The instrument for quantitative cell biology at single-molecule detection.

Stimulated Emission Depletion (STED) is a powerful microscopy technique that allows for the observation of fluorescence structure with spatial resolution below the diffraction limit. The Alba-STED uses the pulsed excitation and pulsed depletion approach (pSTED) in combination with the digital frequency domain fluorescence lifetime imaging (FastFLIM) to record the time-resolved photons which allows for an increase in the image resolution and the separation of two labels with the same excitation wavelength.

Key Features of Alba-STED for FLIM/FFS:

  • pSTED (Pulsed excitation and pulsed STED)
  • FastFLIM for time-resolved pSTED acquisition
  • Improved image resolution using the phasor plot
  • Dual-label excitation
  • Fast image acquisition (5 frames/second at 256x256 pixels)
  • High dynamic range (signal up to 13 million counts/s)

Measurements for Alba-STED for FLIM/FFS

FFS Measurements
  • Fluorescence Correlation Spectroscopy (single- and cross-correlation)
  • Photon Counting Histogram (PCH)
  • FFS measurements at target XYZ locations in an image
  • FLCS, Fluorescence Lifetime Correlation Spectroscopy
  • Scanning FCS
  • Number & Brightness (N&B)
  • Raster Image Correlation Spectroscopy (RICS)
Single-point Module Measurements
  • Intensity
  • Polarization
  • Kinetics
  • Lifetime
Imaging Module Measurements (Single plan and z-stack)
  • Intensity
  • Polarization
  • Ratiometric
  • FLIM
FLIM images (digital frequency-domain) (single plane and z-stack)
  • Acquired in digital frequency-domain (DFD). The routine acquires simultaneously a FLIM image and a steady-state image.
FLIM images time-domain (single plane and z-stack)
  • Acquired in time-correlated single photon counting (TCSPC)
Superresolution
  • Particle tracking
  • Nanoimaging
Single Molecule Module
  • Burst Analysis
  • FRET and Correlation Methods
  • PIE-FRET Methods

Specifications for Alba-STED for FLIM/FFS

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 four channel data acquisition
  • Auxiliary port for camera
Acquisition and Analysis Software
STED laser Pulsed, 775 nmAverage output power: 1 W
Pulsewidth: about 600 ps
Repetition rate: 0-100 MHz; or Ext. CLK
Beam quality: M2 < 1.1, TEM00
Amplitude noise: < 4.0% rms
Excitation laser Pulsed, 640 nmPulsewidth (at medium power): 40-90 ps
Repetition rate: 20, 50, 80 MHz; or Ext. CLK
Power (at 50 MHz): up to 5 mW
Laser Launcher
  • Models for 3, 4, 6 laser diodes. Light is delivered to the microscope through a single-mode fiber optic.
FastFLIM
  • 4 separate input signal
  • Dynamic range: up to 13 million counts/s per channel
  • Lifetime range: from picosecond to second
Microscope
  • Inverted or Upright
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
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
  • APD
  • GaAs PMT
Image acquisition FLIM acquisition: 5 frames/sec (256 x 256 pixels)
OS Requirements
  • Windows 10, 64-bit
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-STED for FLIM/FFS

Measurement Examples from Alba-STED for FLIM/FFS

Confocal (green) vs. pSTED (red) images of 60-nm fluorescent beads, acquired by FastFLIM

Confocal (left) vs. pSTED (right) images of the actin labeled with the SiR dye in fixed glia cells, acquired by FastFLIM.

Increasing the resolution of pSTED images using the Phasor Plot

Confocal images of 60nm fluorescence beads (left); pSTED images (middle); sharpening the pSTED image using a binary filter based on the phasor plots (right).

Separation of the locations of two labels using FastFLIM and the Phasor Plot

Dual labels can be separated using pSTED and FastFLIM. Atto 647N and Atto 655 were used as labels; they both are excited by the 640 nm laser. The two dyes are first separated using the phasor plots, and then assigned with two different false colors (Atto 647N - yellow, Atto 655 - purple) to produce the processed and merged pSTED image of the two labels.

Biochemistry

The Use of FLIM-FRET for the Detection of Mitochondria-associated Protein Interactions
Osterlund, E.J., Liu, Q., Andrews, D.W.
Methods Mol Biol., 2015, 1264, 395-419.
Measuring Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy
Day, R.
Microscopy Today, 2015, 23(3), 44-51.
Cis and Trans Internucleosomal Interactions of H3 and H4 Tails in Tetranucleosomes
Nurse, N.P., Yuan, C.
Biopolymers., 2015, 103(1), 33-40.
A Decoy Peptide That Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.
Piao, W., Shirey, K.A., Ru, L.W., Lai, W., Szmacinski, H., Snyder, G.A., Sundberg, E.J., Lakowicz, J.R., Vogel, S.N., Toshchakov, V.Y.
Cell Rep., 2015, 11(12), 1941-52.
Unexpected Complex Formation Between Coralyne and Cyclic Diadenosine Monophosphate Providing a Simple Fluorescent Turn-on Assay to Detect This Bacterial Second Messenger.
Zhou, J., Sayre, D.A., Zheng, Y., Szmacinski, H., Sintim, H.O.
Anal Chem., 2014, 86(5), 2412-20.
Binding of Fusion Protein FLSC IgG1 to CCR5 Is Enhanced by CCR5 Antagonist Maraviroc.
Latinovic, O., Schneider, K., Szmacinski, H., Lakowicz, J.R., Heredia, A., Redfield, R.R.
Antiviral Res., 2014, 112, 80-90.
Application of Phasor Plot and Autofluorescence Correction for Study of Heterogeneous Cell Population.
Szmacinski, H., Toshchakov, V., Lakowicz, J.R.
J Biomed Opt., 2014, 19(4), 046017.
DNA Methylation Effects on Tetra-nucleosome Compaction and Aggregation
Jimenez-Useche, I., Nurse, N.P., Tian, Y., Kansara, B.S., Shim, D., Yuan, C.
Biophys J., 2014, 107(7), 1629-36.
Unmethylated and Methylated CpG Dinucleotides Distinctively Regulate the Physical Properties of DNA
Jimenez-Useche, I., Shim, D., Yu, J., Yuan, C.
Biopolymers., 2014, 101(5), 517-24.
mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes
Chou, C.-K., Lee, H.-H., Tsou, P.-H., Chen, C.-T., Hsu, J.-M., Yamaguchi, H., Wang, Y.-N., Lee, H.-J., Hsu, J. L., Lee, J.-F., Kameoka, J., Hung, M.-C.
Sci. Signal., 2014, 7(315), rs1 .
Unraveling Transcription Factor Interactions with Heterochromatin Protein 1 Using Fluorescence Lifetime imaging Microscopy and Fluorescence Correlation Spectroscopy
Siegel, A.P., Hays, N.M., Day, R.N.
J. Biomed. Opt., 2013, 18(2).
Chromophore Maturation and Fluorescence Fluctuation Spectroscopy of Fluorescent Proteins in a Cell-free Expression System.
Macdonald, P.J.1., Chen, Y., Mueller, J.D.
Anal Biochem., 2012, 421(1), 291-8.
Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy
Hum, J.M., Siegel, A.P., Pavalko, F.M., Day, R.N.
Int. J. Mol. Sci., 2012, 13, 14385-14400.
HIV-1 Nef Binds a Subpopulation of MHC-I throughout Its Trafficking Itinerary and Down-regulates MHC-I by Perturbing Both Antegrograde and Retrograde Trafficking
Yi, L., Rosales, T., Rose, J.J., Chaudhury, B., Knutson, J.R., Venkatesan, S.
J Biol Chem, 2010, 285(40), 30884-30905.
Familial Hypertrophic Cardiomyopathy Can Be Characterized by a Specific Pattern of Orientation Fluctuations of Actin Molecules
Borejdo, J., Szczesna-Cordary, D., Muthu, P., Calander, N.
Biochemistry, 2010, 49, 5269-5277.
Estrogen Receptor Ineractions and Dynamics Monitored in Live Cells by Fluorescence Cross-Correlation Spectroscopy
Savatier, J., Jalaguier, S., Ferguson, M.L., Cavaille`s, V., Royer, C.A.
Biochemistry, 2010, 49, 772-781.
Monomer-Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination
Fabrini, R., De Luca, A., Stella, L., Mei, G., Orioni, B., Ciccone, S., Federici, G., Lo Bello, M., Ricci, G.
Biochemistry, 2009, 48, 10473-10482.
Fluorescence Correlation Spectroscopy of Phophatidylinositol-specific Phospholipase C Monitors the Interplay of Substrate and Activator Lipid Binding
Pu, M., Roberts, M.F., Gershenson, A.
Biochemistry, 2009, 48(29), 6835-6845.
Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity: Insights Into Phosphatidycholine Activation and Surface Dilution Inhibition
Pu, M., Fang, X., Redfield, A.G., Gershenson, A., Roberts, M.F.
J. of Biol. Chem., 2009, 284(24), 16099-16107.
A Fluorescent Mutant of the NM Domain of the Yeast Prion Sup35 Provides insight into Fibril Formation and Stability
Palhano, F.L., Rocha, C.B., Bernadino, A., Weissmuller, G., Masuda, C.A., Montero-Lomeli, M., Marco Gomes, A., Chien, P., Fernandes, P.M.B., Foguel, D.
Biochemistry, 2009, 48, 6811-6823.
Characterization of the Control Catabolite Protein of Gluconeogenic Genes Repressor by Fluorescence Cross-correlation Spectroscopy and Other Biophysical Approaches.
Zorrilla, S., Ortega, A., Chaix, D., Alfonso, C., Rivas, G., Aymerich, S., Lillo, M.P., Declerck, N., Royer, C.A.
Biophys J., 2008, 95(9), 4403-15.
Applications of Dual-Color Fluorescence Cross-Correlation Spectroscopy in Antibody Binding Studies
Ruan, Q., Tetin, S.Y.
Anal. Biochem., 2008.
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.
Determining the Stoichiometry of Protein Heterocomplexes in Living Cells With Fluorescence Fluctuation Spectroscopy.
Chen, Y.1., Müller, J.D.
Proc Natl Acad Sci U S A., 2007, 104(9), 3147-52.
Fructose-1,6-bisphosphate Acts Both as an Inducer and as a Structural Cofactor of the Central Glycolytic Genes Repressor (CggR)
Zorrilla, S., Chaix, D., Ortega, A., Alfonso, C., Doan, T., Margeat, E., Rivas, G., Aymerich, S., Declerck, N., Royer, C.A.
Biochemistry, 2007, 46(51), 14996-15008.
Escherichia coli Ribosomal Protein L20 Binds as a Single Monomer to its Own mRNA Bearing Two Potential Binding Sites
Allemand, F., Haentjens, J., Chiaruttini, C., Royer, C., Springer, M.
Nucleic Acids Research, 2007, 35(9), 3016-3031.
Monomer Dimer Dynamics and Distribution of GPI-Anchored uPAR are Determined by Cell Surface Protein Assemblies
Caiolfa, V.R., Zamai1, M., Malengo, G., Andolfo, A., Madsen, C.D., Sutin, J., Digman, M.A., Gratton, E., Blasi1, F., Sidenius, N.
J. Cell Biol., 2007, 179, 1067-1082.
Interactions of Two Monoclonal Antibodies with BNP: High Resolution Epitope Mapping Using Fluorescence Correlation Spectroscopy
Tetin, S.Y., Ruan, Q., Saldana, S.C., Pope, M.R., Chen, Y., Wu, H., Pinkus, M.S., Jiang, J., Richardson, P.L.
Biochemistry, 2006, 45(47), 14155-65.
Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus
Quinlan, R.J., Reinhart, G.D.
Biochemistry, 2006, 45, 11333-11341.
Quantitative Detection of the Ligand-dependent Interaction Between the Androgen Receptor and the Co-activator, Tif2, in Live Cells Using Two Color, Two Photon Fluorescence Cross-correlation Spectroscopy
Rosales, T., Georget, V., Malide, D., Smirnov, A., Xu, J., Combs C., Knutson, J.R., Nicolas, J.-C., Royer, C.A.
Eur. Biophys. J., 2006.
The Nuclear Receptor Coactivator PGC-1α Exhibits Modes of Interaction with the Estrogen Receptor Distinct From those of SRC-1
Bourdoncle, A., Labesse, G., Margueron, R., Castet, A., Cavaillčs, V., Royer, C.A.
J. Mol. Biol., 2005, 347, 921-934.
Phosphoinositide Specificity of and Mechanism of Lipid Domain Formation by Annexin A2-p11 Heterotetramer
Gokhale, N.A., Abraham, A., Digman, M.A., Gratton, E., Cho, W.
J. Biol. Chem., 2005, 280(52), 42831-42840.
Vesicle Encapsulation Studies Reveal that Single Molecule Ribozyme Heterogeneities Are Intrinsic
Okumus, B., Wilson, T.J., Lilley, D.M.J., Ha, T.
Biophys. J., 2004, 87, 2798-2806.
Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy
Ruchira, Hink, M.A., Bosgraaf, L., Van Haastert, P.J.M., Visser, A.J.W.G.
The Journal of Biological Chemistry, 2004, 279(11), 10013-10019.
Polarized Fluorescence Correlation Spectroscopy of DNA-DAPI Complexes
Barcellona, M.L., Gammon, S., Hazlett, T., Digman, M.A., Gratton, E.
Microscopy Research and Techniques, 2004, 65, 205-217.
Probing Protein Oligomerization in Living Cells With Fluorescence Fluctuation Spectroscopy.
Chen, Y.1., Wei, L.N., Müller, J.D.
Proc Natl Acad Sci U S A., 2003, 100(26), 15492-7.
Segregation of Saturated Chain Lipids in Pulmonary Surfactant Films and Bilayers
Nag, K., Pao, J.-S., Harbottle, R.R., Possmayer, F., Petersen, N.O., Bagatolli, L.A.
Biophys. J., 2002, 82, 2041-2051.
Molecular Heterogeneity of O-Acetylserine Sulfhydrylase by Two-Photon Excited Fluorescence Fluctuation Spectroscopy
Chirico, G., Bettati, S., Mozzarelli, A., Chen, Y., Müller, J.D., Gratton, E.
Biophys. J., 2001, 80, 1973-1985.

Diagnostics & Sensing

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.

Nanomaterials & Surface Physics

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.

N&B (Number and Brightness)

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.

Particle Tracking

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.

Photon Counting Histogram

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.

Plasmonics

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.

General Theory

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.

Single Molecule Studies and Theory

Measurements of Single Molecules in Solution and Live Cells Over Longer Observation Times Than Those Currently Possible: The Meaningful Time
Földes-Papp, Z.
Curr. Pharm. Biotechnol., 2013, 14.
Fluorescence Molecular Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity
Földes-Papp, Z., Baumann, G.
Curr. Pharm. Biotechnol., 2011, 12(5), pp. 824-833.
Single Actomyosin Motor Interactions in Skeletal Muscle
Földes-Papp, Z., Liao, S.-C., Barbieri, B., Gryczynski Jr., K., Luchowski, R., Gryczynski, Z., Gryczynski, I., Borejdo, J., You, T.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, May 2011, 1813(5), 858-866.
Meaningful Interpretation of Subdiffusive Measurements in Living Cells (Crowded Environment) by Fluorescence Fluctuation Microscopy
Baumann, G., Place, R.F., Földes-Papp, Z.
Current Pharmaceutical Biotechnology, 2010, 11, 527-543.
Anomalous Behavior in Length Distributions of 3D Random Brownian Walks and Measured Photon Count Rates Within Observation Volumes of Single-Molecule Trajectories in Fluorescence Fluctuation Microscopy
Baumann, G., Gryczynski, I., Földes-Papp, Z.
Optics Express, 2010, 18(17), 17883-17896.
Getting High on Single Molecule Biophysics
Block, S.M.
Current Pharmaceutical Biotechnology, 2009, 10(5), 464-466.
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.
Fluorescence Fluctuation Spectroscopic Approaches to the Study of a Single Molecule Diffusing in Solution and a Live Cell without Systemic Drift or Convection: A Theoretical Study
Földes-Papp, Z.
Current Pharmaceutical Biotechnology, 2007, 8, 261-273.
Mobility and Distribution of Replication Protein A in Living Cells at Single Molecule Level
Braet, C., Stephan, H., Dobbie, I., Togashi, D., Ryder, A.G., Földes-Papp, Z., Lowndes, N., Nasheuer., H.P.
Experimental & Molecular Pathology, 2007, 82(2), 156-62.
"True" Single-Molecule Molecule Observations by Fluorescence Correlation Spectroscopy and Two-Color Fluorescence Cross-correlation Spectroscopy
Földes-Papp, Z.
Experimental and Molecular Pathology, 2007, 82, 147-155.
What It Means to Measure a Single Molecule in a Solution by Fluorescence Fluctuation Spectroscopy
Földes-Papp, Z.
Experimental and Molecular Pathology, 2006, 80, 209-218.
How the Molecule Number Is Correctly Quantified in Two-Color Fluorescence Cross-Correlation Spectroscopy: Corrections for Cross-Talk and Quenching in Experiments
Földes-Papp, Z.
Current Pharmaceutical Biotechnology, 2005, 6, 437-444.
Single-Phase Single-Molecule Fluorescence Correlation Spectroscopy (SPSM-FCS)
Földes-Papp, Z., Baumann, G., Kinjo, M., Tamura, M.
Encyclopedia of Medical Genomics and Proteomics, 2005, 1-7.

Super-resolution, Nanoimaging

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 FLIM/FFS

Non Descanned Detection (NDD) Port

Detection through the Non Descanned Detection Port (NDD) is used in conjunction with multiphoton excitation; the fluorescence photons generated in the excitation spot of the laser are scattered back and collected right after the objective (without passing through the optics in the scanning path).

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.

Microscope Stages

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.

XYZ Piezo-controlled Stage

The 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.