Système Hilo / TIRF

This set up is a prototype in the “super-resolution” microscope family. It reaches a resolution above 30nm in 3 colors with standard stainings and also compatible with live imaging.


This setup is a prototype in the “super-resolution” microscope family. It reaches a resolution above 30nm in 3 colors with standard stainings and also compatible with live imaging. Its weakness is to be dedicated to processes in cell culture.
It as been developed jointly between Morpheme team and MICA, in collaboration with Ellen van Obberghen team de l’iBV. this project has been supported by GIS Ibisa and l’Université Nice-Côte d’Azur grants.
 

Objectives

MA-TIRF with and without deconvolutionSuper-resolution techniques were developed (StED, PALM/StORM, SIM) to exceed (or rather “circumvent”) this diffraction limit. Over the years, these different techniques kept on improving and now present the enormous advantage of offering very high performances in both lateral resolution (up to 20 nm for a 2D image) and axial resolution (up to 100 nm for a 3D image). Unfortunately, when using these techniques, enormous quantities of energy are projected on the sample and it must also be stained with very specific fluorescent markers, making it difficult to be used with live samples.

For some applications, it is more crucial to be able to improve the axial resolution, and simultaneously examine several molecules using standard fluorescent molecules or directly fluorescent proteins.

The MA-TIRF (multiple-angle total internal reflection fluorescence) method is particularly suited in this context. Initiated in the 90s, this technique uses one of the properties of light, the "evanescent wave". When a sample is illuminated from a very oblique light source, the evanescent wave only penetrates a few hundred nm (this is the standard TIRF principle). By playing on the angle of illumination, penetration can be made to vary. And by precisely combining several angles of illumination, additional information can be obtained. But this requires a very specific reconstruction algorithm.

Properties

  • The microscope can acquire up to 3 colors sequentially,

  • Axial resolution is above 30nm,

  • Maximal thickness is around 500nm (for the reconstruction)

  • Temporal resolution is around 1s per staining to get 3D image,

  • The axial location is absolute with respect to the coverslip surface. 

MA-TIRF room 

Technical specs

  • Lights sources are 488nm, 561nm, 640nm. 405nm is also available but not for 3d reconstruction.

  • Samples must be in culture medium or PBS on a coverslip #1.5 (170µm thick) on a slide or 35mm Petri dish

  • Microscope is Nikon Ti-E with 100x/1.49O objective

  • Camera is EM-CCD Andor Ultra 2

  • Acquisition is driven by home-made Labview program

  • Analysis is done in a row by a home-made Matlab algorithm with dedicated User-friendly interface 

ApplicationsRed: Extrcellular IntegrinGreen: Intracellular integrinMagenta: Actin

  • Extra-cellular matrix, cell adhesion

  • Endocytosis, Exocytosis

  • Cell traficking

  • Live imaging compatible (running project)

Publications