Interference Microscopy in Cell Biophysics. 1. Principles and Methodological Aspects of Coherent Phase Microscopy

The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this article, we consider the applications of the CPM method to imaging different cells and energy-transducing intracellular organelles (mitochondria and chloroplasts). Experimental data presented below demonstrate that the optical path length difference of the object, which is the basic optical parameter measured by the CPM method, can serve as an indicator of metabolic states of different biological objects at cellular and subcellular levels of structural organization.     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image ('phase thicknesses' Deltah) and cell energization. It was demonstrated that the phase thickness Deltah decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Deltah and cell diameter d demonstrated that a decrease in the phase thickness Deltah could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Coherent phase microscopy in cell biology: visualization of metabolic states

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δh) and cell energization. It was demonstrated that the phase thickness Δh decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δh and cell diameter d demonstrated that a decrease in the phase thickness Δh could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.     

Intracellular Localized Surface Plasmonic Sensing for Subcellular Diagnosis

This paper proposes a method for diagnosing intracellular conditions and organelles of cells with localized surface plasmonic resonance (LSPR) by directly internalizing the gold nanoparticles (AuNPs) into the cells and measuring their plasmonic properties through hyperspectral imaging. This technique will be useful for direct diagnosis of cellular organelles, which have potential for cellular biology, proteomics, pharmaceuticals, drug discovery etc. Furthermore, localization and characterization of citrate-capped gold nanoparticles in HeLa cells were studied, by hyperspectral microscopy and other imaging techniques. Here, we present the method of internalizing the gold nanoparticles into the cells and subcellular organelles to facilitate subcellular plasmonic measurements. An advanced label-free visualization technique, namely hyperspectral microscopy providing images and spectral data simultaneously, was used to confirm the internalization of gold nanoparticles and to reveal their optical properties for possible intracellular plasmonic detection. Hyperspectral technology has proved to be effective in the analysis of the spectral profile of gold nanoparticles, internalized under different conditions. Using this relatively novel technique, it is possible to study the plasmonic properties of particles, localized in different parts of the cell. The position of the plasmon bands reflects the interactions of gold nanoparticles with different subcellular systems, including particle-nucleus interactions. Our results revealed the effect of the different intracellular interactions on the aggregation pattern of gold nanoparticles, inside the cells. This novel technique opens the door to intracellular plasmonics, an entirely new field, with important potential applications in life sciences. Similarly, the characterization of AuNP inside the cell was validated using traditional methods such as light microscopy and scanning electron microscopy. Under the conditions studied in this work, gold nanoparticles were found to be non-toxic to HeLa (cervical cancer) cells.     

Characterizing Organelles in Live Stem Cells Using Label-Free Optical Diffraction Tomography

Label-free optical diffraction tomography (ODT), an imaging technology that does not require fluorescent labeling or other pre-processing, can overcome the limitations of conventional cell imaging technologies, such as fluorescence and electron microscopy. In this study, we used ODT to characterize the cellular organelles of three different stem cells—namely, human liver derived stem cell, human umbilical cord matrix derived mesenchymal stem cell, and human induced pluripotent stem cell—based on their refractive index and volume of organelles. The physical property of each stem cell was compared with that of fibroblast. Based on our findings, the characteristic physical properties of specific stem cells can be quantitatively distinguished based on their refractive index and volume of cellular organelles. Altogether, the method employed herein could aid in the distinction of living stem cells from normal cells without the use of fluorescence or specific biomarkers.     

Electron microscopic studies of the endoplasmic reticulum in whole-mount cultured cells fixed with potassium permanganate.

A method for visualizing the endoplasmic reticulum and other membrane organelles in whole-mount cells with a standard, 60-kV transmission electron microscope has been developed. By use of a new formulation of potassium permanganate as a fixative, intracellular membranes were preserved and stained, while cytosolic proteins were digested, giving a pattern of membranous organelles against a clear background, suitable for transmission EM of whole-mount cells at 60 kV. Mitochondria, lysosomes, and ER were clearly visible in whole-mount cells fixed by this method. We have employed this technique to examine the organization of the ER in a variety of different cell lines. This method also allowed visualization of the three-dimensional organization, relationships, and fine structure of mitochondria. With prolonged permanganate fixation, mitochondrial cristae were clearly visible in whole-mount cells. This method was also useful for fixation and staining of thin sections, and allowed examination of thicker sections than previously possible, thus giving improved imaging of organelle relationships and fine structure. Using this method, we have examined the ER, mitochondria, and Golgi in thin section.     

基于受激发射损耗显微术的活细胞和活体超分辨成像

细胞作为生命体基本的结构和功能单元，在生物、医学等领域有着非常重要的研究意义。随着现代科学和技术的发展，科学家们借助电镜对细胞以及细胞器的空间结构已经有非常清晰的认识，但是对它们的功能以及细胞之间的相互作用却了解得非常少，而这恰恰又是疾病治疗和药物开发亟需了解的信息，因此对离体活细胞（简称活细胞）和活体生物组织细胞（简称活体细胞）中亚细胞器的研究变得非常重要。然而细胞中许多细胞器的结构在纳米量级，传统的光学成像技术由于受到光学衍射极限的限制是无法观察到纳米量级的生物结构，因此光学超分辨成像技术是目前研究亚细胞器结构和功能的有效工具。在所有光学超分辨显微技术中，受激发射损耗显微术（stimulated emission depletionmicroscopy,STED）由于具有实时成像、三维超分辨和断层成像的能力，非常适合用于纳米尺度的活细胞和活体细胞成像研究，而且STED超分辨成像技术经过近几十年的发展，已经广泛用于活细胞甚至活体小鼠细胞的超分辨动态观测。本文总结了近年来活细胞和活体小鼠神经元细胞等领域STED超分辨成像的研究进展，介绍了用于活细胞和活体细胞STED超分辨成像的荧光染料...     

Magnetic particle motions within living cells. Measurement of cytoplasmic viscosity and motile activity.

Submicrometer magnetic particles, ingested by cells and monitored via the magnetic fields they generate, provide an alternative to optical microscopy for probing movement and viscosity of living cytoplasm, and can be used for cells both in vitro and in vivo. We present methods for preparing lung macrophages tagged with magnetic particles for magnetometric study. Interpretation of the data involves fitting experimental remanent-field decay curves to nonlinear mechanistic models of intracellular particle motion. The model parameters are sensitive to mobility and apparent cytoplasmic viscosity experienced by particle-containing organelles. We present results of parameter estimation for intracellular particle behavior both within control cells and after (a) variable magnetization duration, (b) incubation with cytochalasin D, and (c) particle twisting by external fields. Magnetometric analysis showed cytoplasmic elasticity, dose-dependent motion inhibition by cytochalasin D, and a shear-thinning apparent viscosity.     

Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense

African trypanosomes multiply rapidly during the course of infection obtaining nutrients from the host blood and other body fluids. The organelles involved in endocytosis were revealed ultrastructurally using horseradish peroxidase (HRP) and colloidal gold coupled to bovine transferrin (Au-Tf) or bovine serum albumin (Au-BSA). At 0 degree C the markers bound to the cell surface and neither entered the flagellar pocket nor were internalized. Upon warming to 37 degrees C, the markers were found in the flagellar pocket and appeared to enter all the intracellular endocytic organelles within 5 min. Serial sectioning of resin-embedded cells was employed to obtain pseudo three-dimensional views of these organelles. The organelles involved were of three types: (1) small vesicles and cisternae (20-25 nm in diameter), (2) large tubular networks (200 nm diameter) similar to endosomes of mammalian cells, and (3) large lysosome-like vesicles. These organelles were located between the flagellar pocket and the nucleus and were also associated with one face of the Golgi apparatus. In pulse-chase experiments HRP was not detected in intracellular organelles after 410 min but Au-Tf was seen in residual bodies. No exocytosis of Au-Tf from the flagellar pocket was observed. The data suggests that the processes of endocytosis in these parasitic protozoa may be similar to the endocytic processes found in mammalian cells.     

Revealing bacterial cell biology using cryo-electron tomography

Visualizing macromolecules inside bacteria at a high spatial resolution has remained a challenge owing to their small size and limited resolution of optical microscopy techniques. Recent advances in cryo-electron tomography (cryo-ET) imaging methods have revealed the spatial and temporal assemblies of many macromolecules involved in different cellular processes in bacteria at a resolution of a few nanometers in their native milieu. Specifically, the application of cryo-focused ion beam (cryo-FIB) milling to thin bacterial specimens makes them amenable for high-resolution cryo-ET data collection. In this review, we highlight recent research in three emerging areas of bacterial cell biology that have benefited from the cryo-FIB-ET technology - cytoskeletal filament assembly, intracellular organelles, and multicellularity.     

Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.     

Versatile laser‐based cell manipulator

Here we describe a two‐photon microscope and laser ablation setup combined with optical tweezers. We tested the setup on the fission yeast Schizosaccharomyces pombe, a commonly used model organism. We show that long‐term imaging can be achieved without significant photo‐bleaching or damage of the sample. The setup can precisely ablate sub‐micrometer structures, such as microtubules and mitotic spindles, inside living cells, which remain viable after the manipulation. Longer exposure times lead to ablation, while shorter exposures lead to photo‐bleaching of the target structure. We used optical tweezers to trap intracellular particles and to displace the cell nucleus. Two‐photon fluorescence imaging of the manipulated cell can be performed simultaneously with trapping. The combination of techniques described here may help to solve a variety of problems in cell biology, such as positioning of organelles and the forces exerted by the cytoskeleton. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Accumulation of the amyloid-beta precursor protein in multivesicular body-like organelles.

It has been suggested that the successive proteolytic events leading to the production of the amyloid-beta protein from its precursor may take place at different intracellular locations. Using cultured human leptomeningeal smooth muscle cells and brain pericytes, we modulated the intracellular localization of the amyloid-beta precursor protein (APP) to study possible effects on its processing. By using immunofluorescence and immunoelectron microscopy we demonstrated that, under normal conditions, the APP is found in small intracellular vesicles, some of which were characterized as lysosomes. Both the cytokine interferon-gamma and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-alpha (TNF-alpha), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-beta precursor protein was slightly reduced by interferon-gamma or chloroquine. Double-labeling and tracer molecule uptake experiments showed that the multivesicular body-like organelles were part of the endocytic pathway. Our findings suggest that the multivesicular body-like organelles function as an intermediate organelle in the intracellular trafficking of the APP. Accumulation of the APP in this organelle is reflected by its reduced secretion from the cell.     

In situ imaging of agonist-sensitive calcium pools in AR4-2J pancreatoma cells. Evidence for an agonist- and inositol 1,4,5-trisphosphate-sensitive calcium pool in or closely associated with the nuclear envelope.

The activation of phospholipase C by hormones and neurotransmitters activates a complex combination of Ca2+ release and accumulation by intracellular organelles. Previously, we demonstrated that, in some cell types, the fluorescent Ca2+ indicator, fura-2, can be loaded into intracellular, agonist-sensitive Ca2+ pools (Glennon, M. C., Bird, G. St. J., Kwan, C.-Y., and Putney, J. W., Jr. (1992) J. Biol. Chem. 267, 8230-8233). In the current study, we have attempted to exploit this phenomenon by employing digital fluorescence imaging of compartmentalized fura-2 to investigate the localization and function of the major intracellular sites of Ca2+ regulation in AR4-2J pancreatoma cells. By judicious use of a surface receptor agonist together with the Ca(2+)-ATPase inhibitor, thapsigargin, cellular regions were identified whose behavior indicates that they contain the sites of agonist- and inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ release. These regions were located throughout the cell and may include the nuclear envelope. They were distinct in locus and behavior from two other regions, which counterstained with fluorescent markers for nuclei and mitochondria. Fura-2 in mitochondrial regions reported low resting levels of [Ca2+], and revealed that organelles in these regions accumulate and retain Ca2+ after agonist activation. These findings demonstrate that fluorescent Ca2+ indicators can be employed to directly monitor changes in [Ca2+] in the major Ca(2+)-regulating organelles, and provide the first in situ visualization and localization of the major sites of Ca2+ regulation in cells.     

Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy

Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.     

Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

Biological applications of spin pH probes.

The determination of pH is one of the most important problems in the biochemistry of living organisms, since many of the vital processes of cells and cellular organelles depend on the local pH value. Amongst currently used experimental approaches for the measurement of pH, the application of spin pH probes in combination with EPR spectroscopy is a comparatively new and rapidly developing field. In this article we describe the background, advantages and limitations of the method of spin pH probes, and discuss its recent applications. Availability of a wide variety of pH-sensitive nitroxides with different ranges of pH-sensitivity, labeling group and lipophilicity facilitates their application to a variety of biological systems from subcellular organelles to complex organisms. The recent progress in low-field EPR-based imaging and spectroscopy-based techniques allows spin pH probes to be used for non-invasive in vivo pH measurement and pH-sensitive imaging.     

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.     

Cell and nucleus refractive‐index mapping by interferometric phase microscopy and rapid confocal fluorescence microscopy

We present a multimodal technique for measuring the integral refractive index and the thickness of biological cells and their organelles by integrating interferometric phase microscopy (IPM) and rapid confocal fluorescence microscopy. First, the actual thickness maps of the cellular compartments are reconstructed using the confocal fluorescent sections, and then the optical path difference (OPD) map of the same cell is reconstructed using IPM. Based on the co‐registered data, the integral refractive index maps of the cell and its organelles are calculated. This technique enables rapidly measuring refractive index of live, dynamic cells, where IPM provides quantitative imaging capabilities and confocal fluorescence microscopy provides molecular specificity of the cell organelles. We acquire human colorectal adenocarcinoma cells and show that the integral refractive index values are similar for the whole cell, the cytoplasm and the nucleus on the population level, but significantly different on the single cell level.     

Rapid assessment of drug resistance of cancer cells to gefitinib and carboplatin using optical imaging

The overall goal of this study was to evaluate optical molecular imaging approaches to determine the drug response of chemotherapy and molecular targeted agents in drug sensitive and drug resistant cell lines. The optical molecular imaging approaches selected in this study were based on changes in intracellular uptake and retention of choline and glucose molecules. The breast cancer cell lines were treated with a molecular targeted anti-EGFR therapy. The bladder cancer cell lines were treated with a conventional chemotherapy approach. Sensitivity of optical molecular imaging approach was also compared with conventional cell viability and cell growth inhibition assays. Results demonstrate that optical molecular imaging of changes in intracellular uptake of metabolites was effective in detecting drug susceptibility for both molecular targeted therapy in breast cancer cells and chemotherapy in bladder cancer cells. Between the selected metabolites for optical molecular imaging, changes in glucose metabolic activity showed higher sensitivity in discrimination between the drug sensitive and drug resistant cell lines. The results demonstrated that optical molecular imaging approaches more significantly sensitive as compared to the conventional cell viability and growth assays. Overall, the results demonstrate potential of optical molecular imaging of metabolic activity to improve sensitivity of in-vitro drug response assays.