Microscopy refers to the study of objects that are too small to be easily viewed by the unaided human eye. Microscopes are devices that are used to magnify small objects using technology for fixed, live cells, and tissue to obtain optimal high resolution image collection. This process may be carried out by wide-field irradiation, transmitted light of the sample or by scanning of a fine beam over the sample by confocal laser scanning or scanning electron microscopy. The image formed is affected by magnification, image resolution and the contrast generation mechanism. Dimensional information about specimens in 2D and 3D can be obtained.
Confocal Imaging is used for obtaining crisp images with good z-resolution and for 3D imaging.
Real Time Confocal Imaging is used for live cell imaging and monitoring fast dynamics within the cell, by using spinning disk confocal
TIRF Imaging is used for imaging and monitoring events happening on the cell membranes (<100nm depth).
FRET is used to determine when two molecules are in close proximity to one another for monitoring molecular interactions.
Nomarski and fluorescence imaging combination is used to see fluorescence labeling localization within the cell structure.
There are a number of supplications for Microscopy, including, but not limited to:
Fluorescence imaging of a huge range of samples - Different modalities are used for different specimens - Widefield, laser scanning confocal, multi Photon, spinning disk confocal,
Filters/excitation for virtually all fluorophores – Examples: dapi, all fluorescent proteins, Alexa Fluor)
Transmitted light imaging – Imaging can be done using DIC, phase contrast, brightfield, darkfield, polarized light, Colour IHC (eg H&E staining)
Imaging large range of sample sizes – Imaging can take place from macroscopic samples to samples requiring 100x objectives on a widefield, confocal or multiphoton scope
Live-cell imaging –Imaging with temperature, humidity and CO2 regulation on widefield fluorescence, confocal and mutiphoton microscopes with the use of high-speed resonant scanner for rapid less damaging confocal acquisition. High sensitivity EM-CCD camera acquition with time lapse and multipositioning time lapse can be used.
FRAP, FLIP, Photoactivation/conversion – Measurement of protein dynamics in living cells is done with point scanning confocals. They are used for defining regions with slower processes, or point activation, bleaching,for ultra fast, sensitive, experiments.
FRET - Imaging is possible on confocal and widefield systems for sentized emission and acceptor photobleaching. Ratiometric CFP/YFP FRET can be done.
High-throughput microscopy – Imaging and acquisition on a plate format can be done on an inverted microscope with a motorized stage.
Image processing, analysis and quantification – Imaging can be done to measure objects intensity, size, number, etc. with tracking in 2D and 3D and digital deconvolution.