Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae)

The family Orchidaceae comprises the most species of any monocotyledonous family and has interesting characteristics such as seed germination induced by mycorrhizal fungi and flower morphology that co-adapted with pollinators. In orchid species, genomes have been decoded for only a few horticultural species, and there is little genetic information available. Generally, for species lacking sequenced genomes, gene sequences are predicted by de novo assembly of transcriptome data. Here, we devised a de novo assembly pipeline for transcriptome data from the wild orchid Cypripedium (lady slipper orchid) in Japan by mixing multiple data sets and integrating assemblies to create a more complete and less redundant contig set. Among the assemblies generated by combining various assemblers, Trinity and IDBA-Tran yielded good assembly with higher mapping rates and percentages of BLAST hit contigs and complete BUSCO. Using this contig set as a reference, we analyzed differential gene expression between protocorms grown aseptically or with mycorrhizal fungi to detect gene expressions required for mycorrhizal interaction. A pipeline proposed in this study can construct a highly reliable contig set with little redundancy even when multiple transcriptome data are mixed, and can provide a reference that is adaptable to DEG analysis and other downstream analysis in RNA-seq

DEGs and DEG-derived data from RNA-Seq data from pearl millet

Lists of differentially expressed genes (DEGs) detected in leaves and roots of pearl millet plants treated with drought, NaCl, polyethylene glycol (PEG) or abscisic acid (ABA) are provided here. Results of analyses of motifs enriched in the promoters of these DEGs are also provided. Results of analyses of gene ontology (GO) terms enriched in those DEGs are also provided. Matrices of the read counts and transcripts per million (TPMs) derived from the ABA-treated samples are also provided. Matrices of those from the drought-, NaCl- and PEG-stressed samples are provided in https://doi.org/10.6084/m9.figshare.24902100.</p