Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy
(Conference Presentation)

Marcelina Cardoso Dos Santos; Cyrille Vézy; Rodolphe Jaffiol

doi:10.1117/12.2208672

27 April 2016 Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

Marcelina Cardoso Dos Santos, Cyrille Vézy, Rodolphe Jaffiol

Author Affiliations +

Proceedings Volume 9714, Single Molecule Spectroscopy and Superresolution Imaging IX; 97140C (2016) https://doi.org/10.1117/12.2208672
Event: SPIE BiOS, 2016, San Francisco, California, United States

Abstract

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

Conference Presentation

Citation Download Citation

Marcelina Cardoso Dos Santos, Cyrille Vézy, and Rodolphe Jaffiol "Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)", Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 97140C (27 April 2016); https://doi.org/10.1117/12.2208672

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
PRESENTATION

WATCH
PRESENTATION SAVE TO MY LIBRARY

GET CITATION

KEYWORDS

Microscopy

Luminescence

Reflection

Absorption

Cell mechanics

Glasses

Mirrors

Show All Keywords

Keywords/Phrases

Search In:

Publication Years