smears by LED fluorescence microscopy resulted in a diagnostic accuracy that was similar to that of routine methods, while the time needed to identify parasites was shortened significantly. These findings make these new microscopy methods attractive alternatives to procedures that are currently used for diagnosis of T. b. rhodesiense human African trypanosomiasis.

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.

Laser scanning fluorescence microscopic measurement of the movement of cleaving egg surface of Rana Amurensis

By laser scanning fluorescence microscopy for quan-titative measurement of fluorescence intensity changes on egg surface stained with fluorescein isothiocyanate duxing cleavage furrow extending forward, it was found that in area of presumptive cleavage furrow the scanning curve became ∨ shape, indicating dark stripe appeared in that place. Then the fluorescence intensity increased at the place where the botton of ∨ shape had located, and the scanning curve tuxned to ∧ shape, indicating single stripe was formed. While enhanced fluorescence appeared on the borders of ∧ shape, an M shape curve was found, show-ing double stripe occurred. During the distance between two borders of M shape incresing from 50 μm to 100μm,a fluorescence peak came to sight in the middle of the M shape, which being the cleavge furrow bottom. The two lateral sides of furrow bottom with decreasing fluorescence were nascent membrane. At that time the curve became W shape. By the sides of cleavage furrow the the stress folds became conspicous after double stripe stage, showing the stretching of the egg surface being increased. With our[31, 33] and others[32] reports that polylysine could induce the appearance of nascent membrane and phyto-hemagglutinins could decrease or prevent the appearance of nascent membrane, we believed the idea of Schroeder[25] that increasing mechanical stress could initiate nascent membrane formation and thought that the stress lay to the outsides of cleavage furrow.