By Peter J. Verveer
This quantity offers an outline of complicated fluorescence microscopy, overlaying a wide diversity of equipment. every one bankruptcy specializes in a special technique and offers a pragmatic consultant for software in organic platforms. Written within the hugely winning Methods in Molecular Biology sequence layout, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and pointers on troubleshooting and averting recognized 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 resources for Advanced Fluorescence Microscopy: Methods and Protocols
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.
The microendoscopy approach to image deep structures relies on the use of GRIN lenses, which use internal variations in the refractive index (as opposed to curved refractive surfaces of conventional lenses) to guide light. In essence, GRIN lenses act as an optical relay that projects the scanning pattern of the 2PE microscope to a focal plane deep inside the tissue sample. A wide array of microendoscopes is available with varying physical length, optical working distance and numerical apertures, and field of views.
5 Combining 2PE Microscopy with Optogenetics, Glutamate Uncaging Another advantage of 2PE microscopy is that it can easily be combined with optogenetics and be used to uncage neurotransmitters for temporally and spatially precise manipulation and probing of neuronal activity within intact neural circuits. 1 Optogenetics This technique enables researchers to silence or stimulate genetically specified classes of neurons (or other electrically excitable cells) with exquisite temporal precision using light-sensitive molecules [70, 71].
Advanced Fluorescence Microscopy: Methods and Protocols by Peter J. Verveer