Confocal imaging involves point scanning a sample (scanning every point on a sample) using lasers to excite/scan the fluorescent sample. This is usually accomplished via an automated stage to move the sample. The laser is focused through a pinhole on a specific spot on the sample to avoid exciting molecules that are not in the focal plane and only excite those fluorophores that are in the same focal plan as the laser (hence the name Confocal). Because of this specific pinhole excitation, the fluorescence emitted (also going through the pinhole) is also very specific and provides increased resolution, but also decreased signal intensity. In order to get a signal that can be translated into an image, a PMT (photo-multiplier tube) is used.
Confocal scanning also enables optical sectioning, as the sample can be scanned at various focal depths to create several images that replicates imaging a sliced sample (without compromising sample integrity). This enables imaging that gives the user a better idea of the molecular interactions within tissues and provides neat 3D and 4D models of real and live samples.
Fluorescence - the emission of light (lower energy ) from a substance upon excitation, in most single photon cases with a lower wavelength (higher energy) of light. Fluorescence in the life sciences has been a very useful way of tracking and analyzing biological molecules. The fluorescent molecule (also known as a fluorophore), can be used to "label" proteins within a cell or tissue using specific antibodies to target the protein of interest.
FRET (Förster Resonance Energy Transfer) is a quantum mechanical process involving the radiation-less transfer of energy between fluorophores over a small distance (1-10 nm). If the molecules are close enough, the donor fluorophore transfers its energy to the acceptor fluorophore. Monitoring the ratio of the fluorophore emissions provides valuable information to researchers about the spatial localization of the proteins being studied.
Optogenetics is an emerging field of science where genetically modified cells and tissue are controlled by light. Predominantly used to probe neural circuits, optogenetics involves introduction and control of light-activated channels to allow control of neural activity. The key to this is millisecond precision to study the brain and its ultra-fast activities. This is very different from traditional genetics where manipulations take hours-days-months.
Using microscopy to study the presence, absence or co-localization of staining in a sample. Did the cell transfect? Does a cell pick up a certain stain or stain for the particular antigen? Qualitative microscopy can also involve observing a simple increase or decrease in image/structural intensities on drug/chemical treatment. Microscopy applications are moving more and more towards Quantitative applications.
Using Microscopy as an image analysis tool to count or measure intercellular, intracellular or extracellular structures in the case of biological materials. Most image acquisition software packages include these analyses tools. In the case of comparative image analysis, this may be used to compare structure numbers between a healthy and diseased sample or effects of drug/chemical treatments. Microscopy can also be used to measure intensities of staining in brightfield or fluorescence images, depending on the type of staining utilized. In more advanced imaging, the distance between molecules can be measured (FRET) as well as the decay/lifetime of fluorophores (FLIM)
Multi-photon imaging uses the same imaging principles as Confocal imaging. The lasers raster scan the sample and have the ability to optically section the sample.
The difference is in the properties of light used to excite the fluorophores. Multi-photon is named as such because the fluorophore absorbs energy from two long wavelength photons which reach the fluorophore almost simultaneously - the laser is pulsed (femto-nanosecond pulses). The end result is the emission of fluorescence or autofluorescence that is detected by a PMT (photo-multiplier tube) and conversion into an image.
Unlike confocal microscopes, multiphoton systems do not have variable Confocal pinholes. The combining of the pulsed laser beams produces a very small point spread function causing excitation of a very small area in the sample and precluding the need for a pinhole. Multi-photon imaging also allows deeper penetration into tissue due to the infra-red lasers used. The longer wavelength, low energy excitation lasers also allow for long term imaging of live cells and live tissue as they cause less damage and phototoxicity.
Superresolution is the hottest and newest technique that enables imaging with resolution in the tens of nanometers. This relatively new technique breaks the diffraction-limit (factors that limit optical resolution) of systems.
There are several flavours of superresolution: STED (Stimulated Emission Depletion microscopy), PAL-M method (photo-activated localization microscopy) and STORM (stochastic optical reconstruction microscopy). The basic idea of superresolution imaging involves selective activation of molecules to prevent blurring of the image due to simultaneous excitation of fluorescent molecules in close proximity.
Lasers are used in all cases and the imaging requires a lot of computer power and hours of imaging time, but the results produce resolutions encroaching on the territory of electron microscopy!
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