When higher spatial resolution is required, it is preferable to use an objective lens with a higher NA. Use a 100 × objective lens with oil immersion. Note: For imaging of temperature-sensitive mutants such as cdc24-4, cells are grown overnight at room temperature, diluted in prewarmed media (37☌), and shaken for 2 h at 37☌ before imaging in a temperature-controlled environmental chamber at 37☌. Wait for ~ 10 min until the sample temperature is stabilized. Set the 35-mm dish sample into an environmental chamber (DH-35 Warner Instruments) mounted on the sample stage of the inverted microscope. Moreover, whether TLM confers a specific advantage for women of advanced reproductive age has not been systematically investigated. Although TLM is emerging as a useful embryo selection tool, further research is necessary to outline morphometric standards in the setting of various infertility diagnoses and treatment methods (intracytoplasmic sperm injection (ICSI)/IVF). Another study indicated further augmentation of overall IVF pregnancy rates for cleavage-stage transfer using the Eeva (Early Embryo Viability Assessment) tracking software ( Conaghan et al., 2013). An extensive retrospective analysis indicated that the use of TLM instead of standard incubation improved pregnancy rates by 20% ( Meseguer et al., 2012). Early study results, however, are promising. Approximately 20 morphokinetic patterns have been identified in human embryos, but the predictive value of many of these has not been adequately tested ( Kovacs, 2014). TLM has since been applied clinically to monitor human embryo morphokinetics (the precise timing of specific morphologic events) with the goal of defining a profile to identify the best embryo for single embryo transfer ( Kovacs, 2014). Noninvasive time-lapse microscopy (TLM) demonstrated that parameters related to the first mitotic embryonic divisions prior to embryonic genome activation are predictive of development to the blastocyst stage and ploidy status ( Chavez et al., 2012 Wong et al., 2010). Mary Ellen Pavone, in Conn's Handbook of Models for Human Aging (Second Edition), 2018 Time-Lapse Microscopy By repeating this process for images of different time points, spatiotemporal dynamics of fluorescence signal can be determined, demonstrating the activity of a component within the genetic circuit of interest.įrancesca E. Determination of the spatial localization of translational fusions can also be accomplished. Once the cells have been identified, numerous properties of single cells can be measured, such as the mean or maximum of fluorescence signal. Edge detection software and histograms of image intensities, together with constraints on cell size and shape, allow identification of individual bacterial cells in a process called segmentation. For example, in recorded images of bacterial cells with phase contrast optics, cells will appear darker than the background. ![]() The goal of the analysis is to utilize differences in intensities of fluorescence or light images to discriminate cells from background and from each other. Image analysis is a very rapidly developing field, and only the basics are discussed here. Therefore, automated and quantitative image analysis is a critical component of measuring genetic circuit dynamics when using fluorescence time-lapse microscopy. Time-lapse microscopy generates large amounts of image data files. Gürol Süel, in Methods in Enzymology, 2011 3.3 Image analysis
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