LSCM-FCS Combination Sets

The systems are based on a commercial Laser Scanning Confocal Microscope (FV300, Olympus) which is combined with home-built FCS detection units. For the FV300, different optical laser lines between 488 and 640 nm are available. They are coupled into the scanning unit (square box) after passing through an optical fiber, and reflected by an excitation dichroic mirror. A pair of galvanometer scanning mirrors (G120DT, GSI Lumonics) scans the laser beams and directs them into a water immersion objective (60x, NA1.2, Olympus) with the help of a reflective prism. The laser beam is then focused into a small focal volume in the specimen. The fluorescence emitted from the sample is collected by the same objective, is de-scanned, and focused again by a collecting lens into a confocal aperture, which is a rotatable turret of different sizes of pinholes. In the scanning box, a glass slab is used for fluorescence light xy position realignment. An FCS detection unit is mounted on the top of the scanning unit by modifying its cover (Pan et al. Rev. Sci. Instr. 78, 053711 (2007)). The fluorescence light after the confocal pinhole is imaged by a lens (Achromat f = 60 mm, Linos), through a dichroic mirror/beam splitter and emission filters, onto small active areas of avalanche photodiodes (APD) in single photon counting modules (SPCM-AQR-14, Pacer Components). The signals from the detectors are processed online by a hardware correlator (correlator.com) to obtain auto- and/or cross-correlation functions. In addition, all systems possess a PCI6602 card from National Instruments allowing direct read-out of photon arrival time data from the APDs. Curve fitting is performed by a self-written program in Igor Pro (WaveMetrics).

Below is depicted a scheme of a typical setup which shows the configuration for Dual-Color Fluorescence Cross-Correlation Spectroscopy (DC-FCCS). For FCS and SW-FCCS only one laser is used for excitation.

Dual-Color Fluorescence Cross-Correlation Spectroscopy

Confocal set up 1

Time resolution

1.5625 ns

Lasers

488 nm, 543 nm, 633 nm

Number of Detectors

2

Measurement type

FCS/rotational FCS, SW-FCCS, PCH

 

Confocal Set up 1

Confocal set up 2

Confocal Set up 2

This system in built under ‘PicoQuant LSM upgrade kit’ where an Olympus (IX83) CLSM microscope is combined with PicoQuant lasers, Olympus lasers and PicoQuant detector module.

 

Correlator

Software

Lasers

405 nm, 485 nm, 640 nm (pulsed and CW)

Number of Detectors

2 SPAD (for spectroscopy) and 3 PMT (for imaging)

Measurement type

Single spot FCS, FCCS, PIE-FCCS, FLCS, sm-FRET, fluorescence lifetime, PCH

 

Fluorescence lifetime measurements can be done in this machine which is equipped with pulsed excitation sources (405 nm, 485 nm and 640 nm; pulse width < 100 ps and maximum repetition rate 80 MHz) and a sophisticated time correlated single photon counting (TCSPC) card (TimeHarp 260, PicoQuant). The raw data is saved as the ‘Time Tagged Time Resolved’ (TTTR) format in which each recorded photon is tagged with two characteristic times: i) macrotime, i.e., the time relative to the start of the experiment, and ii) microtime, i.e., time relative to the last excitation pulse. The macrotime and microtime are saved with 50 ns and 25 ps time resolution respectively. The distribution of the microtime of all detected photons (photon arrival time distribution) provides the information of average fluorescence lifetime. Fluorescence lifetime is utilized to measure the time-resolved Förster Resonance Energy Transfer (trFRET) between donor-acceptor pair. In addition, one can measure intensity-based single molecule FRET (smFRET) by comparing the fluorescence intensity at donor and acceptor channels.    

More Information on Microscopy Principles

http://micro.magnet.fsu.edu/primer/ (Comprehensive introduction to optical microscopy with many applets and links)
http://www.fcsxpert.com/classroom/ (Site dedicated to FCS with some tutorials)
http://www.olympusconfocal.com/theory/LSCMIntro.pdf (Article about the basics of confocal microscopy)

  • NUS
  • Biophysical Fluorescence Laboratory