- Technical Diagrams
- Measurement Examples
The instrument for quantitative cell biology at single-molecule detection.
Alba is a laser scanning microscope that incorporates several measurement modalities for experimental quantitative biology and material sciences applications requiring the single molecule detection sensitivity.
Key Features of Alba-STED for FFS/FLIM:
- Single- and multi-photon excitation on two separate input ports
- Up to 4 acquisition channels
- Output port for 5th channel acquisition or CCD camera
- Separate, computer-controlled aperture, pinholes on each acquisition channel for high precision FFS measurements
- Fast scanning mirrors
- Polarization module
- Computer-control of filterwheels, shutters, pinholes aperture, lasers intensity, channels alignment
- Powered by VistaVision, a user-friendly software package for the acquisition of confocal images, FLIM/ FRET, RICS, and FFS (FCS, PCH, scanning FCS, N&B)
Separate input ports for one-photon excitation (laser diodes and supercontinuum laser) or multi-photon measurements (Ti:Sapphire laser, fiber laser) are available on the Alba-STED for FFS/FLIM.
Alba-STED for FFS/FLIM can be interfaced with most commercial epifluorescence and upright microscopes.
Fast GaAs detectors, hybrid photomultiplier tubes, avalanche photodiodes.
Measurements for Alba-STED for FFS/FLIM
|Single-point Module Measurements||
|Imaging Module Measurements (Single plan and z-stack)||
|FLIM images (digital frequency-domain) (single plane and z-stack)||
|FLIM images time-domain (single plane and z-stack)||
|Single Molecule Module||
Specifications for Alba-STED for FFS/FLIM
|Acquisition and Analysis Software||
Measurement Examples from Alba-STED for FFS/FLIM
|Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells With a Cu(II)-selective Carbonic Anhydrase-based Biosensor.
McCranor, B.J., Szmacinski, H., Zeng, H.H., Stoddard, A.K., Hurst, T., Fierke, C.A., Lakowicz, J.R., Thompson, R.B.
Metallomics., 2014, 6(5), 1034-42.
|Determining Antibody Stoichiometry Using Time-Integrated Fluorescence Cumulant Analysis
Skinner, J.P., Wu, B., Mueller, J.D., Tetin, S.Y.
J. Phys. Chem. B, 2011, 115, 1131-1138.
|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.
|Antibodies in Diagnostic Applications
Tetin, S.Y., Stroupe, S.D.
Current Pharmaceutical Biotechnology, 2004, 5, 9-16.
|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.
|Interleaflet Diffusion Coupling When Polymer Adsorbs onto One Sole Leaflet of a Supported Phospholipid Bilayer
Zhang, L., Granick S.
Macromolecules, 2007, 40, 1366-1368.
|How to Stabilize Phospholipid Liposomes (Using Nanoparticles)
Zhang, L., Granick, S.
Nano Lett., 2006(6), 4, 694-698.
|Slaved Diffusion in Phospholipid Bilayers
Zhang, L., Granick, S.
PNAS, 2005(102), 26, 9118-9121.
|How Confined Lubricants Diffuse During Shear
Mukhopadyay, A., Bae, S.C., Zhao, J., Granick, S.
Physical Review Letters, 2004, 93, 236105.
|Polymer Lateral Diffusion at the Solid-Liquid Interface
Zhao, J., Granick, S.
J. Am. Chem. Soc., 2004, 126, 6242-6243.
|Trapped Brownian Motion in Single- and Two-Photon Excitation Fluorescence Correlation Experiments
Chirico, G., Fumagalli, C., Baldini, G.
J. Phys. Chem. B, 2002, 106, 2508-2519.
|The Stoichiometry of Scaffold Complexes in Living Neurons - DLC2 Functions as a Dimerization Engine for GKAP.
Moutin, E., Compan, V., Raynaud, F., Clerté, C., Bouquier, N., Labesse, G., Ferguson, M.L., Fagni, L., Royer, C.A., Perroy, J.
J Cell Sci., 2014, 127(Pt 16), 3451-62.
|Reconciling Molecular Regulatory Mechanisms with Noise Patterns of Bacterial Metabolic Promoters in Induced and Repressed States
Ferguson, M.L., Le Coq, D., Jules, M., Aymerich, S., Ovidiu., R., Declerck, N., Royer, C.A.
PNAS, 2012, 109(1), 155-160.
|Absolute Quantification of Gene Expression in Individual Bacterial Cells Using Two-Photon Fluctuation Microscopy
Ferguson, M.L., Le Coq, D., Jules, M., Aymerich, S., Declerck, N., Royer, C.A.
Anal Biochem., 2011, 419, 250-259.
|Efficient Parallel Levenberg-Marquardt Model Fitting Towards Real-Time Automated Parametric Imaging Microscopy
Zhu, X., Zhang, D.
PLoS One., 2013, 8(10), e76665.
|Nanometer-Scale Optical Imaging of Collagen Fibers Using Gold Nanoparticles
Chen, B., Estrada, L.C., Hellriegel, C., Gratton, E.
Biomedical Optics Express, 2011(2), 3, 511-519.
|Characterization of Brightness and Stoichiometry of Bright Particles by Flow-fluorescence Fluctuation Spectroscopy.
Johnson, J.1., Chen, Y., Mueller, J.D.
Biophys J., 2010, 99(9), 3084-92.
|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.
|Unraveling Protein-Protein Interactions in Living Cells with Fluorescence Fluctuation Brightness Analysis
Chen, Y., Wei, L.-N., Müller, J.D.
Biophys. J., 2005, 88, 4366-4377.
|Dual-Color Photon-Counting Histogram
Chen, Y., Tekmen, M., Hillesheim, L., Skinner, J., Wu, B., Müller, J.D.
Biophys. J., 2005, 88, 2177-2192.
|Fluorescence Spectroscopy With Metal-Dielectric Waveguides.
Badugu, R., Szmacinski, H., Ray, K., Descrovi, E., Ricciardi, S., Zhang, D., Chen, J., Huo, Y., Lakowicz, J.R.
J Phys Chem C Nanomater Interfaces., 2015, 119(28), 16245-16255.
|Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates
Szmacinski, H., Toshchakov, V., Piao, W., Lakowicz, J.R.
BioNanoSci., 2013, 3(1), 30-36.
|Confocal Fluctuation Spectroscopy and Imaging
Földes-Papp, Z., Liao, S.-C.J., You, T., Terpetschnig, E., Barbieri, B.
Current Pharmaceutical Biotechnology, 2010, 11, 639-653.
|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.
|Capturing Directed Molecular Motion in the Nuclear Pore Complex of Live Cells
Cardarelli, F., Lanzano, L., Gratton, E.
PNAS, 2012, 109(25), 9863-9868.
|Nanometer-scale Imaging by the Modulation Tracking Method
Lanzano, L., Digman, M.A., Fwu, P., Giral, H., Levi, M., Gratton, E.
J. Biophotonics, 2011, 4(6), 415-424.
Options and Accessories available for Alba-STED for FFS/FLIM
XYZ-stepper motor controlled stage for Microwell plates (8-, 96- and 384-wellplate)The XYZ stage provides high resolution, highly repeatable, and fast controls for the X, Y, and Z position of the microscope stage; it utilizes crossed-roller slides, a high-precision lead screw, and zero-backlash miniature geared DC servomotors for smooth and accurate motion. Controlled through the USB port, it is the ideal stage when measuring samples in a microwell plate.
VistaVision includes protocols for the automatic measurement at single points (FFS, lifetime, polarization); the user can select the sequential measurements on all the wells; alternatively, a set of wells can be selected for the measurements.
XY-stepper motor controlled stage with fast z-axis piezo top plateThe PZ-2150-XY-FT piezo system is fabricated by Applied Scientific Instrumentation. The XY axis range of travel is 120 mm x 110 mm with a resolution of 0.088 µm (encoder step); maximum velocity is 7 mm/sec. The z-axis piezo has a travel range of 150 µm with a step resolution of 2.2 nm. The stage can be fitted with autofocus capability for maintaining the z-plane position for several hours during the measurements. The stage is fully controlled through the VistaVision software which allows for the sequential measurement of each well in the plate or for the selection of target wells. The stage is used for:
- Sequence of FLIM measurements at different wells
- Z-stack of FLIM measurements at different wells
- Particle tracking measurements
- Nanoimaging measurements
XYZ Piezo-controlled StageThe XYZ PZT is an actuated linear nanopositioning stage of exceptional resolution and stability. Manufactured by Mad City Labs, it is packaged for ISS to be utilized in the Alba under the control of VistaVision.
The 66 x 66 mm aperture in the stage center is ideal for applications involving transmitted beams, multiple probes or inverted optics. With its large distance of travel and high stability, the PZT is ideal for the most challenging microscopy and positioning applications. The PZT comes complete with position sensitive detectors for closed loop operation.
The stage is manufactured from a high performance Al alloy. actuators are preloaded within the PZT and supply the driving force for stage movement. The flexure hinges, which form the guidance mechanism, are cut into the stage using electric discharge machining (EDM). EDM is also used to form integrated amplifiers that increase the range of motion of the PZT actuators for the X, Y and Z axis. The PZT actuators are oriented perpendicular to the stage motion direction and within these amplifiers.
Non Descanned Detection (NDD) Port
The Figure displays the NDD port on the Nikon Model Ti microscope coupled to the Alba. A raiser is introduced on the Nikon microscope above the epifluorescence port for connecting the NDD port and adding the filters-cartridge where the dichroic filters for the NDD detection are inserted. The detectors are mounted on an orthogonal mount complete of dichroic and filter holders. The NDD port uses either GaAs PMTs or hybrid detectors. The output of the detectors is diverted either to the data acquisition unit.