Alba v5Time-resolved Confocal Microscopewith Single-Molecule Sensitivity

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Overview of Alba v5

Alba v5 is a laser scanning microscope that incorporates several measurement modalities for experimental quantitative biology and material science applications requiring single molecule detection sensitivity. Capable of acquisition from the violet to the near infrared region, it features two independent laser entry ports. Alba v5 is powered by VistaVision, the comprehensive software package for instrument control, image acquisition and processing.

Key Features of Alba v5

FLIM and FRET in live cells

FLIM FRET in Live Cells

Tissue Imaging

Tissue Imaging

Single Molecule Studies

Single Molecule Studies & Detection of Single Emitters by Antibunching

Multiplex Imaging by Time Resolved Unmixing

Multiplex Imaging by Time Resolved Unmixing Using the Phasor Plot

Multi-modality Imaging

Multi-modality Imaging

Multimodality imaging (Intensity, lifetime and SHG)

Rhizome cross-section of Convallaria (Lily of the Valley). Images were taken simultaneously using an Alba equipped with a multiphoton laser emitting at 800 nm. The standard fluorescence intensity image (first), the fluorescence lifetime image (second) and the second-harmonic generation (SHG) image (third). All images are 256 × 256 pixels, 40 × 40 µm2.

(courtesy of Dr. Zhang, Beckman Institute, Urbana, IL)

Tissue Imaging

Autofluorescence tissue image

Autofluorescence tissue image: Excitation: 375 nm, Emission: 425 nm, Objective: 20X NA0.75

(courtesy of Dr. Aneesh Alex, GSK)

FLIM-FRET in live cells

FRET efficiencies in live cells

FLIM-FRET maps the FRET efficiencies of Cebp/α proteins co-expressing Cerulean and Venus in live cells to localize their dimerization in the cell nuclei (donor-only control: cells expressing Cebp/α-Cerulean only; FRET sample: cells co-expressing Cebp/α-Cerulean and Cebp/α-Venus).

FRET efficiencies in live cells
FRET Efficiency Map overlaid with the Intensity Image (Nature Protocols Vol. 6: 1324-1340, 2011)

Multiplex imaging by Time-Resolved Unmixing using the Phasor Plot

Multiplex image of labeled mitochondria and microtubule in the same cells

Multiplex imaging by time-resolved fluorescence requires using one excitation wavelength, and data is acquired on one detection channel. The figure shows the multiplexing of two labels, mitochondria labeled with Alexa Fluor 647 (AF647) and microtubule labeled with Atto 647 (Atto647) in the same cells. The raw image is acquired with excitation at 640 nm and detection in the same emission channel. Using the analysis routine in the phasor plot, the two structures are separated.

HomoFRET detection by polarization imaging

Venus expressed in a HeLa cell with anisotropy measurements of monomer and hexamer structures.

Venus was expressed in a HeLa cell. Excitation is 514 nm and emission is through a band-pass filter 545/35 nm. A beam-splitter polarizer in the emission splits the fluorescence beam into a perpendicular and parallel orientation beams, with each beam detected by one acquisition channel. The anisotropy image is calculated at each pixel. For the monomer (upper right) the average value of the anisotropy is around 0.2 indicating a prevalent orientation along the parallel axis; for the hexamer is less than 0.1 due to the local homo FRET between different Venus fluorescent proteins on the hexamer structure.

Single-molecule studies: Detection of single emitters by Antibunching

Antibunching data acquired on solution of Rhodamine 110 in water

Antibunching data acquired on a solution of Rhodamine 110 in water with excitation at 488 nm. Measurements are acquired by splitting the signal in two beams following the classic Hanbury-Brown-Twiss set up and sending each of them to a separate detector of the Alba: the acquisition electronics provides a histogram of the time difference between the arrival time of the photons at the detectors. The histogram displays a dip at the coincidence point; the depth of the dip depends on the number of independent emitters in the observation volume while the shape depends on the excited state lifetime.

Product Specifications for Alba v5

Instrument Features
  • Individual pinholes on each acquisition channel
  • Computer-controlled selection of the pinhole variable aperture
  • Computer-controlled positioning of the pinhole in the imaging plane
  • Single-photon or multi-photon excitation
  • Up to 4 channels data acquisition
  • Auxiliary port for camera
  • Inverted and upright
  • Air objectives with 20X, 40X, 60X magnification and 1.5-8.1 working distances
  • Oil immersion objectives, 1.4 NA and 60X (standard); other apertures available
  • Water immersion objectives,1.2 NA 60X (standard), with coverslip correction (for 0.15-0.18 coverslip); other apertures available
Light Sources
  • Single photon lasers housed in a laser launcher with computer-control of beam expander, laser intensity and shutter;
  • Multi-photon excitation with computer-control of beam expander, laser intensity and shutter
Laser Launcher
  • Models for 3-, 4-, 6-lasers. Light is delivered to the microscope through a single-mode fiber optic.
Galvanometer Scanner
  • 2 silver-coated galvanometer scanning mirrors
  • Clear optical surface: 3 mm
  • Maximum scan rate: 5 KHz for 3 mm
  • Scanning resolution: 64 x 64 to 4096 x 4096 pixels
  • Scanning mode: Pt, Xt, XZ, XY, XZt, XYt, XYZ
  • ROI scanning: rectangle, ellipse, polygon, line
Positioning Controls**
  • ISS 3-axis control unit
  • ISS XY galvo scanning mirrors control unit
  • ISS Z-piezo control unit
  • Microscope built-in focusing control module
  • Automatic XY stages (ASI, Prior)
  • XYZ piezo stages (MadCity, PI)
  • Variable-aperture pinhole; diameter from 20 µm to 1000 µm
  • Cooled GaAsP and GaAs PMT
  • Cooled Hybrid PMTs
  • SPADs
Dichroic Filters
  • For single-photon excitation: 1-, 2-, 3-band filters
  • For multi-photon excitation
  • Cube beam splitter, wavelength range: 450 - 1100 nm; extinction ratio: 10,000:1 at ±3 degrees
Data Acquisition Unit
  • FastFLIM (Digital Frequency Domain FLIM)
  • SWISS TCSPC card (Time Domain FLIM)
  • VistaVision
Computer & Monitor
  • High-performance Processor, 32 GB RAM, Windows 10, 64-bit
  • 32" monitor, 2556 x 1440 resolution
Power Requirements
  • 110 - 240 V, 50/60 Hz, 400 VAC
Dimensions (mm)
  • 885 (L) x 600 (W) x 330 (H)
Weight (kg)
  • 40 (with no microscope)

Measurements for Alba v5

Intensity and Lifetime Imaging
  • 1p or 2p confocal images
  • FLIM in frequency-domain or in TCSPC
  • Phosphorescence Lifetime Imaging (PLIM)
  • Polarization images
Steady-state and Time-resolved Polarization Anisotropy Imaging
Fluorescence Fluctuations Spectroscopy (FFS)
  • Fluorescence Correlation Spectroscopy (FCS)
  • Fluorescence Cross-Correlation Spectroscopy (FCCS)
  • Photon Counting Histogram (PCH)
  • Fluorescence Lifetime Correlation Spectroscopy (FLCS)
  • Scanning FCS
  • Number & Brightness (N&B)
  • Raster Imaging Correlation Spectroscopy (RICS)
Particle tracking (optional)
  • 3D reconstruction of molecule trajectory
Super resolution (optional)
  • Nanoimaging reconstruction with 20 nm resolution
Single Molecule FRET Bursts Analysis
  • Burst Analysis
  • FRET and Correlation methods
  • PIE-FRET methods
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Product Accessories for Alba v5

Additional Product Options

  • Perfusion system stabilizes conditions
    Perfusion system

    A peristaltic pump supplies the stage with a solution for keeping the sample conditions (temperature, pH, etc...) stable.

  • Irrigation system prevents drying up samples
    Irrigation system

    When using water objectives for prolonged measurements, it prevents the liquid drying up.

  • Maintain focus automatically
    Auto Focus maintaining

    It keeps the focus position of the objective for hours using an active feedback to counter drifts.

  • Maintain environmental conditions for cell cultures
    Sample temperature control

    Stage top incubator or a full enclosure to maintain the environmental conditions of cell cultures.

  • Epifluorescence lamp
    Epifluorescence lamp

    Visualize your sample with the Epi module. Select as light source either an arc lamp or an LED and the suitable filter cubes to add to the microscope cassette.

  • Connect to an atomic force microscope
    Atomic Force Microscope (AFM)

    Fully integrated for the models:
    NanoWizard by JPK-Bruker
    Resolve by Bruker
    For other models contact ISS.

Product Software for Alba v5

Screenshot showcasing VistaVision


VistaVision is a complete software package for confocal microscopy applications including instrument control, data acquisition and data processing. Easy to use, the software has been developed in modular components that can be activated when a specific instrument configuration is selected. The modules include:

  • FLIM/PLIM Imaging
  • FFS
  • smFRET
  • Particle Tracking
Learn More

Product Resources

Configurations of Alba v5

Alba can be selected in either one of two geometric configurations: the branch configuration where the fluorescence beams for each channel are separated in cascade, and the parallel configuration where the fluorescence beams are separate for the channels 1-2 and 3-4, respectively.

Alba in the Branch ConfigurationAlba in the Parallel Configuration