A simple and stable autofocusing protocol for long multidimensional live cell microscopy

Focus maintenance is a challenging problem in multidimensional wide‐field microscopy. Most automated microscopes use software algorithms, which are applied to z‐sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, autofocus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine autofocus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic‐based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.     

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching‐Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step‐by‐step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2‐fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins.     

The Large-Scale Digital Cell Analysis System: an open system for nonperturbing live cell imaging

The Large-Scale Digital Cell Analysis System (LSDCAS) was designed to provide a highly extensible open source live cell imaging system. Analysis of cell growth data has demonstrated a lack of perturbation in cells imaged using LSDCAS, through reference to cell growth data from cells growing in CO₂ incubators. LSDCAS consists of data acquisition, data management and data analysis software, and is currently a Core research facility at the Holden Comprehensive Cancer Center at the University of Iowa. Using LSDCAS analysis software, this report and others show that although phase-contrast imaging has no apparent effect on cell growth kinetics and viability, fluorescent image acquisition in the cell lines tested caused a measurable level of growth perturbation using LSDCAS. This report describes the current design of the system, reasons for the implemented design, and details its basic functionality. The LSDCAS software runs on the GNU/Linux operating system, and provides easy to use, graphical programs for data acquisition and quantitative analysis of cells imaged with phase-contrast or fluorescence microscopy (alone or in combination), and complete source code is freely available under the terms of the GNU Public Software License at the project website ( http://lsdcas.engineering.uiowa.edu ).     

Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging

Precise measurement of rapid volume changes of substrate‐adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate‐adherent cells based on phase‐contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom‐designed, side‐viewing chamber and subjected to hypotonic swelling. Side‐view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off‐line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (ΔVolume ≤ 14%), but our method has superior temporal resolution limited only by the time of single‐image acquisition, typically ～100 ms. The advantages of using standard phase‐contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.     

Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae

Live cell imaging of protein distributions is an essential tool in modern cell biology. It relies on the functional labelling of a host protein with a fluorophore, which may either be a genetically fused fluorescent protein or an organic dye binding to the host protein. The biarsenical-tetracysteine system or 'FlAsH-labelling', is based on the high affinity interaction between a biarsenical probe and a small protein tag. This approach has been successfully used for live cell imaging in the budding yeast Saccharomyces cerevisiae. However, the established labelling protocols require a lengthy overnight incubation of the cells with the dye under tightly controlled growth conditions, which severely limits the use of this approach. In this study, we characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells using standard electroporation. The labelling time is reduced from more than 12 h to less than 1 h without compromising the labelling efficiency or cell viability. This approach may be used for cells in different growth phases or grown under different conditions. It may be further extended to other small high affinity probes, thus opening up new possibilities for labelling in budding yeast.     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope

Atomic force microscopy has been used to visualize nano‐scale structures of various cellular components and to characterize mechanical properties of biomolecules. In spite of its ability to measure non‐fixed samples in liquid, the application of AFM for living cell manipulation has been hampered by the lack of knowledge of the mechanical properties of living cells. In this study, we successfully combine AFM imaging and force measurement to characterize the mechanical properties of the plasma membrane and the nuclear envelope of living HeLa cells in a culture medium. We examine cantilevers with different physical properties (spring constant, tip angle and length) to find out the one suitable for living cell imaging and manipulation. Our results of elasticity measurement revealed that both the plasma membrane and the nuclear envelope are soft enough to absorb a large deformation by the AFM probe. The penetrations of the plasma membrane and the nuclear envelope were possible when the probe indents the cell membranes far down close to a hard glass surface. These results provide useful information to the development of single‐cell manipulation techniques.     

Influence of the phase effect on gradient‐based and statistics‐based focus measures in bright field microscopy

Autofocusing is essential to high throughput microscopy and live cell imaging and requires reliable focus measures. Phase objects such as separated single Chinese hamster ovary cells are almost invisible at the optical focus position in bright field microscopy images. Because of the phase effect, defocused images of phase objects have more contrast. In this paper, we show that widely used focus measures exhibit an untypical behaviour for such images. In the case of homogeneous cells, that is, when most cells tend to lie in the same focal plane, both gradient‐based and statistics‐based focus measures tend to have a local minimum instead of a global maximum at the optical focus position. On the other hand, if images show inhomogeneous cells, gradient‐based focus measures tend to yield typical focus curves, whereas statistics‐based focus measures deliver curves similar to the case of homogeneous cells. These results were interpreted using the equation describing the phase effect and patch‐wise analysis of the focus curves. Bioprocess engineering experts are also influenced by the phase effect. Forty‐four focus positions selected by them led to the conclusion that they prefer to look at defocused images instead of those at the optical focus.     

3D map distribution of metallic nanoparticles in whole cells using MeV ion microscopy

In this work, a new tool was developed, the MORIA program that readily translates Rutherford backscattering spectrometry (RBS) output data into visual information, creating a display of the distribution of elements in a true three‐dimensional (3D) environment. The program methodology is illustrated with the analysis of yeast Saccharomyces cerevisiae cells, exposed to copper oxide nanoparticles (CuO‐NP) and HeLa cells in the presence of gold nanoparticles (Au‐NP), using different beam species, energies and nuclear microscopy systems. Results demonstrate that for both cell types, the NP internalization can be clearly perceived. The 3D models of the distribution of CuO‐NP in S. cerevisiae cells indicate the nonuniform distribution of NP in the cellular environment and a relevant confinement of CuO‐NP to the cell wall. This suggests the impenetrability of certain cellular organelles or compartments for NP. By contrast, using a high‐resolution ion beam system, discretized agglomerates of Au‐NP were visualized inside the HeLa cell. This is consistent with the mechanism of entry of these NPs in the cellular space by endocytosis enclosed in endosomal vesicles. This approach shows RBS to be a powerful imaging technique assigning to nuclear microscopy unparalleled potential to assess nanoparticle distribution inside the cellular volume.     

High‐resolution wide‐field microscopy with adaptive optics for spherical aberration correction and motionless focusing

Live imaging in cell biology requires three‐dimensional data acquisition with the best resolution and signal‐to‐noise ratio possible. Depth aberrations are a major source of image degradation in three‐dimensional microscopy, causing a significant loss of resolution and intensity deep into the sample. These aberrations occur because of the mismatch between the sample refractive index and the immersion medium index. We have built a wide‐field fluorescence microscope that incorporates a large‐throw deformable mirror to simultaneously focus and correct for depth aberration in three‐dimensional imaging. Imaging fluorescent beads in water and glycerol with an oil immersion lens we demonstrate a corrected point spread function and a 2‐fold improvement in signal intensity. We apply this new microscope to imaging biological samples, and show sharper images and improved deconvolution.