By Peter J. Verveer
This quantity offers an outline of complicated fluorescence microscopy, overlaying a large variety of equipment. every one bankruptcy specializes in a distinct process and offers a pragmatic advisor for software in organic platforms. Written within the hugely profitable Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and pointers on troubleshooting and keeping off recognized pitfalls.
Authoritative and state of the art, Advanced Fluorescence Microscopy: tools and Protocols seeks to supply scientists with tools for organic platforms which are of curiosity.
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Additional info for Advanced Fluorescence Microscopy: Methods and Protocols
Proc Natl Acad Sci U S A 101:7311–7316 9. Akopova I, Tatur S, Grygorczyk M et al (2011) Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells. Poulter et al. Grigoriev I, Splinter D, Keijzer N et al (2007) Rab6 regulates transport and targeting of exocytotic carriers. Berginski ME, Vitriol EA, Hahn KM et al (2011) High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One 6:e22025 12. Partridge MA, Marcantonio EE (2006) Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading.
1× trypsin (Gibco). 4. Lipofectamine 2000 (Invitrogen). 5. Paxillin-RFP construct (courtesy of Dr. Maddy Parsons, King’s College London). 4, with FCS at 5 % (V/V). 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.
3. On day 3, wound the confluent layer of cells using a pipette tip, remove the medium, and replace with CIM that has been prewarmed to 37 °C (see Note 1). Before using the microscope, observing laser safety guidelines, ensure that the TIRF laser is aligned and focused for the objective you are going to use. Place the dishes of cells onto the microscope stage within a microscope incubator that has been pre-warmed to 37 °C for at least several hours before imaging (see Note 2). Ensure that the glass bottom of the dish is clean and it is completely level on the microscope stage (see Note 3).