Fluorescence imaging is limited to superficial structures in biological samples because the signal is severely degraded by optical aberrations in deeper planes. These aberrations are caused by the different refractive index materials within a cell and the optics themselves. Adaptive optics has been applied to various scanning microscopies to correct for these aberrations, but its application to widefield microscopy has been more limited. In super-resolution imaging based on single-molecule detection and localisation, aberrations decrease the resolution achievable by reducing the accuracy and precision when localising single molecules, diminishing the signal-to-noise ratio of the raw data and increasing the proportion of false-positive molecule identifications. In the first part of my talk, I will discuss the extent to which aberrations matter to single molecule techniques and strategies for correcting them.

A number of methods exist to provide axial resolution in SMLM, including biplane imaging and astigmatic and double-helical point spread functions. A common feature of these methods is that their application is limited to a small axial range (1-2 microns). A promising and inexpensive method, first developed at Heriot Watt University in Edinburgh, for imaging larger axial ranges with single-shot acquisition is multifocal microscopy, in which a curved diffraction grating is used to image multiple focal planes simultaneously. It is equally applicable to single particle tracking and flow studies.


Wednesday, May 16, 2016, 12:00. Seminar Room

Hosted by Prof. María García-Parajo