Homebuilt smFRET setup

Homebuilt dedicated 483/635-nm alternating excitation, dual-color dual-polarization detection confocal microscope. This system allows imaging more than 20 parameters per single molecule, making it the ideal system for subnanometer accurate single-molecule FRET studies.

A schematic representation of the light path, including lasers, optics and detectors for the pulsed interleaved exciation confocal burst microscope. A schematic representation of the light path, including lasers, optics and detectors for the pulsed interleaved exciation confocal burst microscope.

Homebuilt dedicated 483/635-nm alternating excitation, dual-color dual-polarization detection confocal microscope. This system allows imaging more than 20 parameters per single molecule, making it the ideal system for subnanometer accurate single-molecule FRET studies.

Schematic overview of the single molecule sensitive confocal microscope with pulsed interleaved excitation and polarization dependent detection.

Microscope

  • Olympus body
  • UPLSAPO 60xW NA1.2 objective lens

Excitation

  • 483/635-nm picosecond pulsed lasers
  • underfilled excitation (large PSF for single molecule imaging)
  • PIE
  • linear polarization

Detection

  • 75-um pinhole
  • blue side: two Laser components COUNT BLUE detectors
  • red side: two Perkin Elmer APDs

Applications

  • FCS
  • single-molecule FRET
  • single molecule fluorescence

Acknowledgements

Thank you for acknowledging our imaging facility. You can use the statement below in all your publications that include data acquired on the homebuilt PIE burst microscope:

We acknowledge the Advanced Optical Microscopy Centre at Hasselt University for support with the microscopy experiments. Microscopy was made possible by the Research Foundation Flanders (FWO, projects G0B4915, G0B9922N and G0H3716N).

Contact

Sam Duwé - AOMC

Location
Agoralaan, Building C, 3590 Diepenbeek
Function
Microscopy expert