Alba FLIM is a computer-controlled instrument specifically designed for confocal imaging applications using either single- or multi-photon excitation. The modular design of the instrument allows it to be easily upgraded to acquire Fluorescence Correlation Spectroscopy (FCS) data.

Besides its most attractive features such as high sensitivity and fast acquisition speed, Alba FLIMT comes with Vista, a powerful and user-friendly data acquisition and analysis software package.

Key Features of Alba FLIM:

  • Acquisition with up to four channels
  • Separate pinholes for each channel for higher resolution
  • Computer-controlled alignment of the confocal pinhole and optics
  • Choice between scanning mirrors or piezo-controlled XYZ stage
  • Single- and multi-photon excitation
  • Powered by Vista, a user-friendly software package for the acquisition of FLIM, FRET, RICS, and FCS
  • Alba FLIM can be combined with Alba FCS to a powerful, fully-equipped Alba - Confocal Spectroscopy and Imaging Workstation

Excitation Modality
One-photon (using an argon ion, krypton ion, or dye-laser) or multi-photon measurements (using a Ti:Sapphire laser) are possible with Alba FLIM

Microscopes
Alba FLIM can be interfaced with either a Nikon or an Olympus epifluorescence microscope.

Detectors
Standard light detectors are cooled photomultiplier tubes.

Upgradeable to Alba FCST
Alba FLIM can be upgraded to acquire FCS data. All in a single instrument.

Specifications for Alba FLIM

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 FLIM

Measurement Examples from Alba FLIM

VistaVision for Alba

VistaVision enables the user to select the size, resolution and type (steady-state/lifetime) of image to be acquired. Residence time at each pixel is selectable. Images of the same X/Y region can be acquired at different z-axis positions. The user can also select a region within the original image and start a new acquisition of the zoomed area. Upon acquisition, the image can be processed using the filters included in VistaVision. Images can be saved and exported in several formats (bmp, gif, jpeg, tiff, png).

Image Acquisition

In this example, 1 nM Rhodamine110 solution was prepared in pH=8 buffer. The solution was placed in a well. After the buffer dried, the crystallized structure was measured with 60X magnification and a N.A. = 0.7 (air objective). The excitation wavelength was 488 nm with 1 W laser power; the emission was collected through a 50 µm pinhole.

Selected publications featuring Alba FLIM

Below is a list of selected publications featuring Alba FLIM 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 Alba FLIM

The following accessories are available for Alba FLIM. 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