Reflection Fluorescence Microscope

When light passes from a medium of higher refractive index to a medium of lower refractive index, the light is refracted (bent) away from the normal at the boundary. At higher angles of incidence there reaches a point where the light will not transmit into the lower refractive index medium and will instead be totally reflected. This phenomenon is known as Total Internal Reflection. We achieve this condition in the microscope with a laser illuminating from the periphery of the back focal plane of an objective lens. The immersion oil and coverslip are the higher refractive index media while the cell and aqueous media are of a lower refractive index. Even though the light is totally reflected according to traditional optical theory, a small amount of energy does pass through this interface into the lower refractive index media in the form of an evanescent wave. The energy wave is termed evanescent due to its penetration of only a few tens of nanometers from the boundary. The penetration depth for a TIRFM evanescent wave is typically 50-100nm. Only the fluorescence molecules within the shallow penetration of the evanescent wave will be visible, allowing very selective, high contrast fluorescence imaging. TIRFM is especially useful in studies of cellular membrane function and single molecule events without the interference of background fluorescence from other areas in the cell body. This results in high contrast images with a signal to noise ratio greatly improved over widefield and even confocal imaging.We are majorly looking for queries from Madhya Pradesh, Chhattisgarh, Maharashtra, Orissa, Andhra Pradesh and Rajasthan.