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

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

Vacuum 1.00Air 1.003Water 1.33Glass 1.52-1.54

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

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

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.

Epi-fluorescence ConfocalTIRF

• 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

TIRF – 110nm

focused atcoverslip

focused atmiddle of

cell

Epi

Epi

Rab11-GFPHEK 293

Confocal

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

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

HEK 293

Neuron

Clathrin coated vesicle

Plasma membrane (~10nm)

Endosomes

90nm 110nm

150nm 200nm

Rab5-GFPHEK 293

TIRF – 150nm

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

Type of Imaging

Acquisition

Speed

Optical Section

MajorAdvantage

Available at UWO

Epi-fluorescence Fast n/aEasy

Inexpensive

Laser Scanning Confocal

Slow 600-900nm Optical sectioning

Multiphoton Slow YesOptical sections in

thick specimen

TIRF Fast50-250nm

from coverslipImage 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