What Is TIRF?
The facility currently has one Total Internal Reflection Fluorescence (TIRF)
Microscope and one TIRF_FLIM system .TIRF is a powerful technique that uses an
evanescent wave to excite flurophores that are present at the bottom of the
specimen adjacent to the glass water interface. The optical section that is
excited is in the range of 100nm-150nm . It is often employed to study cellular membrane
activities, the dynamics of actin, cellular adhesion, cell movement, single
molecular events, vesicle and protein tracking.
TIRF is so named because it involves total internal reflection of the excitation
light. The excitation light must strike the glass- medium interface at the
critical angle (the angle at which no light passes from the glass slide to the
medium). Instead the light is reflected and this results in an evanescent wave
being produced. One of the advantages of TIRF is that the background and out of focus signals are dramatically
reduced which allows you to view very dim fluorescent events.
Above image courtesy of Juliana Schwarz R19 showing
TIRF optical path.
Image confocal optical section verses TIRF
TIRF used to study membrane dynamics at the cell basal membrane. It can only be
used to visualize the bottom plane of the cell. (Optical section of 150nm-200nm
Above comparison of different imaging methods.