called “aperture plane”) is the plane through which the excitatory beam is focused before passing through the objective. Adjusting the distance between the objective and BPF can yield different imaging magnification, as the incident angle will become less or more steep. The beam must be passed through the BPF off-axis in order to pass through the objective at its ends, allowing for the angle to be sufficiently greater than the critical angle. The beam must also be focused at the BPF because this ensures that the light passing through the objective is collimated, interacting with the cover slip at the same angle and thus all totally internally reflecting. Sample The sample should be adsorbed to the surface of the glass cover slide and stained with appropriate fluorophores to resolve the features desired within the sample. This is in protocol with any other fluorescent microscopy technique. Dichroic (Dichromatic) Filter The dichroic filter is an edge filter used at an oblique angle of incidence (typically 45°) to efficiently reflect light in the excitation band and to transmit light in the emission band. The 45° angle of the filter separates the path of the excitation and emission beam. The filter is composed of a complex system of multiple layers of metals, metal salts and dielectrics which have been vacuum-deposited onto thin glass. This coating is designed to have high reflectivity for shorter wavelengths and high transmission for longer wavelengths. While the filter transmits the selected excitation light (shorter wavelength) through the objective and onto the plane of the specimen, it also passes emission fluorescence light (longer wavelength) to the barrier filter and reflecting any scattered excitation light back in the direction of the laser source. This maximizes the amount of exciting radiation passing through the filter and emitted fluorescence beam that is detected by the detector. Barrier Filter The barrier filter mainly blocks off undesired wavelengths, especially shorter excitation light wavelengths. It is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band. More modern microscopes enable the barrier filter to be changed according to the wavelength of the fluorophore's specific emission. Image detection and resolution The image is detected by a charged-coupled device (CCD) digital camera. CCD cameras have photon detectors, which are thin silicon wafers, assembled into 2D arrays of light-sensitive regions. The detector arrays capture and store image information