Real-time Live Cell Imaging Research Articles

Abstract 3982: Super-resolution imaging of drug delivery to live cells using real-time structured illumination microscopy

Abstract Imaging the effects of drug delivery in live cells is most often performed by confocal microscopy, which has a resolution limited by the diffraction of light. Here, we report the development of a novel method for imaging drug delivery to live cells using real-time super-resolution microscopy in wide-field mode. Structured illumination microscopy (SIM) is one of several super-resolving microscopy techniques and is capable of imaging beyond the resolution of conventional microscopy to less than 100 nm. In SIM, each super-resolution (SR) image is computationally constructed from a dataset of numerous raw images. Due to the computational time required for SR image construction, datasets are usually post-processed to avoid slowdowns during the acquisition process. Although post-processing is convenient, this approach has limited use since SR images are not available during image acquisition and thus cannot be utilized for real-time evaluation of the sample. Utilizing SR images in real-time is particularly important when objects of interest are smaller than the resolution limit of conventional microscopy. Applications such as determining optimal focus and finding desirable regions of interest are depending on real-time feedback to user. When performing real-time SIM imaging live cells, image acquisition, processing, and the display of SR images must occur in rapid succession thereby reducing the effects of molecular movement. To achieve the data processing speed necessary for real-time imaging of live cells, we utilized a graphics processing unit (GPU) to perform high-speed SR image construction. Each function for processing SIM data was optimized for parallel operation using GPU kernels. Excluding data transfer overhead, the actual GPU processing time for synthesizing the SR image was 45 milliseconds. Compared with parallel processing on a multi-core computer (CPU), using the GPU achieved a 45-fold increase in processing speed. Overall, data throughput using the GPU attained an 8-fold speed increase over CPU processing, thus allowing real-time high resolution imaging. We applied this SIM platform to record the action of nocodazole on microtubule depolymerization in HeLa cells. We are currently developing this real-time imaging method to investigate the delivery and release mechanism of therapeutic cargoes contained within nanoparticles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3982.

Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis

BackgroundMacrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively.ResultsWe show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest.ConclusionsThe computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without compromise in image quality or information loss in associated spectra. These results motivate further use of label free microscopy techniques in real-time imaging of live immune cells.

Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 microg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca(2+)](i)) and histamine with fluorescent methods.

A Heteroditopic Fluoroionophoric Platform for Constructing Fluorescent Probes with Large Dynamic Ranges for Zinc Ions

A heteroditopic fluoroionophoric platform has been designed for constructing fluorescent probes for zinc ions over large concentration ranges. The responses of the prototype probes 3a and 3b to zinc ions were shown to be consistent with our hypothesis, according to which the modulation of photoinduced electron transfer followed by conformation rigidification or enhanced internal charge transfer of a ditopic ligand upon successive zinc coordination affords a sensitive fluorescence enhancement in one wavelength channel followed by an emission band shift to another wavelength channel. The heteroditopic arylvinyl-bipy platform established in this study provides a lead structure for constructing fluorescent probes for real-time live cell imaging of zinc ions over broad dynamic ranges.

Lights, Camera, Action

![Figure][1] CREDIT: GERALD DONNERT, LARS MEYER, STEFAN W. HELL At the end of a long day in the lab, what we'd all like is an award-winning movie. What makes for a good movie? An engrossing story, deft cinematography, and an extraordinary ensemble of actors. The goal of single-molecule research is to produce a movie of the cell. Biochemistry and biophysics done in the test tube already provide an understanding of the dynamic behavior of molecules; from these studies, what goes on in cells, minute by minute or even second by second, can be inferred. Ultimately, however, the goal is to film single molecules in single cells, focusing in closely enough not only to observe spatial and temporal characteristics but also to decipher molecular mechanisms. We're not there yet, but recent advances in single-molecule techniques bring us tantalizingly close to a molecule-scale movie of cellular life. Because cells are optically transparent, light microscopy is ideal for noninvasive imaging of cells in three dimensions. However, until recently, the resolution of lens-based optical microscopes was constrained by the diffraction barrier, which gave a resolution cutoff at half the wavelength of light. In his Review, Hell (p. [1153][2]) discusses concepts that show how the diffraction barrier can be broken in fluorescence spectroscopy and how these techniques have been applied to achieve nanoscale resolution. His Review gives hope that real-time three-dimensional imaging of live cells with electron-microscopy resolution may not be too far away. Many single-molecule techniques remain in vitro, but take on the challenge of transferring the results into the realm of cellular systems. In their Review, Evans and Calderwood (p. 1148) describe how combining molecular cell biology with single-force spectroscopy provides a tool to explore eukaryotic cell adhesion, revealing how forces applied to cell-surface bonds affect intracellular interactions or chemical reactions. Although these techniques are a powerful probe of mechanical function, understanding molecular mechanisms generally requires molecular dynamics simulations of atomic structural molecules. Sotomayor and Schulten (p. [1144][3]) describe in silico single-molecule experiments that use steered molecular dynamics simulations to explore how macromolecules respond to external forces at an atomic level. Two Reports in this issue also highlight single-molecule techniques. In in vitro experiments, Shiroguchi and Kinosita (p. 1208) provide a clear view of myosin V motion, showing that it walks using a combination of lever action and Brownian motion. Elf et al . (p. 1191) use single-molecule imaging of the lac repressor to directly observe the function of this regulatory system in live Escherichia coli cells. In related online resources, Science 's Signal Transduction Knowledge Environment (STKE) focuses on how single-molecule analysis is increasing understanding of cellular signaling ( ). Chazin describes how x-ray crystallography and nuclear magnetic resonance studies have provided insights into the function of Ca2+-sensing proteins. Navratil et al . discuss the application of microfluidic technology to the counting of low-abundance proteins in single cells, and Ghosh and Wirth present an algorithm for analyzing the movement of receptors in the plasma membrane. We'll be watching with interest as single-molecule approaches move us toward a quantitative and mechanistic understanding of cellular processes. [1]: pending:yes [2]: /lookup/doi/10.1126/science.1137395 [3]: /lookup/doi/10.1126/science.1137591

High speed method for in situ multispectral image registration

High speed data registration is required for the study of fluorescence resonance energy transfer in real time as well as fast dynamic intra- and inter-cellular signaling events. Multispectral confocal spinning disk microscopy provides a high resolution method for performing such real time live cell imaging. However, optical distortions and the physical misalignments introduced by the use of multiple acquisition cameras can obscure spatial information contained in the captured images. In this manuscript, we describe a multispectral method for real time image registration whereby the image from one camera is warped onto the image from a second camera via a polynomial correction. This method provides a real time pixel-for-pixel match between images obtained over physically distinct optical paths. Using an in situ calibration method, the polynomial is characterized by a set of coefficients, using a least squares solver. Error analysis demonstrates optimal performance results from the use of cubic polynomials. High-speed evaluation of the warp is then performed through forward differencing with fixed-point data types. Forward differencing is an iterative approach for evaluating polynomials on the condition that the function variable changes with constant steps. Image reconstruction errors are reduced through bilinear interpolation. The registration techniques described here allow for successful registration of multispectral images in real time (exceeding 15 frame/s) and have a broad applicability to imaging methods requiring pixel matching over multiple data channels.

Epicocconone, A New Cell-Permeable Long Stokes' Shift Fluorescent Stain for Live Cell Imaging and Multiplexing

Epicocconone is a heterocyclic natural product from the fungus Epicoccum nigrum that fluoresces weakly in the green (520 nm). However, cells exposed to epicocconone rapidly absorb the dye and become bright orange fluorescent because the natural product reacts reversibly with proteins. The orange fluorescence is enhanced in lipophilic environments, allowing the visualization of membranous organelles and lipid rafts but does not stain oligonucleotides. As the unconjugated dye has no orange fluorescence, there is no need to wash out the excess fluorophore. Epicocconone is a neutral, non-toxic, small molecule that appears to diffuse readily into live of fixed cells without the need for permeabilization. These features enable the real-time imaging of live cells and the study of organelle movements. Cells stained with epicocconone are excitable by common lasers (UV, 405, 488, and 532 nm) and its long Stokes' shift allows multiplexing applications with more common short Stokes' fluorophores using a single light source.

Direct observation of substrate induction of resistance mechanism in Pseudomonas aeruginosa using single live cell imaging

The resistance mechanism in three strains of Pseudomonas aeruginosa, ΔABM (devoid of MexAB-OprM), WT, and nalB-1 (overexpression of MexAB-OprM), was investigated using real-time single live cell imaging and fluorescence spectroscopy. Time courses of fluorescence intensity of these three strains in ethidium bromide (EtBr) showed that accumulation kinetics and extrusion machinery were highly dependent upon pump substrate (EtBr) concentration. At high substrate concentration (100 μM), the accumulation kinetic profiles in the cells at earlier incubation times were similar to those observed in low concentration. As EtBr accumulated in the cells reached a critical concentration, the fluorescence intensity of ΔABM decreased below the fluorescence intensity of EtBr in buffer solution. This result suggested an inductive mechanism in the development of substrate resistance in P. aeruginosa. Substrates appeared to trigger the degradation of EtBr in ΔABM. Unlike bulk measurements, single live cell imaging overcame the ensemble measurement of bulk analysis and showed that efflux machinery and resistance mechanism in individual cells were not synchronized.

Creating new fluorescent probes for cell biology.

Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno-electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230GRIP-transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230GRIP binds to a specific population of vesicles distinct from those labelled for beta-COP or gamma-adaptin. The GFP-p230GRIP fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulo-vesicular carriers.