Multicolour single molecule imaging in cells with near infra-red dyes.

PloS One
Christopher J TynanStephen E D Webb

Abstract

The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging. A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the opt...Continue Reading

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Citations

Oct 15, 2014·Journal of the American Chemical Society·Ashley R LongstreetD Tyler McQuade
Dec 4, 2012·International Journal of Molecular Sciences·Marisa L Martin-Fernandez, David T Clarke
Jul 4, 2015·Journal of Fluorescence·Abhinav B TatheNagaiyan Sekar
Dec 12, 2012·Journal of Molecular and Cellular Cardiology·Tobias KohlStephan E Lehnart
Sep 15, 2016·Nucleic Acids Research·Anne PlochowietzAchillefs N Kapanidis
Feb 11, 2021·Analytical Chemistry·Cynthia L SchreiberBradley D Smith
Jan 23, 2020·Journal of the American Chemical Society·Suresh SarkarAndrew M Smith

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Methods Mentioned

BETA
fluorescence microscopy
Fluorescence
FRET

Software Mentioned

Origin
STORM

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