Confocal microscope with FCS
Van Noort group
Home built confocal microscope, fitted for fluorescence correlation spectroscopy
Measurement of diffusion rate of fluorescent particles in solution
- 60 water-immersion microscope objective (NA ¼ 1.2, Olympus, Zoeterwoude, The Netherlands)
- excitation sources: 515-nm diode-pumped, solid-state laser (Cobolt, Solna, Sweden) and 636-nm diode laser (Power Technology, Little Rock, AR)
- Lasers are alternated at 20 kHz by analog modulation,either directly (636 nm) or with an AOM (515 nm; Isomet, Springfield, VA).
- Beams are spatially filtered with a single-mode fiber.
- Collected fluorescence is spatially filtered with a 50-mm pinhole in the image plane, and split into a donor and an acceptor channel by a dichroic mirror (640dcxr, Chroma, Rockingham, VT).
- The fluorescence iss filtered with emission filters (hq570/100m for the donor channel, hq700/75m for the acceptor channel; Chroma) to minimize crosstalk, and was imaged on the active area of single photon avalanche photodiodes (model No. SPCM AQR-14; Perkin-Elmer, Waltham, MA).
Photodiodes are read out with a TimeHarp 200-photon counting board (Picoquant, Berlin, Germany).