17 Mar 2011 Confocal microscopy compared to conventional wide-field microscopes includes a depth-specific imaging technique whereby micrographs are produced from a specific focal plane often beneath the surface of a semi-transparent sample such as a biological cell specimen. Only light from within the focal plane is transmitted with the depth of the focal plane being the “focal plane thickness”. Confocal microscopy therefore enables the reconstruction of three-dimensional structures from the obtained images. The setup includes a point light source such as a laser, low-light detector such as a single photon counting module, beam splitter, converging lens and pin-holes for spatial filtration. The fluorescence only occurs at a specific point as well as the transmission of light from the sample. As the fluorescence is from a single-point the three-dimensional image is built up by scanning across the sample and adjustments of the focal plane.
Confocal microscopy is becoming increasingly popular fueling investment in development such as the introduction of solid-state single photon counting modules instead of PMTs in particular the COUNT modules from LASER COMPONENTS achieving a cutting-edge dark-count-rate of 10 photons per second compared to the previously available 25cps greatly improving the potential resolution of microscopy systems.
Fluorescence can be achieved by exposing a substance with light of a certain wavelength that causes electrons in the valance band to jump up to a higher energy band. Being unstable the electron decays causing the emission of light of a different wavelength, with lower energy. Consequently, the excitation wavelength is typically shorter, e.g. 440nm, than the emission wavelength, e.g. 480nm.
Fluorophores are compounds used as a dye to mark substances, e.g. proteins, with a specific fluorescent marker. Selecting the correct excitation wavelength and fluorophore allows for precise inspection of specific substances. However, as the excitation wavelength can be close to the emission wavelength, any imaging system, like a confocal microscope, will not be able to easily differentiate between the two wavelength signals, particularly if the emission wavelength intensity is significantly lower than the excitation wavelength.
To separate the wavelengths very steep cut-on and cut-off optical filters are used. Typically the two light paths are separated using a dichroic filter, which will reflect the excitation wavelength but transmit the emission wavelength. Selecting the correct optical filters, fluorophores, and detection system, such as one using the COUNT single photon counting module, precise 3D micrographs can be obtained.
LASER COMPONENTS supplies a complete range of optical fluorescence filters, for FRET experiments, emission filter wheels, filter cubes, Pinkel filters, multi-band, and more.
For more information visit our website at Confocal Microscopy
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