1. TIRF Total Internal Reflection Fluorescence Microscopy specialized fluorescence microscopy technique specifically images a very thin optical section (50-250nm) adjacent to the coverslip using conditions to create total internal reflection that generates an evanescent wave

2. Refractive Index: A measure in the reduction of the speed of light inside the medium (compared to the speed of light in a vacuum) Refraction of Light: the bending or change in direction of light as it travels from medium into another with different refractive indexes refraction of light only occurs when the incident light meets the interface at an angle light will travel straight through with no change of direction when crossing perpendicular to the interface the degree of refraction increases as angle of the incident light increases Vacuum1.00 Air1.003 Water1.33 Glass1.52-1.54 Critical Angle: the angle of the incident light where the refraction angle is 90 degrees Total Internal Reflection: Occurs when the incident angle is greater than the critical angle Majority of the light is reflected

3. Evanescent Wave: During total internal reflection a small portion of the reflected light penetrates through the interface This creates a very thin electromagnetic field (<250nm) adjacent to the interface (evanescent wave) Identical frequency to the incident light Propagates parallel to the interface Intensity decreases exponentially with increasing distance away from the interface This evanescent wave is used for excitation in TIRF microscopy

4. TIRF Microscopy Laser angled within the objective past the critical angle resulting in total internal reflection of the laser and the generation of an evanescent wave the evanescent wave travels along the coverslip exciting the entire sample simultaneously the intensity of the evanescent wave decreases exponentially away from the coverslip The evanescent wave only has sufficient energy for excitation within close proximity of the coverslip. therefore only fluorophores within this close proximity of the coverslip produce emission.

5. Epi-fluorescence Confocal TIRF entire sample exposed to excitation light simultaneously fast image acquisition with CCD camera laser passes over the sample point by point Slow image acquisition (25-30 sec/image) evanescent wave travels along the coverslip exciting the entire sample simultaneously fast image acquisition with CCD camera (30 frames /sec) No out-of-focus emission generated Very thin optical section (50-250nm) decrease in signal-to-noise improving the contrast May improve resolution at the cell surface compared to confocal Both in-focus and out-of- focus emission collected No optical sectioning (“fuzzy” image) High signal-to-noise (poor contrast) Out-of-focus emission blocked by pinhole optical section (600-900nm) decrease in signal-to-noise improving the contrast

6. TIRF – 110nm focused at coverslip focused at middle of cell Epi Rab11-GFP HEK 293 Confocal

7. Resolution: the minimum distance between two points required to identify them as separate points The resolution limit of a microscope is determined by: - wavelength of light used for excitation - numerical aperture (NA) of the objective R=0.61 /NA Example: R = (0.61x488nm)/1.4 = ~200nm Resolution MAY be improved with TIRF microscopy compared to confocal microscopy

8. Resolution: the minimum distance between two points required to identify them as separate points The resolution limit of a microscope is determined by: - wavelength of light used for excitation - numerical aperture (NA) of the objective R=0.61 /NA Example: R = (0.61x488nm)/1.4 = ~200nm Resolution MAY be improved with TIRF microscopy compared to confocal microscopy

9. HEK 293 Neuron Clathrin coated vesicle Plasma membrane (~10nm) Endosomes

10. 90nm110nm 150nm200nm Rab5-GFP HEK 293 TIRF – 150nm

11. Advantages of TIRF: Leica AM TIRF MC Fast image acquisition Very thin optical section Decrease signal-to-noise (increase in contrast) May improve resolution TIRF microscopy is a tool to study the molecular events at or near the cell surface at a speed and resolution that is not possible with other imaging techniques Applications of TIRF: Distribution Colocalization Trafficking movement on the surface endocytosis exocytosis

12. Type of Imaging Acquisition Speed Optical Section Major Advantage Available at UWO Epi-fluorescenceFastn/a Easy Inexpensive Laser Scanning Confocal Slow600-900nmOptical sectioning MultiphotonSlowYes Optical sections in thick specimen TIRFFast 50-250nm from coverslip Image events at or near the membrane X TIRF microscopy is…. Summary specialized fluorescence microscopy technique images a very thin optical section (50-250nm) adjacent to the coverslip uses conditions that create total internal reflection that generates an evanescent wave study molecular events at or near the cell surface speed and resolution that is not possible with other imaging techniques