Automated live cell imaging systems reveal dynamic cell behavior

Automated time‐lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time‐dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time‐lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high‐throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time‐lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man‐hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

Practical guide of live imaging for developmental biologists

Time-lapse imaging of fluorescent proteins in living cells has become an indispensable tool in biological sciences. However, its application at the organismal level still faces a number of obstacles, such as large specimen sizes preventing illumination of internal tissues, high background fluorescence and uncontrollable movement of target tissues or embryos. Here we describe our solutions for these issues to obtain 4-D fluorescent images from living Drosophila embryos using confocal microscopes. A computational procedure that detects and corrects the shift of moving objects to virtually stabilize them in time-lapse movies (iSEMS) is presented. We discuss the importance of postimaging treatment of raw image stacks for the discovery of novel phenotypes that have previously escaped attention from the analyses of fixed specimens.     

Thermo-responsive non-woven scaffolds for "smart" 3D cell culture

The thermo‐responsive polymer poly(N‐isopropylacrylamide) has received widespread attention for its in vitro application in the non‐invasive, non‐destructive release of adherent cells on two dimensional surfaces. In this study, 3D non‐woven scaffolds fabricated from poly(propylene) (PP), poly(ethylene terephthalate) (PET), and nylon that had been grafted with PNIPAAm were tested for their ability to support the proliferation and subsequent thermal release of HC04 and HepG2 hepatocytes. Hepatocyte viability and proliferation were estimated using the Alamar Blue assay and Hoechst 33258 total DNA quantification. The assays revealed that the pure and grafted non‐woven scaffolds maintained the hepatocytes within the matrix and promoted 3D proliferation comparable to that of the commercially available Algimatrix? alginate scaffold. Albumin production and selected cytochrome P450 genes expression was found to be superior in cells growing on pure and grafted non‐woven PP scaffolds as compared to cells grown as a 2D monolayer. Two scaffolds, namely, PP‐g‐PNIPAAm‐A and PP‐g‐PNIPAAm‐B were identified as having far superior thermal release capabilities; releasing the majority of the cells from the matrices within 2 h. This is the first report for the development of 3D non‐woven, thermo‐responsive scaffolds able to release cells from the matrix without the use of any enzymatic assistance or scaffold degradation. Biotechnol. Bioeng. 2012; 109:2147–2158. © 2012 Wiley Periodicals, Inc.     

Trypanosoma cruzi: single cell live imaging inside infected tissues

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single‐cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed‐CL or GFP‐G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.     

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py₃-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py₃-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py₃-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py₃-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py₃-FITC solution. Therefore, Py₃-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.     

Genetic reporter for live tracing fluid flow forces during cell fate segregation in mouse blastocyst development

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应用活体骨髓瘤细胞制备单克隆抗体

【目的】用活体骨髓瘤细胞SP2/0做融合提高融合率制备单克隆抗体,并与常规方法比较效果。【方法】将SP2/0细胞打到8周龄的SPF级BALB/c小鼠皮下,待实体瘤生长到直径达2～3cm时无菌解剖取实体瘤,分离出骨髓瘤细胞进行融合。同时用培养基培养SP2/0细胞进行融合做比较,分两组进行。比较两种方法的融合率以及两种方法制备出来的单克隆抗体的相对亲和力。【结果】做了6次融合,实体瘤融合组融合率为70.4％,常规法融合组44.6％,两种方法制备单抗的相对亲和力均达到1:100000以上。【结论】利用活体实体瘤细胞进行融合能明显提高细胞融合率。     

CRISPR/dCas9系统在活细胞成像领域的研究进展

研究不同基因、染色体以及基因与染色体之间的时空关系在遗传学、发育生物学和生物医学等领域具有重要意义。CRISPR/Cas9基因编辑技术具有优异的靶向性,已经成为应用最广泛的基因编辑工具。近年来,研究人员基于Cas9的核酸酶失活突变体dCas9发展了一系列先进的活细胞成像技术,为染色质、基因组特定位点的高分辨成像提供了快速、方便的研究工具。文中从细胞递送方式、荧光信号优化以及正交多色成像3个方面对CRISPR/dCas9系统在活细胞成像中的研究进展进行了综述,并对该领域的发展趋势进行了展望。     

Analyzing cell fate control by cytokines through continuous single cell biochemistry

Cytokines are important regulators of cell fates with high clinical and commercial relevance. However, despite decades of intense academic and industrial research, it proved surprisingly difficult to describe the biological functions of cytokines in a precise and comprehensive manner. The exact analysis of cytokine biology is complicated by the fact that individual cytokines control many different cell fates and activate a multitude of intracellular signaling pathways. Moreover, although activating different molecular programs, different cytokines can be redundant in their biological effects. In addition, cytokines with different biological effects can activate overlapping signaling pathways. This prospect article will outline the necessity of continuous single cell biochemistry to unravel the biological functions of molecular cytokine signaling. It focuses on potentials and limitations of recent technical developments in fluorescent time‐lapse imaging and single cell tracking allowing constant long‐term observation of molecules and behavior of single cells. J. Cell. Biochem. 108: 343–352, 2009. © 2009 Wiley‐Liss, Inc.     

A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07‐07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07‐07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07‐07059 only marginally inhibits mitochondrial respiration and function. V07‐07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07‐07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)‐diacetate N‐succinimidyl ester, V07‐07059 proved superior regarding toxicity and photostability.

     

Dynamic behavior of clathrin in Arabidopsis thaliana unveiled by live imaging

Clathrin-coated vesicles (CCV) are necessary for selective transport events, including receptor-mediated endocytosis on the plasma membrane and cargo molecule sorting in the trans-Golgi network (TGN). Components involved in CCV formation include clathrin heavy and light chains and several adaptor proteins that are conserved among plants. Clathrin-dependent endocytosis has been shown to play an integral part in plant endocytosis. However, little information is known about clathrin dynamics in living plant cells. In this study, we have visualized clathrin in Arabidopsis thaliana by tagging clathrin light chain with green fluorescent protein (CLC-GFP). Quantitative evaluations of colocalization demonstrate that the majority of CLC-GFP is localized to the TGN, and a minor population is associated with multivesicular endosomes and the Golgi trans-cisternae. Live imaging further demonstrated the presence of highly dynamic clathrin-positive tubules and vesicles, which appeared to mediate interactions between the TGNs. CLC-GFP is also targeted to cell plates and the plasma membrane. Although CLC-GFP colocalizes with a dynamin isoform at the plasma membrane, these proteins exhibit distinct distributions at newly forming cell plates. This finding indicates independent functions of CLC (clathrin light chains) and dynamin during the formation of cell plates. We have also found that brefeldin A and wortmannin treatment causes distinctly different alterations in the dynamics and distribution of clathrin-coated domains at the plasma membrane. This could account for the different effects of these drugs on plant endocytosis.     

ELF exposure system for live cell imaging

A programmable system has been developed for the study of both transient and persistent effects of extremely low frequency (ELF) magnetic field exposure of cell cultures. This high‐precision exposure system enables experimental blinding and fully characterized exposure while simultaneously allowing live cell imaging. It is based on a live imaging cell around which two asymmetrical coils are wound in good thermal contact to a temperature‐controlled water jacket, and is mounted on a microscope stage insert. The applied B‐field uniformity of the active volume is better than 1.2% with an overall exposure uncertainty of less than 4.3% with very low transient field levels. The computer‐controlled apparatus allows signal waveforms that are sinusoidal or composed of several harmonics, blind protocols, and monitoring of exposure and environmental conditions. B‐fields up to 4 mT root mean square amplitude are possible with minimal temperature variation and no recognizable temperature differences between exposure and sham states. Sources of artifacts have been identified and quantified. There are no visible vibrations observable even at the highest magnifications and exposure levels. Bioelectromagnetics 34:231–239, 2013. © 2012 Wiley Periodicals, Inc.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.     

Spectral imaging and its applications in live cell microscopy

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.     

A poly(dimethylsiloxane)-based device enabling time-lapse imaging with high spatial resolution

We have developed a regulator-free device that enables long-term incubation of mammalian cells for epi-fluorescence imaging, based on a concept that the size of sample to be gassed and heated is reduced to observation scale. A poly(dimethylsiloxane) block stamped on a coverslip works as a long-lasting supplier of CO₂-rich gas to adjust bicarbonate-containing medium in a tiny chamber at physiological pH, and an oil-immersion objective warms cells across the coverslip. A time-lapse imaging experiment using HeLa cells stably expressing fluorescent cell-cycle indicators showed that the cells in the chamber proliferated with normal cell-cycle period over 2 days.     

Characterization of the KG1a cell line for use in a cell migration based screening assay

High-throughput screening has become a popular method used to identify new “leads” for potentially therapeutic compounds. Further screening of these lead compounds is typically done with secondary assays which may utilize living, functioning cells as screening tools. A problem (or benefit) with these cell-based assays is that living cells are very sensitive to their environment. We have been interested in the process of stem cell migration and how it relates to the cellular therapy of bone marrow transplantation. In this study we describe a secondary, cell-based assay for screening the effects of variousin-vitro conditions on Immature Hematopoietic Cell (IHC) migration. Our results have revealed many subtle factors, such as the cell's adhesive characteristics, or the effect of a culture's growth phase, that need to be accounted for in a screening protocol. Finally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly suggest that KG1a cells secrete a chemokinetic factor during the exponential growth phase of a culture.     

Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains

We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel‐wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)