adjusted by changing the angle the excitation beam enters the objective lens. Finally, the reflective indices () of the solution and cover slip can be experimentally found or reported by manufacturers. Excitation Beam For complex fluoroscope microscopy techniques, lasers are the preferred light source as they are highly uniform, intense, and near-monochromatic. However, it is noted that ARC LAMP light sources and other types of sources may also work. Typically the wavelength of excitation beam is designated by the requirements of the fluorophores within the sample, with most common excitation wavelengths being in the 400-700 nm range for biological samples. In practice, a lightbox will generate a high intensity multichromatic laser, which will then be filtered to allow the desired wavelengths through to excite the sample. For objective-based TIRFM, the excitation beam and fluoresced emission beam will be captured via the same objective lens. Thus, to split the beams, a dichromatic mirror is used to reflect the incoming excitation beam towards the objective lens, and allow the emission beam to pass through into the detector. Additional filtering may be required to further separate emission and excitation wavelengths. Emission Beam When excited with specific wavelengths of light, fluorophore dyes will reemit light at longer wavelengths (which contain less energy). In the context of TIRFM, only fluorophores close to the interface will be readily excited by the evanescent field, while those past ~100 nm will be highly attenuated. Light emitted by the fluorophores will be undirected, and thus will pass through the objective lens at varying locations with varying intensities. This signal will then pass through the dichromatic mirror and onward to the detector. Cover Slip & Immersion Oil Glass cover slips typically have a reflective index around , while the immersion oil refractive index is a comparable . The medium of air, which has a refractive index of , would cause refraction of the excitation beam between the objective and the coverslip, thus the oil is used to buffer the region and prevent superfluous interface interactions before the beam reaches the interface between coverslip and sample. Objective Lens The objective lens numerical aperture (NA) specifies the range of range of angles over which the system can accept or emit light. To achieve the greatest incident angles, it is desirable to pass light at an off-axis angle through the peripheries of the lens. Back focal plane (BPF) The back focal plane (also