Alba FCS is a dual-channel spectrometer that combines a confocal scanning microscope with fluorescence correlation spectroscopy (FCS), and is optimized for both single-photon and multi-photon excitation.

Measurement Parameters when using auto-correlation and cross-correlation functions:

  • Diffusion coefficient
  • Diffusion time
  • Concentration
  • Triplet state decay time constant
  • Triplet function
  • Flow rate
  • Size of excitation volume
  • Number of molecules

When using Photon Counting Histogram (PCH):

  • Number of molecules
  • Molecular brightness

Acquisition Modes
Alba acquires data in time-mode (the classical way: photons acquired in time intervals are counted) or in photon-mode (time delay between one photon and the next one is measured and an histogram is built). A dual-channel data acquisition card receives the data stream from the detectors and passes them on to the computer.

Raw Data Stream
Raw data are stored in a data file for further processing. No need anymore to reject an autocorrelation function because of large particle interference. Raw data can be purged from large particles presence and the autocorrelation function can be recalculated.

Excitation Modality
Single photon excitation (using an argon ion, krypton ion, or dye-laser); or multi-photon excitation (using a Ti:Sapphire laser).

Microscopes
Alba can be interfaced with Leica, Nikon, Olympus or Zeiss epifluorescence microscopes.

Detectors
Light detectors are cooled photomultiplier tubes or avalanche photodiodes. The system is provided for single channel or dual channel data acquisition for cross-correlation measurements. Alba FCS can be combined with Alba FLIM to a powerful, fully-equipped Alba - Confocal Spectroscopy and Imaging Workstation.

Specifications for Alba FCS

Instrument Features • Computer-controlled selection of the confocal pinhole size
• Computer-controlled alignment of the confocal pinhole and optics
• Computer-controlled positioning of the objective (z-axis)
• Computer-controlled XYZ microscope stage
• Single-photon or multi-photon excitation
• One channel or dual-channel data acquisition
• Avalanche photodiodes or cooled photomultiplier tubes detectors
Measurements

• Fluorescence Correlation Spectroscopy (single- and cross-correlation)
• Photon Counting Histogram (PCH)
• Phase- and modulation-resolved spectra (up to 3 components in a mixture)
• Polarization measurements
• Particle tracking
• Scanning FCS

Parameters That Can Be Derived Using VistaVision Software Autocorrelation and Cross-Correlation Functions
Vista resolves 1 or 2 species using:
• Diffusion coefficient
• Diffusion time
• Sample concentration
• Triplet state decay time constant
• Triplet function
• Flow rate
• Size of excitation volume
• Number of molecules

Photon Counting Histogram (PCH)
Vista resolves 1 or 2 species using:
• Number of molecules
• Molecular brightness
Light Sources • Single photon lasers housed in a laser launcher with computer-control of beam expander, laser intensity and shutter; or,
• Multi-photon excitation with computer-control of beam expander, laser intensity and shutter
Optics • An adapted Nikon microscope with lens revolver for objectives, bottom and side ports and computer-controlled XY-stage for sample slides and for 384-well plates
• Available for Leica, Olympus and Zeiss inverted and upright microscopes
Objectives • Air objectives with 20X, 40X, 60X magnification and 1.5-8.1 working distances
• Oil immersion objectives, 1.4 NA and 60X (standard); other aperture available • Water immersion objectives,1.2 NA 60X (standard), with coverslip correction (for 0.15-0.18 coverslip); other apertures available
Dichroic Filters •For single-photon excitation: 1-, 2-, 3-band filters
• For multi-photon excitation
Polarizer Cube beam splitter, wavelength range: 450-1100 nm; extinction ratio: 10,000:1 at +/- 3degrees
Stages • Large distance movement (100x100x10 mm)
• Stepper motor-controlled XYZ stage
• Micro-distance movements
• XYZ piezo-controlled stage, 100x100x50 µm with 5 nm step resolution.
Sample Holders • Microwell plates
• Petri dishes
• Coverslip
Light Detectors • Both detectors feature pre-amplifier discriminators with TTL output avalanche photodiodes (APDs)
• Cooled photomultiplier tubes
Data Acquisition Modes • Time mode
• Photon mode
Statistical Functions Utilized for Data Analysis • Autocorrelation function
• Cross-correlation function
• Photon Counting Histogram (PCH)
Single Set and Global Fitting Models for Fitting Autocorrelation and cross-correlation functions
Vista resolves 1 or 2 species using:
• 2D- or 3D-Gaussian PSF
• 3D-Gaussian-Lorentzian PSF
• One-photon excitation
• Two-photon excitation
• Presence of flow
• Input of user-defined equation

Photon Counting Histogram (PCH)
Vista resolves 1 or 2 species using:
• 2D- or 3D uniform
• 2D- or 3D-Gaussian PSF
• 3D-Gaussian-Lorentzian PSF
• One-photon excitation
• Two-photon excitation
Software VistaVision for Alba
OS Requirements Windows XP operating system
Power Requirements Universal power input: 110-240 V, 50/60 Hz, 400 VAC
Dimensions 885 mm (L) x 600 mm (W) x 330 mm (H)
Weight 40 Kg

Schematic Diagram of Alba FCS

Measurement Examples from Alba FCS

Rhodamine 110 in Water

Auto-correlation curves and 3D-Gaussian fits for a concentration series of 1.25, 2.5, 5.0, 10 and 20 nM Rhodamine 110 in water.

Autocorrelation Function and the Photon Counting Histogram (PCH)

Alba FCS provides two fundamental and complementary features to obtain information about the molecular dynamics of the sample under observation: the autocorrelation function and the photon counting histogram (PCH). In the measurement on the left, solutions of different concentrations of Coumarin and Rhodamine110 were prepared and exposed to 2-photon excitation at 780-nm. A global PCH analysis was then performed on the data. From this data, the following parameters can be determined: brightness and number of molecules in the excitation volume.

Selected publications featuring Alba FCS

Below is a list of selected publications featuring Alba FCS from ISS. Click here to browse our complete library of publications.

Getting High on Single Molecule Biophysics
Block, S.M.
Current Pharmaceutical Biotechnology, 2009, 10(5), 464-466
Fluorescence Fluctuation Spectroscopy: Ushering in a New Age of Enlightenment for Cellular Dynamics
Jameson, D.M., Ross, J.A., Albanesi, J.P.
Biophys. Rev., 2009.
Fluorescence Correlation Spectroscopy Assay for Gliadin in Food
Varriale, A., Rossi, M., Staiano, M., Terpetschnig, E., Barbieri, B., Rossi, M., D'Auria, S.
Anal Chem. 2007, 79(12), 4687-4689.
Application of Fluorescence Correlation Spectroscopy to Hapten-Antibody Binding
Hazlett, T.L., Ruan, Q., Tetin, S.Y.
Methods in Molecular Biology, 2005, 305, 415-438.
How Confined Lubricants Diffuse During Shear
Mukhopadyay, A., Bae, S.C., Zhao, J., Granick, S.
Physical Review Letters, 2004, 93, 236105.
How to Stabilize Phospholipid Liposomes (Using Nanoparticles)
Zhang, L., Granick, S.
Nano Lett., 2006(6), 4, 694-698.
Fluorescence Correlation Spectroscopy and Photon Counting Histogram on Membrane Proteins: Functional Dynamics of the Glycosylphosphatidylinositol-Anchored Urokinase Plasminogen Activator Receptor
Malengo, G., Andolfo, A., Sidenius, N., Gratton, E., Zamai, M., Caiolfa, V.R.
J. Biomed. Opt., 2008, 13(3), 031215.
Reducing Background Contributions in Fluorescence Fluctuation Time-Traces for Single-Molecule Measurements in Solution
Földes-Papp, Z., Liao, S.-C.J., You, T., Barbieri, B.
Current Pharmaceutical Biotechnology, 2009, 10, 532-542.
Endothelial Adhesion Receptors Are Recruited to Adherent Leukocytes by Inclusion in Preformed Tetraspanin Nanoplatforms
Barreiro, O., Zamai, M., Yáńez-Mó, M., Tejera, E., López-Romero, P., Monk, P.N., Gratton, E., Caiolfa, V.R., Sánchez-Madrid, F.
J. Cell Biol., 2008, 183(3), 527-542.

Accessories available for FCS

The following accessories are available for Alba FCS. For more information please visit our Microsopy Accessories page.

  • XYZ Piezo-controlled Stage
  • Galvo-controlled Scanning Mirrors
  • Coverslip on XYZ Piezo-controlled Stage
  • 8-well on XYZ Piezo-controlled Stage
  • 35 mm Diameter Petri Dish on XYZ Piezo-controlled Stage
  • 96-wellplate on XY Stepper-motor Stage
  • Polarizer and Beam Splitter
  • Filters