Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

Eli Slenders; Marco Castello; Mauro Buttafava; Federica Villa; Alberto Tosi; Luca Lanzanò; Sami Valtteri Koho; Giuseppe Vicidomini

doi:10.1038/s41377-021-00475-z

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Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

Articles

- Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector
- Light: Science & Applications Vol. 10, Issue 2, Pages: 289-300(2021)
- Affiliations：
  
  1.Molecular Microscopy and Spectroscopy, Istituto Italiano di Tecnologia, Genoa, Italy
  2.Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Milan, Italy
  3.Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy
  4.Dipartimento di Fisica e Astronomia, Università di Catania, Catania, Italy
- Author bio：
  
  Giuseppe Vicidomini (giuseppe.vicidomini@iit.it)
- Funds：
- DOI：10.1038/s41377-021-00475-z
  CLC：
- Published：2021，
  
  Published Online：05 February 2021，
  
  Received：23 July 2020，
  
  Revised：17 December 2020，
  
  Accepted：14 January 2021
- Accepted：
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Slenders, E. et al. Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector. Light: Science & Applications, 10, 289-300 (2021).
DOI：

Slenders, E. et al. Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector. Light: Science & Applications, 10, 289-300 (2021). DOI： 10.1038/s41377-021-00475-z.

摘要

Abstract

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast

sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques

as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However

the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques

such as fluorescence lifetime imaging. Here

we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy

pair-correlation function analysis

and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

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references

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