Direct observation of chemokine receptors 5 on T-lymphocyte cell surfaces using fluorescent metal nanoprobes 2: Approximation of CCR5 populations

Metal nanoparticle probes were used as molecular imaging agents to detect the expression levels and spatial distributions of the CCR5 receptors on the cell surfaces. Alexa Fluor 647-labeled anti-CCR5 monoclonal antibodies (mAbs) were covalently bound to 20 nm silver nanoparticles to synthesize the mAb–metal complexes. We measured the single nanoparticle emission of the mAb–metal complexes, showing that the complexes displayed enhanced intensities and reduced lifetimes in comparison with the metal-free mAbs. Six HeLa cell lines with various CCR5 expressions were incubated with the mAb–metal complexes for the target-specific binding to the cell surfaces. Fluorescence cell images were recorded on a time-resolved confocal microscope. The collected images expressed clear CCR5 expression-dependent optical properties. Two regression curves were obtained on the basis of the emission intensity and lifetime over the entire cell images against the number of the CCR5 expression on the cells. The emission from the single mAb–metal complexes could be distinctly identified from the cellular autofluorescence on the cell images. The CCR5 spatial distributions on the cells were analyzed on the cell images and showed that the low-expression cells have the CCR5 receptors as individuals or small clusters but the high expression cells have them as the dense and discrete clusters on the cell surfaces.     

Direct observation to chemokine receptor 5 on T-lymphocyte cell surface using fluorescent metal nanoprobes

Chemokine receptor 5 (CCR5) is a cell surface protein required for HIV-1 infection. It is important to detect the amount and observe the spatial distribution of the CCR5 receptors on the cell surfaces. In this report, we describes the metal nanoparticles which were specially designed as molecular fluorescent probes for imaging of CCR5 receptors on the T-lymphocytic PM1 cell surfaces. These CCR5 monoclonal antibodies (mAbs) metal complexes were prepared by labeling mAbs with Alexa Fluor 680 followed by covalent binding the labeled mAbs on the 20 nm silver nanoparticles. Compared with the labeled mAbs without metal, the mAb-metal complexes were found to display enhanced emission intensity and shortened lifetime due to interactions between fluorophores and metal. The mAb-metal complexes were incubated with the PM1 cell lines. The confocal fluorescent intensity and lifetime cell images were recorded on single cells. It was observed that the mAb-metal complexes could be clearly distinguished from the cellular autofluorescence. By analyzing a pool of cell images, we observed that most CCR5 receptors appeared as clusters on the cell surfaces. The fluorophore-metal complexes developed in this report are generally useful for detection of cell surface receptors and provide a new class of probe to study the interaction between the CCR5 receptors with viral gp120 during HIV infections.     

Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10⁹ in a single automated experimental run.     

Imaging single virus particles on the surface of cell membranes by high-resolution scanning surface confocal microscopy

We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 ± 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell.     

Imaging of the Blue,Green, and Red Fluorescence Emission of Plants: An Overview

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

Concerted Antibody and Antigen Discovery by Differential Whole-cell Phage Display Selections and Multi-omic Target Deconvolution

Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm² is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.     

近场扫描光学成像结合量子点标记的纳米技术在细胞生物学中的应用

近场扫描光学显微镜（NSOM）对传统的光学分辨极限产生了革命性的突破,可在超高光学分辨率下无侵人性和无破坏性地对生物样品进行观测。量子点（QDs）具有极好的光学性能,如荧光寿命长、激发谱宽、生物相容性强、光稳定性好等优点,适合先进的生物成像。NSOM结合QDs标记的纳米技术被应用在细胞生物学中。通过纳米量级NSOM免疫荧光成像（50nm）对特定蛋白分子在细胞表面的动态分布进行可视化研究和数量化分析,阐明了蛋白分子在不同细胞过程中的作用机制。因此,NSOM／QD基成像系统提供了单个蛋白分子最高分辨率的荧光图像,为可视化研究蛋白分子机制的提供了一种强有力的工具。     

Nanoaperture Fluorescence Enhancement in the Ultraviolet

We study fluorescence excitation and emission enhancement in the ultraviolet regime for molecules confined within sub-wavelength metal apertures. Calculations are performed across a range of excitation wavelengths for individual apertures constructed in gold, silver, and aluminum. As expected, enhancement in the ultraviolet is greatest with aluminum. Using excitation and emission wavelengths appropriate for tryptophan, we find that more than 10× net increase in fluorescence count rate should be obtainable for aluminum apertures of ~75 nm diameter. These results suggest that many applications utilizing native protein fluorescence could become practical, even to the single molecule level.     

Direct observation of ligand colocalization on individual receptor molecules.

We have exploited the novel methodology of far-field fluorescence microscopy at the single molecule level to study colocalization of two different ligand molecules on an individual receptor. The use of dual-wavelength single molecule imaging allows discrimination between isolated and colocalized ligands with an accuracy of 40 nm. In the case of very close proximity of the two ligands, below 7 nm, single pair Forster energy-transfer was observed. The latter finding unequivocally demonstrates colocalization of two ligands on an individual receptor.     

Molecular beacons with intrinsically fluorescent nucleotides

We report the design, synthesis and characterization of a novel molecular beacon (MB-FB) which uses the fluorescent bases (FB) 2-aminopurine (AP) and pyrrolo-dC (P-dC) as fluorophores. Because the quantum yield of these FB depend on hybridization with complementary target, the fluorescent properties of MB-FB were tuned by placing the FB site specifically within the MB such that hybridization with complementary sequence switches from single strand to double strand for AP and vice versa for P-dC. The MB-FB produces a ratiometric fluorescence increase (the fluorescence emission of P-dC over that of AP in the presence and absence of complementary sequence) of 8.5 when excited at 310 nm, the maximum absorption of AP. This ratiometric fluorescence is increased to 14 by further optimizing excitation (325 nm). The fluorescence lifetime is also affected by the addition of target, producing a change in the long-lived component from 6.5 to 8.7 ns (Exc. 310 nm, Em. 450 nm). Thermal denaturation profiles monitored at 450 nm (P-dC emission) show a cooperative denaturation of the MB-FB with a melting temperature of 53°C. The thermal denaturation profile of MB-FB hybridized with its target shows a marked fluorescence reduction at 53°C, consistent with a transition from double stranded helix to random coil DNA.     

Study of effects of cholesterol on the polar groups of the lipid bilayer using a fluorescent probe, 4-dimethylaminochalcone

Fluorescence parameters of a probe 4-dimethylaminochalcone (DMAC) in egg phosphatidylcholine bilayer lipid membranes are extremely sensitive to cholesterol, since the latter influences the solvation shells of DMAC formed by lipid molecules. The membrane population of DMAC is heterogeneous and is represented by two populations of molecules. The first of them contains about 60% of DMAC molecules, which fluoresce with a short (0.3 ns) decay time at 485 nm; their solvation shells are insensitive to 30 mol. % cholesterol. The second DMAC population is responsible for more than 50% of total fluorescence. Reorientation of the probe solvation shell dipoles is accompanied by a noticeable Stokes shift of the DMAC fluorescence spectrum; in the absence of cholesterol the Stokes shift amounts to 50 nm (i.e., from 485 to 535 nm). In the presence of 30 mol. % cholesterol, the effect of relaxation of the solvation shells on the DMAC fluorescence shift is reduced to 22 nm. At the same time, cholesterol reduces fluorescence quenching in the second population of DMAC molecules due to a twofold increase in their fluorescence quantum yield. Reorientation of solvation shell dipoles inducing the Stokes shift is complete within less than 0.1 ns, apparently, at the expense of fast-relaxing dipoles, viz., water molecules penetrating into the lipid bilayer. The data obtained suggest that cholesterol induces only partial modification of the lipid bilayer; some membrane regions are not exposed to its effect even when the cholesterol fraction constitutes up to 30% of the total lipid content.     

Single cell measurement of micro-viscosity by ratio imaging of fluorescence of styrylpyridinium probe

In aqueous solution, compounds containing the styrylpyridinium group showed dual fluorescence, in which excitation at either 469 or 360 nm each produced an emission band around 600 nm. The ratio of fluorescence intensities of the two bands (R = I469/I360) was sensitive to local viscosity. The N-carboxymethyl butyl ester of DMASP was found to be able to irreversibly load into a living cell; presumably by hydrolysis involving cellular lipases it was transformed to a membrane-impermeable fluorescent carboxylate. A map of the ratio, R, from a single cell was generated using fluorescence imaging microscopy with a spectrofluorimeter in dual-excitation single-emission mode. After calibrating the ratio for the probe in water/glycerol solutions, the intracellular viscosities were obtained for a single cell of smooth muscle of a rat embryonic thoracic aorta. The intracellular viscosity is differentiated inside the cell and the obtained values 18-7 cP obey all the values reported by other laboratories. Fluorescence emission of the probe (500-650 nm) is in a very favourable region for its use with visible fluorescence microscopy, without interferences from cell or tissue auto-fluorescence. The results present ability to detect and follow small changes in the ratio of fluorescence intensities, and apparently of the micro-viscosity.     

Single-Molecule Imaging on Living Bacterial Cell Surface by High-Speed AFM

Advances in microscopy have contributed to many biologic discoveries. Electron microscopic techniques such as cryo-electron tomography are remarkable tools for imaging the interiors of bacterial cells in the near-native state, whereas optical microscopic techniques such as fluorescence imaging are useful for following the dynamics of specific single molecules in living cells. Neither technique, however, can be used to visualize the structural dynamics of a single molecule at high resolution in living cells. In the present study, we used high-speed atomic force microscopy (HS-AFM) to image the molecular dynamics of living bacterial cell surfaces. HS-AFM visualizes the dynamic molecular processes of isolated proteins at sub-molecular resolution without the need for complicated sample preparation. In the present study, magnetotactic bacterial cells were anchored in liquid medium on substrate modified by poly-l-lysine and glutaraldehyde. High-resolution HS-AFM images of live cell surfaces showed that the bacterial outer membrane was covered with a net-like structure comprising holes and the hole rims framing them. Furthermore, HS-AFM captured the dynamic movement of the surface ultrastructure, showing that the holes in the net-like structure slowly diffused in the cell surface. Nano-dissection revealed that porin trimers constitute the net-like structure. Here, we report for the first time the direct observation of dynamic molecular architectures on a live cell surface using HS-AFM.     

Photoelectron imaging of cells: photoconductivity extends the range of applicability.

Photoelectron imaging is a sensitive surface technique in which photons are used to excite electron emission. This novel method has been applied successfully in studies of relatively flat cultured cells, viruses, and protein-DNA complexes. However, rounded-up cell types such as tumor cells frequently are more difficult to image. By comparing photoelectron images of uncoated and metal-coated MCF-7 human breast carcinoma cells, it is shown that the problem is specimen charging rather than a fundamental limitation of the electron imaging process. This is confirmed by emission current measurements on uncoated monolayers of MCF-7 carcinoma cells and flatter, normal Wi-38 fibroblasts. We report here that sample charging in photoelectron microscopy can be eliminated in most specimens by simultaneous use of two light sources--the standard UV excitation source (e.g., 254 nm) and a longer wavelength light source (e.g., 325 nm). The reduction in sample charging results largely from enhanced photoconduction in the bulk sample and greatly extends the range of cells that can be examined by photoelectron imaging. The contributions of photoconductivity, the electric field of the imaging system, and the short escape depths of the photoelectrons combine to make photoelectron imaging a uniquely sensitive technique for the study of biological surfaces.     

Assessing the poplar photochemical response to high zinc concentrations by image processing and statistical approach

Exposure of plants to high-heavy metals concentration inhibits multiple metabolic processes in plants and leads to an oxidative stress commonly referred as heavy metal ion toxicity. Chlorophyll a fluorescence has enhanced understanding of heavy metal ion action on the photosynthetic system. A rapid and non-invasive technique involving imaging of chlorophyll fluorescence is a useful tool for early detection of plant responses to heavy metal ion toxicity. In this work chlorophyll fluorescence emission and photochemical parameters in plants of Populus x euramericana clone I-214 were investigated by the portable Imaging PAM fluorometer at different days after soil treatment with zinc. Custom software for analysis of the photochemical parameters images has been developed in order to gain a better assessing of the plant performance in response of metal stress. The imaging analysis allowed visualizing heterogeneity in plant response to high zinc concentrations. The heterogeneity of images suggests spatial differences in photochemical activity and changes in the antenna down-regulation.     

Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells.

A new method of fluorescence microscopy for cell imaging has been developed that takes advantage of the spatial variations of fluorescence lifetimes in single cells as a source of image contrast, and thus it is named "fluorescence lifetime imaging microscopy (flimscopy)". Since time-resolved fluorescence measurements are sensitive to molecular dynamics and interactions, flimscopy allows the molecular information to be visualized in single cells. In flimscopy measurements, several (nanosecond) time-resolved fluorescence images of a sample are obtained at various delay times after pulsed laser excitation of the microscope's entire field of view. Lifetimes are calculated pixel-by-pixel from these time-resolved images, and the spatial variations of the lifetimes are then displayed in a pseudocolor format (flimscopy image). The total data acquisition time needed to obtain a flimscopy image with the diffraction-limited spatial resolution (approximately 250 nm) is decreased to just approximately 30 s for approximately 300 fluorescent molecules/micron2. This was achieved by developing a high-frequency (400 kHz) nanosecond-gating (9 ns full width at half height)-signal accumulation system. This technique allows the extent of resonance energy transfer to be visualized in single living cells, and is free from the errors due to variations in path length, light scattering, and the number of fluorophores that necessitate complex corrections in steady-state microfluorometry and fluorescence ratio imaging microscopy. Flimscopy was applied here to observe the extent of fusion of individual endosomes in single cells. Results revealed the occurrence of extensive fusion between primary endocytic vesicles and/or sorting endosomes, thereby raising the possibility that the biogenesis of sorting endosomes involves multiple fusions of primary endocytic vesicles.