LSM upgrade kit

Compact FLIM and FCS Upgrade Kit for Laser Scanning Microscopes


Confocal Laser Scanning Microscopes (LSMs) are widely used tools in biochemistry, cell biology, and other related life sciences. The capabilities of these microscopes can be further enhanced by using time-resolved techniques, granting following advantages:

  • Lifetime FRET for quantitative measurements of FRET efficiency
  • Time-resolved imaging reveals environmental parameters such as ion concentrations or pH
  • Lifetime measurements are independent from fluorophore concentration
  • Separation of molecules with spectrally overlapping emission by lifetime fingerprinting
  • Reduced number of needed detectors – one detector is sufficient for simultaneous detection of different fluorophores based on their specific lifetimes by pattern matching
  • Discrimination of fluorescence light against elastic and Raman scattering and other background contributions by temporal resolution
  • Decay time as a further parameter enhances the accuracy of analytical measurements

The compact FLIM and FCS upgrade kit enables multiple time-resolved applications with Laser Scanning Microscopes (LSMs), such as:

  • Time-Resolved Fluorescence
  • NEW – rapidFLIM – Redefining standards for dynamic FLIM imaging
  • Fluorescence Lifetime Imaging (FLIM)
  • Phosphorescence Lifetime Imaging (PLIM)
  • Fluorescence Correlation Spectroscopy (FCS)
  • Fluorescence Lifetime Correlation Spectroscopy (FLCS)
  • Fluorescence Cross-Correlation Spectroscopy (FCCS)
  • Foerster Resonance Energy Transfer (FRET)
  • Pulsed Interleaved Excitation (PIE)
  • Laser Cutting/Ablation
  • Pattern Matching Analysis
  • Time-Resolved Photoluminescence (TRPL)
  • TRPL Imaging
  • Antibunching
  • Anisotropy
Excitation System
  • Picosecond diode lasers with adjustable output power and repetition rates up to 80 MHz inside a compact laser combining unit
  • Wavelengths between 375 and 900 nm
  • Single or multi-channel laser driver
  • Optional: external laser (e.g. Titanium:Sapphire laser)
  • 560 nm picosecond pulsed excitation with the LDH-D-TA-560

Supported LSMs
  • Nikon: A1, C2+, C2, C1si
  • Olympus: FluoView FV3000, FVMPE-RS, FluoView FV1200 (MPE), FluoView FV1000 (MPE)
  • Scientifica: VivoScope, HyperScope
  • Zeiss: LSM 980, LSM 880, LSM 780, LSM 710
Detection
  • Up to four parallel detection channels
  • Descanned or non-descanned configuration
  • Connection via optical fibers to the LSM
Detectors
  • Hybrid-Photomultiplier Tubes
  • Single Photon Avalanche Diodes
  • Photomultiplier Tubes
Data acquisition
  • Based on the method of Time-Correlated Single Photon Counting (TCSPC) in the unique Time-Tagged Time Resolved (TTTR) measurement mode
  • Simultaneous data acquisition in up to four channels
Software
  • SymPhoTime 64

rapidFLIM – Redefining standards for dynamic FLIM imaging

rapidFLIM measurements enable the imaging of dynamics in fluorescence lifetime. This new approach allows for fast FLIM acquisition with several frames per second as well as imaging of dynamic processes (e.g., protein interaction, chemical reaction, or ion flux), highly mobile species (e.g., mobility of cell organelles or particles, cell migration), and investigating FRET dynamics. More than 10 frames per second can be acquired, depending on sample brightness and image size. Unilamellar vesicles can be produced in a size regime ranging from giant (GUVs) to large (LUVs), and even to small (SUVs). The flexibility in membrane composition and possibility to introduce specifically labeled lipids increase the importance of unilamellar vesicles for studies in cell biology. Thus turning such vesicles a very powerful model to investigate e.g., membrane domain formation as well as lipid organization in membrane micro-domains. Until now, acquiring FLIM images took up to several minutes and due to the high mobility of GUV’s, imaging them accurately is difficult. Applying the rapidFLIM approach significantly decreases the acquisition time, allowing to record up to several frames per seconds. Thus even highly mobile GUVs can be precisely tracked. In this example, two fluorophore labeled lipids (C6-NBD-PC and N-Rhd-DOPE) were incorporated into GUVs. In non-phase separated GUVs, the lifetime of NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) is strongly quenched (down to ~2 ns) due to FRET to the acceptor rhodamine. The video shown here contains 300 frames that were recorded with a frame rate of 5.6 fps.

Sample details:

  • GUVs with NBD and Rhodamin labeled lipids (no phase separation): DOPC + 0.5 mol % Palmitoyl-C6-NBD-PC + 0.5 mol % N-Rhd-DOPE
  • NBD fused to Palmitoyl-C6-NBD-PC (phosphatidyl choline)
  • Rhodamine fused to N-Rhd-DOPE (Di-oleyl-phosphatidyl-ethanolamin)

Set-up:

  • Excitation: 485 nm, 40 MHz
  • Long pass filter: 488 nm
  • 75 x 75 µm, 300 x 300 pixel, 1 µs/pixel
  • 300 frames with 5.6 fps

Lipid Order Determination Using Fluorescence Lifetime

FLIM measurements facilitate the differentiation between ordered and disordered membrane phases. The membrane dyes Laurdan and di-4-ANEPPDHQ can be used to image membrane order due to a spectral blue-shift in the fluorescence emission as well as a lifetime shift between the liquid-ordered and liquid-disordered phases. These images typically take the form of a normalized intensity ratio image known as a generalized polarization (GP) plot. Here, the known excited state photo physics is exploited via Time-Correlated Single-Photon Counting (TCSPC) to demonstrate GP contrast enhancement for these two probes. The image shows a GP plot of a Laurdan stained, fixed BAEC cell that combines lifetime and spectral changes. The plasma membrane at the cell surface shows higher order (red) compared to the inner cell compartments (blue).

Set-up:

Imaging specific newly synthesized proteins within cells by fluorescence resonance energy transfer

Shen L., Cai L., Liu J., Zhang S., Xu J.-J., Zhang X., Chen H.-Y. Chemical Science, Vol.008, p.748-754 (2017)

Reference to: LSM Upgrade Kit Related to: FLIM, FRET


Silver-coated nanoporous gold skeletons for fluorescence amplification

Lee M.-J., Yang W.-G., Kim J.H., Hwang K., Chae W.-S. Microporous and Mesoporous Materials, Vol.237, p.60-64 (2017)

Reference to: MicroTime 200, SymPhoTime Related to: FLIM


Intracellular fate of polymer therapeutics investigated by fluorescence lifetime imaging and fluorescence pattern analysis.

Panek J., Koziolova E., Stepanek P., Etrych T., Janouskova O. Physiological Research, Vol.065, p.217-224 (2016)

Reference to: LSM Upgrade Kit Related to: FLIM


A comparative study of the photophysics of phenyl, thienyl, and chalcogen substituted rhodamine dyes

Sabatini R.P., Mark M. F., Mark D.J., Kryman M.W., Hill J.E., Brennessel W.W., Detty M.R., Eisenberg R., McCamant D.W. Photochemical & Photobiological Sciences, Vol.015, p.1417-1432 (2016)

Reference to: PicoHarp 300, SymPhoTime


Mechanistic determinants of MBNL activity

Sznajder L.J., Michalak M., Taylor K., Cywoniuk P., Kabza M., Wojtkowiak-Szlachcic A., Matloka M., Konieczny P., Sobczak K. Nucleic Acids Research, Vol.044, p.10326-10342 (2016)

Reference to: LSM Upgrade Kit Related to: FLIM, FRET


Structure and dynamics of polyelectrolyte surfactant mixtures under conditions of surfactant excess

Hoffmann I., Simon M., Farago B., Schweins R., Falus P., Holderer O., Gradzielski M. The Journal of Chemical Physics, Vol.145, 124901 (2016)

Reference to: PicoHarp 300, LSM Upgrade Kit Related to: FCS


Investigating the DNA-binding interactions of small organic molecules utilizing ultrafast nonlinear spectroscopy

Doan P. Dissertation University of Michigan (2016)

Reference to: TimeHarp 100/200, LSM Upgrade Kit Related to: FLIM


Control of spontanous emission from quantum emitters using hyperbolic metamaterial substrates

Galfsky T. Dissertation City University of New York (2016)

Reference to: PicoHarp 300, SymPhoTime


Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

Andersen T.G., Nintemann S.J., Marek M., Halkier B.A., Schulz A., Burow M. Scientific Reports, Vol.006, 27766 (2016)

Reference to: MicroTime 200, LSM Upgrade Kit, SymPhoTime Related to: FLIM, FRET


Exploring the HYDRAtion method for loading siRNA on liposomes: the interplay between stability and biological activity in human undiluted ascites fluid

Dakwar G.R., Braeckmans K., Ceelen W., De Smedt S.C., Remaut K. Drug Delivery and Translational Research, Vol.007, p.241-251 (2016)

Reference to: LSM Upgrade Kit, SymPhoTime Related to: FCS