Single-molecule Fluorescence Microscopy

The great advantage of single-molecule approach in structural biology is in detection of true statistical distributions (as opposed to an average value) and also in the absence of requirement of synchronization of action of individual processes.

Using fluorescence of individual molecules, we can extract plethora of useful information from one measurement, e.g. cellular localization, distance along one or more reaction coordinates (FRET), hydrated radius, translational diffusion coefficient (FCS) and rotational diffusion coefficient (time-resolved anisotropy).

To observe individual molecules, we have to measure extremely diluted samples with high spatio-temporal resolution. Molecules can be either immobilized, and then we can observe their fate for prolonged time (from milliseconds to several minutes) or freely diffusing in aqueous buffer or in the most relevant environment in living cell. Main advantage of analysis on freely diffusing molecules is that we can collect thousands of events within several minutes of observation and thus statistically describe behaviour of distinct species (including rare species) of observed population.

Our single-molecule set-ups are homemade, and to assure their state-of-the-art status, they are constantly under development.

Currently we have following detection capabilities on freely diffusing samples: PAX FRET [1], RASP [2], lifetimes, F(C)CS, time-resolved anisotropy.  Soon, we want to expand this list for four-colour PIE [3].

We can detect fluorescence from immobilized molecules using TIRF excitation scheme, this offers information about localization and FRET.

Simultaneous operation of AFM with all abovementioned advanced optical techniques (FRET, TIRF, FLIM, FCS…) and with optical super-resolution microscopy (STORM/PALM/SRRF [4]) should be possible later during 2018 and 2019.

[1] Eur Biophys J. 2007 Jul; 36(6): 669–674. Published online 2007 Feb 6. doi:  10.1007/s00249-007-0133-7
Phys Chem Chem Phys. 2011 Feb 7; 13(5): 1857–1871.Published online 2011 Jan 7. doi:  10.1039/c0cp01911a
Proc Natl Acad Sci U S A. 2004 Jun 15; 101(24):8936-41. 2004 Jun 15;101(24):8936-41. Epub 2004 Jun 2.
. 2016; 7: 12471. Published online 2016 Aug 12. doi:  10.1038/ncomms12471