By Peter J. Verveer
This quantity presents an summary of complicated fluorescence microscopy, overlaying a huge diversity of equipment. each one bankruptcy specializes in a unique technique and offers a pragmatic consultant for program in organic structures. Written within the hugely profitable Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and tips about troubleshooting and keeping off identified pitfalls.
Authoritative and state-of-the-art, Advanced Fluorescence Microscopy: equipment and Protocols seeks to supply scientists with equipment for organic platforms which are of curiosity.
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Extra info for Advanced Fluorescence Microscopy: Methods and Protocols
7. Neubauer cell counting chamber. 35 mm glass-bottomed imaging dishes (MatTek Corporation, USA). 49, Nikon). l) set at 37 °C for live-cell imaging. 3. Green diode 561 nm laser. 4. 12-bit iXon 1 M EM-CCD camera. 5. 2 software. Trypsinise a T75 flask of MDA-MB-231 cells and count the number of cells per 1 ml using a counting chamber. Seed 6 × 105 cells onto each 35 mm glass bottom dish and incubate in complete medium at 37 °C, 5 % CO2 for 24 h. On day 2, transiently transfect cells with paxillin-RFP using Lipofectamine 2000 according to the manufacturer’s instructions using a ratio of 4 μg DNA to 10 μl Lipofectamine 2000 per 35 mm dish of cells.
Webb RL, Rozov O, Watkins SC et al (2009) Using total internal reflection fluorescence (TIRF) microscopy to visualize cortical actin and microtubules in the drosophila syncytial embryo. Dixit R, Ross JL (2010) Studying plus-end tracking at single molecule resolution using TIRF microscopy. Gardner MK, Charlebois BD, Jánosi IM et al (2011) Rapid microtubule self-assembly kinetics. Cell 146:582–592 19. Graham-Smith SF, King TA (2000) Optics and photonics: an introduction. Millis BA (2012) Evanescent-wave field imaging: an introduction to total internal reflection fluorescence microscopy.
Tet-ON/Tet-OFF) provides further spatial and temporal control of gene expression . Transfection methods: In the context of live cell imaging with 2PE, nucleic acids (DNA or shRNA constructs) are most commonly introduced in neurons using plasmid electroporation or viral infection. These methods can be used in Cre mice for conditional gene activation to obtain higher labeling specificity . For in vivo electroporation, the delivery of a plasmid into neurons of living mice is achieved by injecting a DNA solution into the brain (or into a single cell with patch-clamp electrophysiology) followed by short electric pulses that permeabilize cell membranes temporarily and allow plasmid entry .