Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

Abstract

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…The SW-FCCS instrumentation was described in Pan et al (39). Briefly, the setup consists of an FV300 confocal microscope (Olympus, Melville, NY) equipped with two avalanche photodiodes (SPCM-AQR-14; PerkinElmer, Waltham, MA) as light detectors for FCS measurements.…”

Molecular Diffusion Measurement in Lipid Bilayers over Wide Concentration Ranges: A Comparative Study

“…[26] The excitation beam of a 543-nm argon laser (25 mW, Melles Griot, Singapore) was reflected by a dichroic mirror (560DCLP, Omega, VT) and a scanning mirror, and focused by a water-immersion objective (60 , NA 1.2, Olympus) into the fluorescent sample. The emitted light after the confocal pinhole was focused by a lens (Achromats f = 60 mm, Linos, Goettingen, Germany), and separated from the excitation light by an emission filter (595AF60, Omega, VT).…”

Probing events with single molecule sensitivity in zebrafish and Drosophila embryos by fluorescence correlation spectroscopy

“…FCS measurements were performed on a modified commercial confocal laser scanning microscope (FV300, Olympus, Singapore) as described previously (Pan et al, 2007a). Briefly, the laser beam (30 lW, 488 nm, Melles Griot, Singapore) is reflected by a long-pass excitation dichroic mirror (488/543/633, Olympus, Singapore) into the scanning unit (G120DT, GSI Lumonics).…”

“…A custombuilt slider then directs the light to either the FV300 photomultipliers for imaging, or to the avalanche photodiode (SPCM-AQR-14, Berkshire, UK) for FCS analysis. The single pinhole for both imaging and spectroscopy guarantees the accurate positioning of the FCS observation volume after confocal image acquisition (Pan et al, 2007a). In the FCS part, the fluorescence light is focused by a lens (f ¼ 60 mm, Achromats, Linos, Goettingen, Germany) and pass through a 510AF23 emission filter to the photodiode.…”

“…FCS analyzes fluorescence signals from a very small observation volume ($10 À15 L) and determines local concentrations and diffusion coefficients of fluorescently labeled molecules. The combination of imaging techniques with FCS thus allows quantitative analysis of protein dynamics within subcellular compartments (Terry et al, 1995;Pan et al, 2007a). Up to now, FCS application in living animals is still limited due to the thick tissue induced light scattering.…”