LSM Upgrade Kit

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 Upgrade Kit is an external addition to the LSM and grants easy-to-use, versatile, and comfortable performance without time-consuming adjustments. The turn-key setup consists of three major parts: picosecond pulsed excitation, single molecule sensitive detection, and Time-Correlated Single Photon Counting (TCSPC) data acquisition.

Flexible excitation system

All samples containing commonly used fluorophores can be examined with the LSM Upgrade Kit. For this purpose, the excitation subsystem consists of a pulsed diode laser driver of the PDL Series and different laser heads with pulses in the picosecond time regime (additional CW mode is available as an option).

The available wavelengths range from 375 to 900 nm. The laser heads are integrated along with multiple optical components in one Laser Combining Unit (LCU) for comfortable handling and coupling into an optical fiber enabling single or multicolor excitation. The sample background is significantly reduced due to the narrow excitation spectra of the lasers.

The repetition rate and laser power can be adapted via the laser driver to the fluorescence lifetime which minimizes bleaching of the sample. As an alternative to pulsed diode lasers, especially for multi-photon excitation schemes, short pulse laser systems such as Titanium:Sapphire lasers can be integrated as well.

Excellent timing with picosecond resolution

Fluorescence lifetimes down to a few picoseconds or even up to milliseconds for phosphorescence and luminescence studies are easily resolvable with the LSM Upgrade Kit. This broad measurement range covers almost all samples that are analyzed in life as well as material sciences. Time-resolved microscopy requires the registration of not only the photons themselves, but also their position in time and, for imaging, in space. The ideal technique for that purpose is the Time-Tagged Time-Resolved (TTTR) data acquisition developed by PicoQuant, which is a variation of the classical method of Time-Correlated Single Photon Counting (TCSPC). Using TTTR data acquisition vastly different measurement procedures, like FLIM, lifetime based FRET, FCS, or even coincidence correlation ("antibunching") can be performed, based on just one fundamental data format. The TTTR format ist supported by all available TCSPC electronics from PicoQuant. Different modules with up to eight independent TCSPC channels for fast and parallel detection are available which provide highest data throughput, multi-stop possibility, and low dead time to realize short acquisition times.

Single photon sensitive detection for highest sensitivity

To ensure optimal experimental conditions, four different detector types are available for the LSM upgrade kit:

• Hybrid PMT, the best allrounder for FLIM, FCS, and NDD deep tissue imaging
• PDM SPAD, the fastest detector for FLIM and FCS
• SPCM-AQRH SPAD, the highest sensitivity detector best for FCS
• Photomultiplier tubes (PMTs) of the PMA series, the economic variant for FLIM

Hybrid PMTs and SPADs offer highest sensitivity as needed for single molecule experiments and are suited for, e.g., cellular FLIM and FCS studies at low fluorescence intensities. The detectors differ in their efficiency, temporal resolution, spectral range, or active area. Thus, the choice of the detector depends on several factors such as the targeted application or interface to the microscope for confocal or NDD (non-descanned) detection.

more...

Intuitive software

The system software SymPhoTime 64 provides multiple, easy-to-use acquisition and analysis tools for all kind of samples. Based on the sophisticated data collection and handling, the system software SymPhoTime 64 supports a multitude of methods, such as Fluorescence Lifetime Imaging (FLIM), Förster Resonance Energy Transfer (FRET), Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Correlation Spectroscopy (FLCS), intensity time trace, burst analysis, lifetime histogramming, and anisotropy, to name only a few.
SymPhoTime 64 data handling maintains a transparent data structure where all derived data is stored in one workspace, including a log file to keep track of all measurement and analysis steps. Image data can be processed further or exported to standard formats. A large number of algorithms for those methods are already integrated in the software, providing a analysis platform for ready-to-publish data. At the same time, SymPhoTime 64 offers enhanced flexibility for the integration of novel, cutting edge algorithms by the user. A dedicated scripting language interface allows to modify and expand the analysis routines according to the experimental needs.

Advanced offline image analysis is possible with the NovaFLIM software package. NovaFLIM features an extremely fast GPU-accelerated analysis of FLIM, FLIM-FRET, and anisotropy data. Efficient batch analysis of z-stacks, time-lapse series and stitched images as well as advanced, flexible and reproducibl ROI handling enables high quality image analysis.

A specially developed interface between the SymPhoTime 64 and the LSM software (Nikon and Leica) strongly facilitates data acquisition and enables straightforward recording of, e.g., FLIM images up to 4096 × 4096 pixels, time series FLIM, z-stacks as well as single and multipoint measurements for FCS. Online previews of the main parameters (Fast FLIM, FCS, time-traces, or TCSPC histograms) allow to quickly optimize data acquisition.

Examples and tutorials

Supported LSMs
 

AX
A1
C1si, C1
C2+, C2

FluoView FV4000
FluoView FV3000
FVMPE-RS
FluoView FV1200 (MPE)
FluoView FV1000 (MPE)

HyperScope
VivoScope
 

LSM 980
LSM 880
LSM 780
LSM 710
 

 

All Information given here is reliable to our best knowledge. However, no responsibility is assumed for possible inaccuracies or omissions. Specifications and external appearances are subject to change without notice.

• Excitation wavelengths: 375 nm - 900 nm
• Parallel attachment of pulsed lasers from PicoQuant and MPE laser (Nikon A1)
• Simultaneous usage of pulsed lasers from PicoQuant and CW lasers from Nikon A1
• Special fiber switch for incoupling of the pulsed lasers (Nikon C series)
• Attachment of PicoQuant detectors to a special exit port of the LSM (Nikon A1)
• Alternative coupling of PicoQuant detectors and Nikon´s spectral detection unit (Nikon C1si, C2) or Nikon´s PMT detection unit (Nikon C1, C2)
• Parallel setup for confocal and NDD imaging as well as polarization measurements (Nikon A1)
• SymPhoTime 64 integration into the LSM NIS software to facilitate FLIM and FCS data acquisition
• Data analysis using the powerful SymPhoTime 64 software
• Full control of PicoQuant pulsed laser in NIS elements to enable high content FLIM screening using JOBS

For details, please check the selection chart for Nikon A1, C1si and C2+, C1 and C2.

• Excitation wavelengths: 405 - 900 nm (FV3000) and 375 - 900 nm (FV1200 / FV1000)
• Simultaneous usage of pulsed lasers from PicoQuant and CW lasers from Evident, independent of excitation wavelength
• Usage of pulsed lasers with normal or SIM scanner
• Attachment of PicoQuant detectors to a special exit port of the LSM
• Parallel setup for confocal and NDD imaging (FV1000MPE, FV1200MPE) as well as polarization measurements
• Data analysis using the powerful SymPhoTime 64 software
• Usage of up to four internal NDD PMTs for deep-tissue FLIM (FV1000MPE, FV1200MPE)

For details, please check the selection chart for FluoView FV4000, FluoView FV3000, FluoView FV1000 (MPE) / FV1200 (MPE), and FVMPE-RS.

• Usage of MPE laser
• Usage of up to four internal NDD detectors for deep-tissue FLIM
• Usage of PicoQuant single photon detectors for standard NDD imaging
• Parallel set-up for FLIM and intensity based NDD imaging
• SymPhoTime 64 integration into the ScanImage software from VidrioTechnologies to facilitate FLIM data acquisition
• Data analysis using the powerful SymPhoTime 64 software

• Excitation wavelengths: 405 nm - 640 nm
• Parallel attachment of pulsed lasers from PicoQuant and Zeiss
• Parallel attachment of pulsed VIS lasers from PicoQuant and MPE laser
• Attachment of PicoQuant detectors to a special exit port of the LSM
• Simultaneous coupling of PicoQuant detectors and the ConfoCor Unit (LSM 710)
• NDD in combination with NLO for upright and inverse microscopes
• Single as well as dual channel NDD (LSM 710 NLO / LSM 780 NLO/ LSM 980 NLO)
• Parallel setup for confocal and NDD imaging as well as polarization measurements
• Data analysis using the powerful SymPhoTime 64 software
• SymPhoTime 64 integration into the LSM ZEN software to facilitate FLIM and FCS data acquisition

For details, please check the selection chart for Zeiss LSM980, Zeiss LSM 710/780/880/980.

• Excitation wavelengths: 405 nm, 440 nm, 470 nm and 640 nm (TCS SP5 / SP8)
• Simultaneous usage of pulsed lasers from PicoQuant and CW lasers from Leica
• Parallel attachment of the pulsed 405 nm laser from PicoQuant and MPE laser

Intracellular Ca2+ signalling upon mechanical stimulation

Intracellular Ca²⁺ signals induced by mechanical stimulation of HEK-cells loaded with Oregon-Green-Bapta-1. In this video, the cells were imaged before, during, and after mechincal stimulation of the top two cells. The mechanical stimulation leads to an uptake of calcium ions in the cells. This increase in Ca2+ concentration results in a change of the fluorescence lifetime of OGB-1 AM, and the top two cells light up in green for a short time. The rapidFLIM^HiRes method enabled imaging a sample area of 128 x 128 pixels with a speed of 5 FLIM frames per second, which makes it possible to quantitatively observe the rise of calcium ion concentration in the cells over a short time period.

Sample details:

• Human Embryonic Kidney (HEK) cells loaded with Oregon-Green-Bapta-1 (OGB-1 AM) kept in artificial cerebrospinal fluid

Set-up:

• 128 x 128 pixel, 3.81 µs pixel dwell time
• 5 frames per second

Sample and data courtesy of Prof. Rose (Institute of Neurobiology, Heinrich Heine University Düsseldorf, Germany)

Monitoring changes in membrane tension with rapidFLIM^HiRes

This video shows MDCK cells whose membranes were stained with the fluorescent probe Fliper-TR^® (Fluorescent lipid tensor Reporter). This probe displays a significant change in fluorescence lifetime when embedded in a confining environment. If lateral pressure is applied, Fliper-TR^® will undergo a planarization which leads to a change in fluorescence lifetime, which allows imaging lipid composition and membrane tension in both live cell and artificial membranes.

Under isoosmotic conditions, Fliper-TR^® exhibits a fluorescence lifetime of ca. 5.1 ns. After inducing an hyperosmotic shock, the membrane tension decreases rapidly and a shorter lifetime is observed. Imaging with the rapidFLIM^HiRes approach is fast enough so that this lifetime change and by extension the change in membrane tension can be recorded in real time.

Sample details:

• Madin-Darby Canine Kidney (MDCK) cells, membranes stained with Fliper-TR^®

Set-up:

• Excitation: 485 nm
• Image size: 512 x 256 pixels
• 1.0 frames per second

Sample courtesy of A. Colom, University of Geneva, Switzerland

Reference: Colom, A., Derivery, E., Soleimanpour, S. et al. A fluorescent membrane tension probe. Nature Chem 10, 1118–1125 (2018). https://doi.org/10.1038/s41557-018-0127-3

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

GUV preparation by Ivan Haralampiev, Lab of Molecular Biophysics, Humboldt Universität zu Berlin

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:

• Combined MicroTime 200 and LSM Upgrade Kit (Leica TCS SP5)
• Excitation: two-photon excitation at 800 nm, SpectraPhysics MaiTai
• Analysis: SymPhoTime

Courtesy of Katharina Gaus, Centre for Vascular Research, University of New South Wales, Sydney, Australia

Reference: Owen et al., Microsc. Res. Tech. 73(6), (2010)

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

• Time-Resolved Fluorescence
• NEW – rapidFLIM^HiRes - 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

The following documents are available for download:

• Brochure LSM Upgrade Kit
• Selection chart for Nikon AX
• Selection chart for Nikon A1
• Selection chart for Nikon C1
• Selection chart for Nikon C1si and C2 with Lu4 Laser Combiner
• Selection chart for Nikon C2+ with LUN Laser Combiner
• Selection chart for Olympus FV1000 and FV1200
• Selection chart for Olympus FV3000
• Selection chart for Olympus FVMPE-RS
• Selection chart for Zeiss LSM 710/780/880/980
• NIS-Elements PicoQuant FLIM & FCS Software Control Plug-in
• Poster: Quick Reference for confocal time-resolved microscopy (FLIM, FRET, FCS)
• Practical Manual for Fluorescence Microscopy Techniques

Presentations (as PDF files)

• Fluorescence Lifetime Correlation Spectroscopy (FLCS)

Related technical and application notes:

• Technical note: Combination of pulsed and cw lasers on a Olympus LSM
• Technical note: FLIM NDD Upgrade Kit for the Olympus FluoView FV1000MPE
• Technical note: Polarization Extension Unit for LSM Upgrade Kits
• Technical note: ROI scanning using pulsed lasers on a Olympus FluoView
• Technical note: Time-Correlated Single Photon Counting (TCSPC)
• Application note: Fluorescence Lifetime Imaging (FLIM) in Confocal Microscopy
• Application note: Imaging of molecular distances using FLIM-FRET
• Application note: FRET analysis with Pulsed-Interleaved Excitation (PIE)
• Application note: Time-gated Fluorescence Correlation Spectoscopy
• Application note: Fluorescence Lifetime Correlation Spectroscopy (FLCS)
• Technical note: Phosphorescence Lifetime Imaging Microscopy (PLIM) Measurements - Practical Aspects
• Application note: Visualize dynamic processes with rapidFLIM^HiRes, the ultra fast FLIM imaging method with outstanding time resolution