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Spectrally-resolved single-molecule fluorescence microscopy

The Lee lab developed spectrally-resolved PAINT (points accumulation for imaging in nanoscale topography) or sPAINT, which simultaneously records the spatial position and emission spectrum of single dye molecules to super-resolve an image. For example, the fluorescence emission wavelength of the phenoxazone-based dye nile red (NR) is known to be sensitive to the hydrophobicity of its environment, and we have exploited this with sPAINT to super-resolve biological structures in the hydrophobicity domain. sPAINT is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

Principles of the spectral-PAINT technique. a) Optical set-up for sPAINT showing the use of a diffraction grating to obtain spectral information and b) a typical image of nile red data. c) Coparison of fluorescent spectrum recorded by sPAINT and a fluorimeter. d) Representative sPAINT hydrophobicity images of oligomers and fibrials using the dye nile red (scale bar is 100 nm). e) Locialisation precision and f) spectral precision as a function of detected photons per molecule. 

Related Publications 

Multi-dimensional super-resolution imaging enables surface hydrophobicity mapping.
MN Bongiovanni, J Godet, MH Horrocks, L Tosatto, AR Carr, DC Wirthensohn, RT Ranasinghe, JV Fritz, CM Dobson, D Klenerman, SF Lee – Nat Commun (2016) 7, 13544
Spectrally-Resolved Photodynamics Of Individual Emitters In Large-Area Monolayers Of Hexagonal-Boron Nitride.
HL Stern, R Wang, Y Fan, R Mizuta, JC Stewart, L-M Needham, TD Roberts, R Wai, NS Ginsberg, D Klenerman, S Hofmann, SF Lee – ACS nano (2019) 13, 4538
Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale.
J-E Lee, JC Sang, M Rodrigues, AR Carr, MH Horrocks, S De, MN Bongiovanni, P Flagmeier, CM Dobson, DJ Wales, SF Lee, D Klenerman – Nano Letters (2018) 18, 7494