Search

A major challenge in ART is to select high-quality oocytes and embryos. The metabolism of oocytes and embryos has long been linked to their viability, suggesting the potential utility of metabolic measurements to aid in selection. Here, we review recent work on noninvasive metabolic imaging of cumulus cells, oocytes, and embryos. We focus our discussion on fluorescence lifetime imaging microscopy (FLIM) of the autofluorescent coenzymes NAD(P)H and flavine adenine dinucleotide (FAD+), which play central roles in many metabolic pathways. FLIM measurements provide quantitative information on NAD(P)H and FAD+ concentrations and engagement with enzymes, leading to a robust means of characterizing the metabolic state of cells. We argue that FLIM is a promising approach to aid in oocyte and embryo selection. [ABSTRACT FROM AUTHOR]

Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics. [ABSTRACT FROM AUTHOR]

Time-lapse imaging of live cells using multiple fluorescent reporters is an essential tool to study molecular processes in single cells. However, exposure to even moderate doses of visible excitation light can disturb cellular physiology and alter the quantitative behavior of the cells under study. Here, we set out to develop guidelines to avoid the confounding effects of excitation light in multi-color long-term imaging. We use widefield fluorescence microscopy to measure the effect of the administered excitation light on growth rate (here called photomorbidity) in yeast. We find that photomorbidity is determined by the cumulative light dose at each wavelength, but independent of the way excitation light is applied. Importantly, photomorbidity possesses a threshold light dose below which no effect is detectable (NOEL). We found, that the suitability of fluorescent proteins for live-cell imaging at the respective excitation light NOEL is equally determined by the cellular autofluorescence and the fluorescent protein brightness. Last, we show that photomorbidity of multiple wavelengths is additive and imaging conditions absent of photomorbidity can be predicted. Our findings enable researchers to find imaging conditions with minimal impact on physiology and can provide framework for how to approach photomorbidity in other organisms. [ABSTRACT FROM AUTHOR]

TI Workbench is a software package that serves as a control and analysis center for cellular imaging and electrophysiological experiments. It is unique among general-purpose software packages where it integrates the control of cellular imaging and electrophysiological devices, as well as sophisticated data analyses, which provides superior usability in imaging experiments combined with electrophysiology. During the development over the last 20 years, the range of supported image acquisition devices has expanded from cooled charge-coupled device (CCD) cameras to multi-photon microscope systems. In this review, I outline the concept of TI Workbench together with its unique functions and features derived from ideas emerging during daily experiments in my own lab and in those of my collaborators over the last 20 years. TI Workbench includes standard functions required for time-lapse multicolor fluorescence imaging and electrophysiological experiments, in addition to specialized functions such as random-scan or conventional raster-scan two-photon microscopy packages and fluorescence life time imaging (FLIM) utilities. Data analysis modules, e.g. digital data filters for temporal waveforms of timelapse image data and electrophysiology and for 2-D image data, and fluorescence correlation spectroscopy (FCS) analysis functions, are well integrated with data acquisition functions. A notebook function holds formatted text, graphs, image and movie data altogether, which are linked to the actual data files. TI Workbench uses Igor Pro software as a back-end output for publishing. In addition, TI Workbench imports several different formats of image and electrophysiology data, serving as a general-purpose data analysis software package. [ABSTRACT FROM AUTHOR]

Motivation: Present research on gene expression using live cell imaging and fluorescent proteins or tagged RNA requires accurate automated methods of quantification of these molecules from the images. Here, we propose a novel automated method for classifying pixel intensities of fluorescent spots to RNA numbers.Results: The method relies on a new model of intensity distributions of tagged RNAs, for which we estimated parameter values in maximum likelihood sense from measurement data, and constructed a maximum a posteriori classifier to estimate RNA numbers in fluorescent RNA spots. We applied the method to estimate the number of tagged RNAs in individual live Escherichia coli cells containing a gene coding for an RNA with MS2-GFP binding sites. We tested the method using two constructs, coding for either 96 or 48 binding sites, and obtained similar distributions of RNA numbers, showing that the method is adaptive. We further show that the results agree with a method that uses time series data and with quantitative polymerase chain reaction measurements. Lastly, using simulated data, we show that the method is accurate in realistic parameter ranges. This method should, in general, be applicable to live single-cell measurements of low-copy number fluorescence-tagged molecules.Availability and implementation: MATLAB extensions written in C for parameter estimation and finding decision boundaries are available under Mozilla public license at http://www.cs.tut.fi/%7ehakkin22/estrna/.Contact: andre.ribeiro@tut.fi [ABSTRACT FROM AUTHOR]

Brain function is based on proper connectivity between neuronal cells. In the developing brain, neurons extend axons and form synaptic connections with appropriate postsynaptic neurons. Molecular mechanisms underlying establishment of proper synaptic connections are one of the most important topics in the field of developmental neurobiology. Dynamics of synaptic structure and local recruitment of synaptic molecules can be studied by live-cell imaging of neurons expressing fluorescent probes of synaptic molecules. In this review, examples of live-cell fluorescence imaging are presented and their contributions to our understanding about the molecular mechanisms of synapse formation and remodeling are discussed. Imaging of synaptic proteins in living neurons revealed rapid formation of individual synapses within hours and extensive remodeling of synaptic connections. Different types of neurons express unique protrusions from dendrites and axons, which play important roles in synapse formation and maturation. Rapid formation of synaptic structure is associated with continual assembly and disassembly of synaptic scaffolding proteins, which are essential building blocks of the presynaptic active zone and the postsynaptic density (PSD). Quantitative analyses of PSD scaffolding proteins further confirmed their essential roles in maintenance of the synaptic structure. These examples clearly indicate that fluorescence-based live-cell imaging is an indispensable technique in the research on synapse development and its impact will further increase in combination with development of new light microscopic techniques in the future. [ABSTRACT FROM AUTHOR]

Lead ion (Pb2+) is one of the most hazardous heavy metals to almost all life forms. The components of store-operated Ca2+ entry as a molecular gateway have been previously found to participate in the cytotoxic entry of Pb2+. However, the safe levels of intracellular Pb2+ hiding in blood Pb2+ levels are still not determined with full certainty. The present study aimed to construct protein-based Pb2+ indicators to help establish a reliable setting for the content monitoring of intracellular Pb2+. A series of Pb2+ indicators based on fluorescence resonance energy transfer, Met-leads, were developed. The Pb2+-binding protein PbrR (from Cupriavidus metallidurans CH34) was applied between the fluorescent protein pair ECFP(ΔC11) and cp173Venus. The spectral patterns and sensing ranges of all Met-leads were characterized in vitro. Among these constructs, Met-lead 1.59 had relatively high ion selectivity and broad dynamic range (3.3–5.7). Consequently, this Met-lead was adopted in the cellular Pb2+ biosensing. The intracellular Pb2+ content in human embryonic kidney cells was successfully monitored using Met-lead 1.59 under both short- and long-term treatments. The existence of intracellular Pb2+ can be significantly sensed using Met-lead 1.59 after 3 h 0.5μM (10 μg/dl) exposure, which is 200 times more improved than previous live-cell indicators. In summary, a new Pb2+ indicator, Met-lead 1.59, was successfully developed for advanced research on Pb2+ toxicology. [ABSTRACT FROM PUBLISHER]

Background Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM. [ABSTRACT FROM AUTHOR]