High Probe Intensity Photobleaching Measurement of Lateral Diffusion in Cell Membranes

Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on cell membrane molecules' size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to weakly expressed proteins of either of two types: fusions of membrane receptors with visible fluorescent proteins or membrane molecules on autofluorescent cells. To achieve adequate sensitivity in these cases, techniques such as interference fringe FPR are needed. However, in such measurements, cytoplasmic species contribute to the fluorescence recovery signal and thus yield diffusion parameters not properly representing the small number of surface molecules. A new method helps eliminate these difficulties. High Probe Intensity (HPI)-FPR measurements retain the intrinsic confocality of spot measurements to eliminate interference from fluorescent cytoplasmic species. However, HPI-FPR methods lift the previous requirement that FPR procedures be performed at probe beam intensities low enough to not induce bleaching in samples during measurements. The high probe intensities now employed provide much larger fluorescence signals and thus more information on molecular diffusion from each measurement. We report successful measurement of membrane dynamics by this technique.     

Synthesis of Novel Fluorogenic L-Fmoc Lysine Derivatives as Potential Tools for Imaging Cells

Two coumarin-labeled lysines were conveniently prepared as fluorescent probes. 7-Methoxy and 7-diethylamino coumarin-3-carboxylic acids were synthesized according to a modification of known procedures. Labeling at lysine was achieved in solution via the active N-hydroxysuccinimide ester of the carboxylic acid coumarin derivatives to give the target compounds in good yield. Spectroscopic data (UV-vis and fluorescence) were recorded for all compounds.     

Laser-Induced Fluorescence Imaging and Spectroscopy of GFP Transgenic Plants

Green fluorescent protein (GFP) and other fluorescent protein bioreporters can be used to monitor transgenes in plants. GFP is a valuable marker for transgene presence and expression, but remote sensing instrumentation for stand-off detection has lagged behind fluorescent protein marker biotechnology. However, both biology and photonics are needed for the monitoring technology to be fully realized. In this paper, we describe laser-induced fluorescence imaging and laser-induced fluorescence spectroscopy of GFP-transgenic plants in ambient light towards the application of remote sensing of transgenic plants producing GFP.     

Electroporative Adjustment of pH in Living Yeast Cells: Ratiometric Fluorescence pH Imaging

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.     

Ultrasensitive Microscopy of the Plasma Membrane of Living Cells

The view of the plasma membrane of biological cells was dramatically changed due to the discovery of lipid domains. Initially found as structurally distinct areas characterized by a specific protein content, the concept of lipid domains was rapidly taken over as a new scheme for explaining membrane targeted cellular processes. In this review, we discuss the capabilities of imaging methodologies to study lipid domains and their contributions to the current model of the cellular plasma membrane.     

用于活体人眼视网膜观察的自适应光学成像系统

利用自适应光学技术,研制了两套活体人眼视网膜高分辨力成像系统,在实时校正人眼波前误差的基础上,实现活体人眼视网膜细胞尺度的高分辨力成像。这两套系统分别采用19和37单元小型压电变形反射镜作为波前校正元件,哈特曼-夏克(Hartmann-Shack)波前传感器测量波前误差,用眼底反射的半导体激光作为波前探测的信标。在用计算机控制自适应光学系统实现人眼波前误差校正后,触发闪光灯照明视网膜,用CCD相机记录视网膜的高分辨力图像。校正后的残余波前误差的均方根值已分别小于1／6和1／10波长,相当于视网膜上成像分辨力分别为3．4μm和2．6μm,接近衍射极限。试验表明37单元系统的成像质量更好。     

一种示踪干细胞的磁共振-荧光双模成像探针

本文设计并合成了Gd基磁共振-荧光双模成像探针——Gd-DOTA-PEG-GA,通过电穿孔的方式标记人源间充质干细胞（hMSCs）.电穿孔标记诱导细胞将探针组装成团簇状纳米粒子进入细胞质,显著延长其与细胞结合的时间,并呈现出明显的T₂信号减弱效应,且信号减弱效应可以持续7天以上.在水溶液中,该探针的发射带集中在498 nm,并且荧光强度在一周内无明显衰减.该探针标记的细胞在荧光倒置显微镜下呈现绿色荧光.这些结果表明该探针可以作为磁共振-荧光双模成像探针用于干细胞示踪.     

Membrane Organization and Dynamics of the G-Protein-Coupled Serotonin<Subscript>1A</Subscript> Receptor Monitored Using Fluorescence-Based Approaches

The G-protein-coupled receptor (GPCR) superfamily represents one of the largest classes of molecules involved in signal transduction across the plasma membrane. Fluorescence-based approaches have provided valuable insights into GPCR functions such as receptor–receptor and receptor–ligand interactions, real-time assessment of signal transduction, receptor dynamics on the plasma membrane, and intracellular trafficking of receptors. This has largely been possible with the use of fluorescent probes such as the green fluorescent protein (GFP) from the jellyfish Aequoria victoria and its variants. We discuss the potential of fluorescence-based approaches in providing novel information on the membrane organization and dynamics of the G-protein-coupled serotonin_1A receptor tagged to the enhanced yellow fluorescent protein (EYFP). These authors contributed equally to the work.     

Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores. Authors contributed equally to this article.     

Fluorescence Methods to Probe Nanometer-Scale Organization of Molecules in Living Cell Membranes

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods which are capable of resolving structures at nanometer-scale resolution in living cells. In this review we survey fluorescence imaging methodologies capable of nanometer-scale resolution. We then critically examine specific biological applications of these methods, in the context of understanding membrane protein conformation and dynamics, intracellular signaling, organization of lipid rafts, and cell surface topology.     

A Depolarized Dynamic Light Scattering Method to Calculate Translational and Rotational Diffusion Coefficients of Nanorods

A new analysis of depolarized dynamic light scattering data is presented, which allows the unambiguous determination of rotational and translational diffusions coefficients of nanorods in suspension. By visualizing data scaling, purely translational diffusive motions can be isolated from vertically polarized scattering, allowing the unique determination of rotational diffusion from the depolarized scattering. The method is applied to nanorods with four different aspect ratios, and compared with theoretical predictions. Diffusion coefficients obtained show good agreement with calculations based on the direct measurements of rod length and diameter. Where the theories are shown to be valid, the method allows the measurement of statistically meaningful particle sizes and aspect ratios.     

Facile One‐Pot Conversion of Petroleum Asphaltene to High Quality Green Fluorescent Graphene Quantum Dots and Their Application in Cell Imaging

Benefiting from the natural nano‐size graphene‐structure in natural asphaltene material, a facile one‐pot route, mild chemical oxidation of low‐value petroleum asphaltene followed by routine ammonium neutralization, is presented to produce high quality graphene quantum dots (GQDs). The asphaltene‐derived GQDs possess a variety of oxygen‐containing and nitrogen‐containing functional groups such as carboxyl, hydroxyl, amine, and nitro groups. They present such excellent fluorescence properties as stable ability to retain strong green fluorescence within a relative broad excitation range in a bio‐suitable pH range of 4–7, high photoluminescence quantum yield of 18% and good fluorescent stability against photobleaching. And they are much smaller and thinner than most reported GQDs, displaying good biocompatibility with low cytotoxicity, effective cellular uptake, and excellent fluorescent probe performance for cancer cell imaging.     

Live Imaging of Glucose Homeostasis in Nuclei of COS-7 Cells

Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells. As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels. Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light. These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume. This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus. The glucose-sensor was targeted to nuclei of COS7-cells. To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels. Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration. Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.     

Fourier Interferometry as Applied to Microspectrofluorometry and Fluorescence Imaging of Living Cells

Excitation-emission fluorescence spectroscopy and imaging are applied to studies of cellular metabolism and at the convergence of cellular differentiation, detoxification, transformation, and senescence. Metabolic activity, intracellular redox levels, and compartmentation are probed by coenzyme [NAD(P)H] transients and monochlorobimane for glutathione dehydrogenase. Gene expression or its failure in lysosomal disorders is identified with fluorogenic probes. The "multiorganelle detoxification complex" is visualized and investigated with cytotoxic agents. A kind of photoactivated "accelerated cellular senescence" is recognized by accumulation of Schiff bases. Conventional and novel mitochondrial probes are used to localize these organelles in Saccharomyces cerevisiae as a model for future studies in mammalian cells and to detect in these very cells the organelle interactions resulting from the action of mitochondria-toxic drugs. The potential of these studies for biotechnology and instrumentation relying on fibers and integrated optics is considerably enhanced by Fourier interferometry.     

Imaging through scattering media using characteristics of the temporal distribution of transmitted laser pulses

An object consisting of small inhomogeneities embedded in a highly scattering solution was imaged using measurements of the time-resolved transmitted intensity of picosecond pulses of near-infrared light. Data acquisition involved translating the object in two orthogonal directions across the beam, and recording the temporal distribution of transmitted light at a series of discrete positions. Images were constructed from the total transmitted light, the first four moments of the temporal distribution, and from parameters derived from a comparison of the distribution with an analytical model, based on the diffusion approximation to the radiative transfer theory. The results show that the optical properties along a line-of-sight between source and detector influence some of these characteristics more than others.     

NMR Imaging of Thermally Polarized Helium-3 Gas

It is shown that thermally polarized 3He gas can be used to measure important physical parameters and to design, test, and tune imaging sequences. The bulk values of T1, T2, and the diffusion coefficient were measured in a glass cell containing a mixture of helium-3 (0.8 bar) and oxygen (0.2 bar). They were found to be T1 = 7 s, T2 = 2.4 s, and D = 1.6 cm2 s(-1). The relaxation times T2* and T1 and the apparent diffusion coefficient of thermally polarized helium-3 gas were measured in the rat lung, and these parameters were used to design a helium-3 optimized multi-spin-echo sequence which was shown to increase the signal-to-noise ratio sufficiently to obtain the first NMR-images of thermally polarized helium-3 in the rat lung.     

Ultrasmall Red Fluorescent Gold Nanoclusters for Highly Biocompatible and Long-Time Nerve Imaging

A rapidly growing area of neuroscience demands next-generation neurofluorescent probes are fulfilling several stringent criteria, including water solubility, distinct signal-to-background ratio, anti-photobleaching, and low toxicity. Herein, a novel neurofluorescent probe based on gold nanoclusters capped with glutathione (Au-GSH) is introduced and characterized by advanced fluorescence photophysical properties composed of comparative high quantum yield (8.9%), negligible blinking, and bright fluorescence in the red spectral range (E_m = 650 nm) with sub-millisecond-scale lifetime (0.62 ms). Fluorescent performance is tested and demonstrated negligible photobleaching under exposure to ultraviolet light (365 nm, 30 W) over 4 h, immunity to variation of the microenvironment characterized by pH range of 4–10, and colloidal stability in serum over 24 h during the blood circulation. Coupled with 2.4 ± 0.9 nm ultrasmall size and good water solubility, they are superior to fluorescent proteins, quantum dots, and organic fluorescent dyes. Au-GSH are further confirmed that they can be used as a fluorescent label for in vivo nerve and brain imaging, and even after injecting Au-GSH into the rat sciatic nerve for 21 d, the red fluorescence is still preserved. This combination of favorable properties makes Au-GSH a promising candidate for neurofluorescent probes.     

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.     

Cross‐Linked Proteins with Gold Nanoclusters: A Dual‐Purpose pH‐Responsive Material for Controllable Cell Imaging and Antibiotic Delivery

This report describes the development of a facile method for the synthesis of cross‐linked proteins with gold nanoclusters (CP‐GNC). The synthesis reaction is completed within 15 min at 97 °C. The synthesized CP‐GNC are characterized by using UV–vis absorption, fluorescence, X‐ray photoelectron spectroscopy, and transmission electron microscopy. CP‐GNC are approximately 100 nm in diameter and 700 nm in length, whereas AuNCs within the nanorods are approximately 6 nm in size. These materials are highly fluorescent with quantum yield of 7.2% and can be absorbed onto and release from bacterial cells in a pH‐dependent and reversible manner. The recent data show that CP‐GNC can be a useful, new tool with potential applications in fluorescent cell imaging and antibiotic targeting.