Bioluminescence microscopy using a short focal‐length imaging lens

Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low‐light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo‐toxicity associated with fluorescence imaging and permits the long‐term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.     

Quantitative and qualitative methods using fluorescence microscopy for the study of modified low density lipoproteins uptake

Atherosclerosis and heart disease are the main cause of death in United States. The development of atherosclerosis includes lipid deposition and foam cell formation in the artery wall. Scavenger Receptors A‐I and II (SRA‐I/II) have an important role of in foam cell formation and atherogenesis. Most of the SRA‐I/II studies had been performed using Iodine‐125‐radiolabeled modified low‐density lipoprotein. This report attempts to validate the use of fluorescence microscopy techniques as an alternative to obtain qualitative and quantitative information of the uptake of fluorescence‐labeled acetylated low‐density lipoprotein (AcLDL) in adherent CHO cells expressing SRA‐I/II. After verifying the protein expression of SRA‐I and II, uptake was quantified using a Laser Scan Cytometer, and images of cells containing fluorescent AcLDL were obtained. A significant increase in fluorescence was found in the cells transfected with SRA‐I/II vs. those with empty vector. When SRA‐I/II competitive ligands were used, the uptake of AcLDL was significantly decreased. In conclusion, the use of fluorescence microscopy techniques in obtaining qualitative and quantitative information of the uptake of fluorescence‐labeled AcLDL by adherent cells, such as CHO cells, is an alternative to the traditional use of radiolabeled iodine. SCANNING 31: 167–173, 2009. © 2009 Wiley Periodicals, Inc.     

Real‐time monitoring of the penetration of amphiphilic acrylate copolymer in leather using a fluorescent copolymer as tracer

A fluorescent tracer, poly (acrylic‐co‐stearyl acrylate‐co?3‐acryloyl fluorescein) [poly (AA‐co‐SA‐co‐Ac‐Flu)], used for real‐time monitoring the penetration of amphiphilic acrylate copolymer, poly (acrylic‐co‐stearyl acrylate) [poly (AA‐co‐SA)], in leather was synthesized by radical polymerization of acrylic, stearyl acrylate and fluorescent monomer, 3‐acryloyl fluorescein (Ac‐Flu). The structure, molecular weight, introduced fluorescent group content and fluorescent characteristics of the fluorescent tracer and target copolymer, amphiphilic acrylate copolymer, were also characterized. The results show that the tracer presents the similar structural characteristics to the target and enough fluorescence intensity with 1.68 wt % of the fluorescent monomer introduced amount. The vertical section of the leather treated with the target copolymer mixing with 7% of the tracer exhibits evident fluorescence, and the change of fluorescence intensity along with the vertical section with treating time increasing can reflect the penetration depth of the target copolymer. The introduction of the fluorescent group in polymer structure through copolymerization with a limited amount of fluorescent monomer, Ac‐Flu, is an effective way to make a tracer to monitor the penetration of the target in leather, which provides a new thought for the penetration research of syntans such as vinyl copolymer materials in leather manufacture. Microsc. Res. Tech. 78:1146–1153, 2015. © 2015 Wiley Periodicals, Inc.     

Rapid single‐molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface‐based single‐molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single‐molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single‐molecule imaging in off‐the‐shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin‐coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody–sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single‐molecule imaging assays without time‐consuming coating procedures. Microsc. Res. Tech. 78:309–316, 2015. © 2015 Wiley Periodicals, Inc.     

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF‐1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.     

Signal analysis of total internal reflection fluorescent speckle microscopy (TIR-FSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells

Fluorescent speckle microscopy (FSM) uses low levels of fluorescent proteins to create fluorescent speckles on cytoskeletal polymers in high‐resolution fluorescence images of living cells. The dynamics of speckles over time encode subunit turnover and motion of the cytoskeletal polymers. We sought to improve on current FSM technology by first expanding it to study the dynamics of a non‐polymeric macromolecular assembly, using focal adhesions as a test case, and second, to exploit for FSM the high contrast afforded by total internal reflection fluorescence microscopy (TIR‐FM). Here, we first demonstrate that low levels of expression of a green fluorescent protein (GFP) conjugate of the focal adhesion protein, vinculin, results in clusters of fluorescent vinculin speckles on the ventral cell surface, which by immunofluorescence labelling of total vinculin correspond to sparse labelling of dense focal adhesion structures. This demonstrates that the FSM principle can be applied to study focal adhesions. We then use both GFP‐vinculin expression and microinjected fluorescently labelled purified actin to compare quantitatively the speckle signal in FSM images of focal adhesions and the actin cytoskeleton in living cells by TIR‐FM and wide‐field epifluorescence microscopy. We use quantitative FSM image analysis software to define two new parameters for analysing FSM signal features that we can extract automatically: speckle modulation and speckle detectability. Our analysis shows that TIR‐FSM affords major improvements in these parameters compared with wide‐field epifluorescence FSM. Finally, we find that use of a crippled eukaryotic expression promoter for driving low‐level GFP‐fusion protein expression is a useful tool for FSM imaging. When used in time‐lapse mode, TIR‐FSM of actin and GFP‐conjugated focal adhesion proteins will allow quantification of molecular dynamics within interesting macromolecular assemblies at the ventral surface of living cells.     

Laser‐induced shockwave paired with FRET: A method to study cell signaling

Cells within the body are subject to various forces; however, the details concerning the way in which cells respond to mechanical stimuli are not well understood. We demonstrate that laser‐induced shockwaves (LIS) combined with biosensors based on fluorescence resonance energy transfer (FRET) is a promising new approach to study biological processes in single live cells. As “proof‐of‐concept,” using a FRET biosensor, we show that in response to LIS, cells release intracellular calcium. With the parameters used, cells retain their morphology and remain viable. LIS combined with FRET permits observation of the cells immediate response to a sudden shear force. Microsc. Res. Tech. 78:195–199, 2015. © 2015 Wiley Periodicals, Inc.     

三维培养结肠癌细胞的微流控芯片荧光成像研究

改良了一种微流控芯片,可用于对结肠癌细胞进行三维培养并实现实时荧光成像。在结肠癌细胞内植入内源性的红色荧光蛋白,使用激光共聚焦显微镜对芯片中三维培养的细胞进行成像。通过细胞内部红色荧光蛋白的表达,可以观测到细胞的生长状态,实现对细胞的实时监测和高分辨率荧光成像。同时,通过免疫荧光染色来表征反映细胞活性的特征蛋白,其荧光强度和蛋白表达呈正相关。研究结果提示,细胞活性相关蛋白的表达受到微环境的影响,其在芯片三维培养中的活性强于二维培养,表明芯片内环境更加接近真实的人体微环境。该方法为进一步探究肿瘤细胞转移机制及相关药物的筛选研究提供了一种新的技术手段及实验平台。     

MRT letter: Nanoscopy of protein colocalization in living cells by STED and GSDIM

We report the use of superresolution fluorescence microscopy for studying the nanoscale distribution of protein colocalization in living mammalian cells. Nanoscale imaging is attained both by a targeted and a stochastic fluorescence on-off switching superresolution method, namely by stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecular return (GSDIM), respectively. Analysis of protein colocalization is performed by bimolecular fluorescence complementation (BiFC). Specifically, a nonfluorescent fragment of the yellow fluorescent protein Citrine is fused to tubulin while a counterpart nonfluorescent fragment is fused to the microtubulin-associated protein MAP2 such that fluorescence is reconstituted on contact of the fragment-carrying proteins. Images with resolution down to 65 nm prove a powerful new way for studying protein colocalization in living cells at the nanoscale.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.     

Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass‐based surfaces

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass‐based materials are due to altered mRNA and protein levels. Primary rat‐derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real‐time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals. Microsc. Res. Tech. 78:1046–1053, 2015. © 2015 Wiley Periodicals, Inc.     

The role of the gastrointestinal epithelium as a possible pathway for the transfer of nutrients to the embryo's circulation

The endodermal cells of the human yolk sac (YS) produce non‐nucleated erythrocytes (NNEs) and numerous serum proteins that are transiently storage within the YS cavity. After their transfer via the vitelline duct to the embryo gastrointestinal lumen, the nutrients’ final fate is unknown. With the aim of investigate how erythroid cells and nutrients are conveyed to embryo circulation, we studied, using a morphological and immunohistochemical approach, the embryo anatomy and the serum protein α‐fetoprotein (AFP) presence, in 15 human embryos and their YS, collected from tubal pregnancies from 4 to 8 wpf. We observed at 5 wpf, a strong AFP staining in the endodermal cells of the YS, thereafter AFP was only present in the YS cavity and the gastrointestinal lumen. During 7 wpf, AFP expression declined and disappeared, concomitant with YS regression. Between 5 and 7 wpf, NNEs were observed in the gastrointestinal cavity, where they accumulate in the stomach. Here, the cells were attached to the endodermal epithelial cells or were free in the lumen. By scanning electron microscopy, we identified signs of NNEs phagocytized by endodermal cells. Those NNEs free in the lumen, after hemolysis, were probably removed by endocytosis (cell debris). Taking all together, we postulate that after reaching the endodermal epithelial cells of the stomach, nutrients are transferred to the embryo by a phagocytic/endocytic mechanism that is operative until the end of 6 wpf. After absorption, NNEs are probably degraded within phagosomes, nutrients delivered to the cell cytoplasm and then transported towards the embryonic circulation. Microsc. Res. Tech. 78:500–507, 2015. © 2015 Wiley Periodicals, Inc.     

Early history,discovery, and expression of Aequorea green fluorescent protein,with a note on an unfinished experiment

The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (λ_max = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. Microsc. Res. Tech. 73:785–796, 2010. © 2010 Wiley‐Liss, Inc.     

Direct visualization of the dynamics of membrane-anchor proteins in living cells

Dynamic properties of proteins have crucial roles in understanding protein function and molecular mechanism within cells. In this paper, we combined total internal reflection fluorescence microscopy with oblique illumination fluorescence microscopy to observe directly the movement and localization of membrane‐anchored green fluorescence proteins in living cells. Total internal reflect illumination allowed the observation of proteins in the cell membrane of living cells since the penetrate depth could be adjusted to about 80 nm, and oblique illumination allowed the observation of proteins both in the cytoplasm and apical membrane, which made this combination a promising tool to investigate the dynamics of proteins through the whole cell. Not only individual protein molecule tracks have been analyzed quantitatively but also cumulative probability distribution function analysis of ensemble trajectories has been done to reveal the mobility of proteins. Finally, single particle tracking has acted as a compensation for single molecule tracking. All the results exhibited green fluorescence protein dynamics within cytoplasm, on the membrane and from cytoplasm to plasma membrane.     

Light and electron microscopy study of the spermatheca of Eupholidoptera chabrieri bimucronata (Ramme, 1927) and Uromenus brevicollis trinacriae La Greca 1964 (Orthoptera: Tettigoniidae)

A study by both optical and electron microscopy has been carried out on the spermatheca of Eupholidoptera chabrieri bimucronata and Uromenus brevicollis trinacriae (Orthoptera: Tettigoniidae). In both the examined species, the spermatheca consists of a sac/kidney‐shaped seminal receptacle and a more or less tortuous spermathecal duct that opens into the common oviduct. The wall of both the organs consists of a pseudostratified epithelium surmounted by a cuticular intima; the latter is made up of a thicker endocuticle and an epicuticle. The epithelium shows two different cell types, irregularly arranged and with well differentiated functions: cuticle‐forming and gland cells. In both the species, the cuticle‐forming cells perform other functions, in addition to producing the cuticular intima. The gland cells never come in contact with the cuticular intima, have inside the reservoir a secretion whose appearance can diversify also in contiguous zones of the seminal receptacle. Based on our findings in both the species, the functions of the seminal receptacle would differ from those of the spermathecal duct. In the latter, some areas of the wall of the connecting tract show an activity of lysis, by contiguous epithelial cells, that could play a role in control and selection of spermatozoa. As for the feather‐shaped spermatodesms, similar in both the species, freeze‐fracture observations have shown that the acrosome of each spermatozoon regularly covers three‐quarters of the extension of the acrosome of the following spermatozoon. Finally, the significance of our findings, compared with what is known in literature, is discussed. Microsc. Res. Tech. 78:577–586, 2015. © 2015 Wiley Periodicals, Inc.     

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH‐6 clone 5). We directly observed the cultured cells on a pressure‐resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co‐localization of CD133 with F‐actin. An antibody against CD133 decreased cell migration. Methyl‐β‐cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration. Microsc. Res. Tech. 76:844–852, 2013. © 2013 Wiley Periodicals, Inc.     

Mesoporous iron oxide nanoparticles prepared by polyacrylic acid etching and their application in gene delivery to mesenchymal stem cells

Novel monodisperse mesoporous iron oxide nanoparticles (m‐IONPs) were synthesized by a postsynthesis etching approach and characterized by electron microscopy. In this approach, solid iron oxide nanoparticles (s‐IONPs) were first prepared following a solvothermal method, and then etched anisotropically by polyacrylic acid to form the mesoporous nanostructures. MTT cytotoxicity assay demonstrated that the m‐IONPs have good biocompatibility with mesenchymal stem cells (MSCs). Owing to their mesoporous structure and good biocompatibility, these monodisperse m‐IONPs were used as a nonviral vector for the delivery of a gene of vascular endothelial growth factor (VEGF) tagged with a green fluorescence protein (GFP) into the hard‐to‐transfect stem cells. Successful gene delivery and transfection were verified by detecting the GFP fluorescence from MSCs using fluorescence microscopy. Our results illustrated that the m‐IONPs synthesized in this work can serve as a potential nonviral carrier in gene therapy where stem cells should be first transfected and then implanted into disease sites for disease treatment. Microsc. Res. Tech. 76:936–941, 2013. © 2013 Wiley Periodicals, Inc.     

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT₁) and Endothelin 1 type A (ET_A) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT₁ and ET_A receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT₁ receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ET_A receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.     

A simple,straightforward correlative live‐cell‐imaging‐structured‐illumination‐microscopy approach for studying organelle dynamics

Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two‐fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high‐speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high‐speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells. Microsc. Res. Tech. 78:777–783, 2015. © 2015 Wiley Periodicals, Inc.