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The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy.

Anand Pratap Singh1, Jan Wolfgang Krieger, Jan Buchholz

  • 1Department of Biological Sciences and NUS Centre for Bio-Imaging Sciences, National University of Singapore, 14 Science Drive 4, 117557, Singapore.

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|April 11, 2013
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Summary

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) enables 3D imaging of molecular dynamics. This study compares detector technologies, finding SPAD arrays and sCMOS cameras offer superior temporal resolution for enhanced SPIM-FCS applications.

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Area of Science:

  • Biophysics
  • Microscopy
  • Spectroscopy

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is crucial for analyzing molecular dynamics in biological samples.
  • Traditional confocal FCS is limited in spatial coverage and speed.
  • Single Plane Illumination Microscopy (SPIM) offers optical sectioning and reduced phototoxicity, making it suitable for 3D imaging.

Purpose of the Study:

  • To evaluate and compare the performance of different low-light array detectors for Single Plane Illumination Microscopy based Fluorescence Correlation Spectroscopy (SPIM-FCS).
  • To determine how detector characteristics influence the effective FCS observation volume and the accuracy of measurements.
  • To assess the suitability of SPIM-FCS for quantitative analysis of molecular diffusion, transport, and concentration in 3D samples.

Main Methods:

  • Implementation of SPIM-FCS using array detectors: Single-Photon Avalanche Diode (SPAD) arrays, Electron Multiplying Charge Coupled Device (EMCCD) cameras, and scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras.
  • Characterization of detector properties including quantum efficiency, noise level, read-out speed, and effective pixel area.
  • Validation of SPIM-FCS results through parallel measurements using confocal FCS, Single Particle Tracking (SPT), and determination of concentration gradients.

Main Results:

  • SPIM-FCS, particularly with light sheet illumination, accurately determines absolute diffusion coefficients in 3D samples.
  • SPAD arrays and sCMOS cameras provide significantly higher temporal resolution (down to 10 μs) compared to EMCCD cameras (millisecond range).
  • Detector choice critically impacts the effective FCS observation volume and the overall performance of SPIM-FCS.

Conclusions:

  • SPIM-FCS is a powerful imaging technique for quantitative analysis of molecular dynamics in 3D.
  • SPAD arrays and sCMOS cameras are superior choices for high-speed SPIM-FCS applications requiring precise temporal resolution.
  • The study provides a framework for selecting optimal detectors for SPIM-FCS based on specific experimental needs.