A Programmable Light Engine For Quantitative TIRF And HILO Imaging

• Published in: Biophysical journal 2009-02, Vol.96 (3), p.17a-18a
• Main Authors: van't Hoff, Marcel, de Sars, Vincent, Oheim, Martin
• Format: Article
• Language: English
• Subjects: Charge coupled devices; Correlation; Dynamical systems; Fluorescence; Illumination; Imaging; Ligands; Mathematical models
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2008.12.992

We report on a simple yet powerful implementation of objective type total internal reflection fluorescence (TIRF) and highly inclined laminated optical sheet (HILO) illumination microscopy. Both rely on an asymmetric oblique illumination to confine spatially fluorescence excitation to a dielectric boundary or a thin sheet of light, respectively. Undesired side effects are intensity variations across the field of view, due to scattering and interference. Utilizing an acousto-optical deflector (AOD) based scanner, we restore the symmetry of illumination by generating a hollow cone of light, impinging at the sample at an angle adjustable within mu s. Parameters relevant for quantitative image analysis are measured on-line, during fluorescence image acquisition by capturing an objective back-focal plane image on a second small CCD detector. Our device affords background-free variable-angle TIRF/HILO even for the shortest practical exposure times. We expect it to be useful for evanescent-wave imaging involving multiple excitation wavelengths, multiple penetration depths (Variable-angle TIRF) and experiments using objectives with different magnification (and hence different back pupils). We illustrate the system performance by demonstrating ultra-low background TIRF imaging of 200 Hz Qdot blinking, vinculin-EGFP labeled cellular adhesion sites and lysosomal dynamics in cortical astrocytes. [MathML equation] relative to [MathML equation]. To understand the role of site-site interactions on mu super(ex) sub(flux,X), we decompose P sub(X)([var epsilon]) into n-dependent distributions, where n is the number of ion-coordinating ligands within a distance lambda from the ion. For lambda comparable to typical ion-oxygen bond distances, investigations building on this multistate model reveal an inverse correlation between favorable ion-site and site-site interactions: the ion-coordination states that most influence the thermodynamics of the ion are also those for which the binding site is energetically less strained and vice versa. This correlation motivates understanding entropic effects in ion binding to the site and leads to the finding that mu super(ex) sub(flux,X) is directly proportional to the average site-site interaction energy, a quantity that is sensitive to the chemical type of the ligand coordinating the ion. Increasing the coordination number around Na super(+) only partially accounts for the observed magnitude of selectivity; acknowledging the chemical typ