Localization of single biological molecules out of the focal plane

L. Gardini; M. Capitanio; F. S. Pavone

doi:10.1117/12.2039369

4 March 2014 Localization of single biological molecules out of the focal plane

L. Gardini, M. Capitanio, F. S. Pavone

Proceedings Volume 8950, Single Molecule Spectroscopy and Superresolution Imaging VII; 895019 (2014) https://doi.org/10.1117/12.2039369
Event: SPIE BiOS, 2014, San Francisco, California, United States

Abstract

Since the behaviour of proteins and biological molecules is tightly related to the cell’s environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.

Citation Download Citation

L. Gardini, M. Capitanio, and F. S. Pavone "Localization of single biological molecules out of the focal plane", Proc. SPIE 8950, Single Molecule Spectroscopy and Superresolution Imaging VII, 895019 (4 March 2014); https://doi.org/10.1117/12.2039369

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