Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.     

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.     

Reversibility of coated vesicle dissociation

The dissociation of the coated vesicles to clathrin and uncoated vesicles and their reassociation have been studied under various conditions. The extent of reassociation is pH dependent and increases slightly with increasing concentrations of the components. Unlike the self-association of clathrin which is strongly salt dependent, the reassociation of clathrin and uncoated vesicles is practically independent of salt concentration. The coated vesicle gradually loses its coat with increasing pH, and the dissociation process is not an all or none reaction. Ca2+ inhibits dissociation of the coated vesicles and enhances the reassociation of clathrin and uncoated vesicles. Our results show that, although many conditions result in reassociation of protein and lipid vesicle, few conditions result in vesicles of both the same size and composition as native coated vesicles.     

Molecular live cell bioimaging in stem cell research

Functional heterogeneity within stem and progenitor cells has been shown to influence cell fate decisions. Similarly, intracellular signaling activated by external stimuli is highly heterogeneous and its spatiotemporal activity is linked to future cell behavior. To quantify these heterogeneous states and link them to future cell fates, it is important to observe cell populations continuously with single cell resolution. Live cell imaging in combination with fluorescent biosensors for signaling activity serves as a powerful tool to study cellular and molecular heterogeneity and the long-term biological effects of signaling. Here, we describe these methodologies, their advantages over classical approaches, and we illustrate how they could be applied to improve our understanding of the importance of heterogeneous cellular and molecular responses to external signaling cues.     

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     

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.     

Self-adhesive microculture system for extended live cell imaging

Abstract

Gas permeable and biocompatible soft polymers are convenient for biological applications. Using the soft polymer poly(dimethylsiloxane) (PDMS), we established a straightforward technique for in-house production of self-adhesive and optical grade microculture devices. A gas permeable PDMS layer effectively protects against medium evaporation, changes in osmolarity, contamination and drug diffusion. These chip-based devices can be used effectively for long term mammalian cell culture and support a range of bioassays used in pharmacological profiling of anti-cancer drugs. Results obtained on a panel of hematopoietic and solid tumor cell lines during screening of investigative anti-cancer agents corresponded well to those obtained in a conventional cell culture on polystyrene plates. The cumulative correlation analysis of multiple cell lines and anti-cancer drugs showed no adverse effects on cell viability or cell growth retardation during microscale static cell culture. PDMS devices also can be custom modified for many bio-analytical purposes and are interfaced easily with both inverted and upright cell imaging platforms. Moreover, PDMS microculture devices are suitable for extended real time cell imaging. Data from the multicolor, real time analysis of apoptosis on human breast cancer MCF-7 cells provided further evidence that elimination of redundant centrifugation/washing achieved during microscale real time analysis facilitates preservation of fragile apoptotic cells and provides dynamic cellular information at high resolution. Because only small reaction volumes are required, such devices offer reduced use of consumables as well as simplified manipulations during all stages of live cell imaging.     

Optical imaging fiber-based live bacterial cell array biosensor

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.     

Nanogel-quantum dot hybrid nanoparticles for live cell imaging

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.     

Dye selection for live cell imaging of intact siRNA

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.     

Functional reassembly of the coated vesicle proton pump

We have shown previously that treatment of the coated vesicle proton-translocating adenosine triphosphatase (H(+)-ATPase) with chaotropic agents results in the release of a set of peripheral polypeptides which includes the 73-, 58-, 40-, 34-, and 33-kDa subunits (Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) J. Biol. Chem. 265, 967-973), with a coordinate loss of H(+)-ATPase activity. In the present paper we report the functional reassembly of the coated vesicle proton pump following dissociation of the peripheral subunits. Reassembly was demonstrated by restoration of ATP-driven proton transport using both native membranes and reconstituted vesicles and by Western blot analysis using a monoclonal antibody specific for the 73-kDa subunit. Reassembly occurs by attachment of a peripheral subcomplex containing the 73-, 58-, 34-, and 33-kDa subunits together with the 40-kDa polypeptide. The reassembled H(+)-ATPase, like the native proton pump, is inhibited by N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and N,N'-dicyclohexylcarbodiimide. Reassociation shows a biphasic time dependence, with restoration of 50-60% of the starting proton transport activity in the 1st h followed by recovery of a further 20-30% of the activity after 24 h. Reassembly also shows a marked dependence on protein concentration but, unlike solubilization of the intact H(+)-ATPase complex, does not require the presence of glycerol. Despite the ability of nucleotides to promote dissociation of the peripheral complex by chaotropic agents, reassociation is not blocked by the presence of 1 mM ATP. These results thus provide the first evidence for functional reassembly of a vacuolar H(+)-ATPase complex and should be useful in further analysis of the role of individual subunits in the assembly and activity of these ATP-driven proton pumps.     

Immunofluorescent localization of 100K coated vesicle proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.     

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.     

Three-dimensional visualization of coated vesicle formation in fibroblasts

Fibroblasts apparently ingest low density lipoproteins (LDL) by a selective mechanism of receptor-mediated endocytosis involving the formation of coated vesicles from the plasma membrane. However, it is not known exactly how coated vesicles collect LDL receptors and pinch off from the plasma membrane. In this report, the quick-freeze, deep- etch, rotary-replication method has been applied to fibroblasts; it displays with unusual clarity the coats that appear under the plasma membrane at the start of receptor-mediated endocytosis. These coats appear to be polygonal networks of 7-nm strands or struts arranged into 30-nm polygons, most of which are hexagons but some of which are 5- and 7-sided rings. The proportion of pentagons in each network increases as the coated area of the plasma membrane puckers up from its planar configuration (where the network is mostly hexagons) to its most sharply curved condition as a pinched-off coated vesicle. Coats around the smallest vesicles (which are icosahedrons of hexagons and pentagons) appear only slightly different from "empty coats" purified from homogenized brain, which are less symmetrical baskets containing more pentagons than hexagons. A search for structural intermediates in this coat transformation allows a test of T. Kanaseki and K. Kadota's (1969. J. Cell Biol. 42:202--220.) original idea that an internal rearrangement in this basketwork from hexagons to pentagons could "power" coated vesicle formation. The most noteworthy variations in the typical hexagonal honeycomb are focal juxtapositions of 5- and 7-sided polygons at points of partial contraction and curvature in the basketwork. These appear to precede complete contraction into individual pentagons completely surrounded by hexagons, which is the pattern that characterizes the final spherical baskets around coated vesicles.     

Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.     

Genetically encoded fluorescent indicators for live cell pH imaging

Background

Intracellular pH underlies most cellular processes. There is emerging evidence of a pH-signaling role in plant cells and microorganisms. Dysregulation of pH is associated with human diseases, such as cancer and Alzheimer's disease.