field. Typically objective-based TIRFM are more popularly used, however have lowered imaging quality due to stray light noise within the evanescent wave. Prism-based Less extraneous scattering Much cheaper (hundreds instead of thousands of dollars) Best for low-mid range magnification and water immersion objectives Easiest with free collimated laser sources Larger range of incidence angles Desirable to achieve smallest evanescent field depth Objective-based High magnification and aperture Stable, easy to set up and align Works with free collimated laser, optical fiber, or conventional arc sources Methodology Fundamental Physics behind TIRFM TIRFM is predicated on the optical phenomena of Total Internal Reflection, in which waves arriving at a medium interface do not transmit into medium 2 but are completely reflected back into medium 1. Total internal reflection requires medium 2 to have a lower refractive index than medium 1, and for the waves must be incident at sufficiently oblique angles on the interface. An observed phenomena accompanying total internal reflection is the evanescent wave, which spatially extends away perpendicularly from the interface into medium 2, and decays exponentially, as a factor of wavelength, refractive index, and incident angle. It is the evanescent wave which is used to achieve increased excitation of the fluorophores close to the surface of the sample, and diminished excitation of superfluous fluorophores within solution. For practical purposes, in objective based TIRF, medium 1 is typically a high refractive index glass coverslip, and medium 2 is the sample in solution with a lower refractive index. There may be immersion oil between the lens and the glass coverslip to prevent significant refraction through air. Evanescent Wave The critical angle for excitatory light incidence can be derived from Snell's law: For the refractive index of sample, the refractive index of the cover slip. Thus, as the angle of incidence reaches , we begin observing effects of total internal reflection and evanescent wave, and as it surpasses these effects are more prevalent. The intensity of the Evanescent Wave is given by: With penetration depth given by: Typically, ≤~100 nanometers, which is typically much smaller than the wavelength of light, and much thinner than a slice from confocal microscopes. For TIRFM imaging the wavelength of the excitation beam within the sample can be selected for by filtering. Additionally, the range of incident angles is determined by the numerical aperture (NA) of the objective, and requires that NA > . This parameter can be