Total Internal Reflection Fluorescence (TIRF)

  • Irit Shoval (Manager)

Equipment/facility: Equipment

Equipments Details


Total Internal Reflection Fluorescence (TIRF) system with infinity laser scanner system for photo-activation, bleaching, FRET and more.
TIRF is a highly sensitive technique in fluorescence microscopy that delivers images with an outstandingly high axial resolution - below 100nm. Especially used for the observation of membrane-associated processes.

The high signal to noise ratio and high resolution in the z-direction above the coverslip/water interface allows to visualize and to analyze vesicle transport and signaling events, as well as kinetic studies and single molecule detection.

The basis of TIRF is the total reflection of a laser beam at the interface of glass and water that creates an electromagnetic wave, the so-called evanescent field. This evanescent field is what excites the fluorophores. Because the energy of the evanescent field decreases exponentially with the distance of the interface coverslip/water, the method is exceptional for fluorophors near the interface, but not efficient at greater distances.

Other techniques that this machine is especially suited for:

FRAP - we have 4 lasers with which we can perform bleaching: 405, 488, 514, 552
FRET - we have dedicated CFP and YFP filters
Photoactivation - using the scanning capabilities, we can choose specific ROIs
Links: Leica TIRF

Examples for paper reference: (edit for your specific application, magnification and numbers):

Images were acquired on a DMi8 Leica wide-field inverted microscope (Leica Microsystems, Mannheim, Germany), equipped with an Infinity scanner with standard laser lines, an sCMOS DFC9000GT Leica camera and driven by LasX software, using a 63X 1.4NA objective.

FRAP: After image acquisition parameter setup, ROIs were chosen and the 515nm laser was set on maximum power for ROI bleaching. 5 images were acquired pre-bleach and then 40 images post-bleach every 30 seconds. Images were then processed within the LasX program as part of the FRAP workflow, and data was exported to excel and then plotted.

DAPI BP 320-380 BP 420-470 DAPI, Hoechst, Alexa350, Dylight405

GFP BP 450-490 BP 500-550 GFP, eGFP, FITC, Alexa488

RFP BP 530-580 LP >600 RFP, mCherry, Rhodamin, Alexa594


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