TIRF Microscopy

Total Internal Reflection Fluorscence (TIRF) microscopy is an advanced imaging technique that allows for small (less than 100nm thick) regions of a sample to be examined while excluding noise from the rest of the sample. TIRF is unique when compared to other biological imaging techniques in that it is capable of producing particularly sharp images, due to its ability to block out noise. Dr. Weihong Qiu uses TIRF to study the behavior of single motor proteins walking along microtubule tracks.

TIRF microscopy is based on the phenomenon known as the evanescent wave. When excitation light is shone on an interface between two different media at an angle greater than what is known as the critical angle, the excitation light undergoes total internal reflection and is not transmitted into the sample. However, at the critical angle a small exponentially-decaying light called the evanescent wave does get transmitted into the sample. This wave typically penetrates less than 100nm of the sample. The part of the sample inside this evanescent wave region is then viewed through the microscope. By only illuminating the first 100nm of a sample, the TIRF system achieves the high signal-to-noise ratio necessary for creating precise biological images.

Epifluorescent microscopy uses direct laser light, whereas TIRF uses light shone at the “critical angle”. This allows TIRF to illuminate a smaller area and pick up less background noise.

Applications of TIRF Microscopy for studying Molecular Motors

Microtubule Gliding Assays
Labeled microtubules “crowd surf” across a field of moving motor protein feet
Single Molecule Assays
Labeled motor proteins walk along fixed microtubule tracks
Microtubule Sliding Assays
Two microtubules slide across each other by the walking of motor proteins sandwiched between them
Dynamic Microtubule Assays
Microtubules dynamically grow and shrink in the presence of tubulin proteins