PicoQuant - It's about time

Fluorescence Microscopes

LSM Upgrade Kit

Compact FLIM and FCS Upgrade Kit for Laser Scanning Microscopes

  • FLIM, FRET, and FCS in one turn-key system
  • Compact, easy-to-use and maintenance-free kit, customized to all major LSMs in various configurations for unlimited flexibility
  • Highest sensitivity with up to 4 detection channels
  • Fluorescence lifetimes from < 100 ps up to µs
  • Advanced and user-friendly data analysis software with multiple analysis tools
  • Options for anisotropy measurements and deep-tissue FLIM imaging
  • NEW: rapidFLIM - Redefining standards for dynamic FLIM imaging
  • NEW brochure available

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

General setup scheme of a LSM Upgrade KitThe 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

Laser Combining Unit (LCU)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 Directly coupled laser diode headto 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

PicoHarp 300 - Stand-alone TCSPC Module with USB 2.0 InterfaceFluorescence 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

Multichannel detection unit for LSMsTo 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.

Confocal and NDD detection
The detectors can be attached in confocal mode using a pinhole and fitting to all detector types, as well as in non-descanned detection (NDD) for multi photon excitation.

In the standard confocal mode, the detectors are connected via an optical fiber to the fiber exit port of the microscope. In this confocal configuration set-ups with up to 4 detectors are available. Integrated filter holders allow quick change of emission filters in order to adapt to different experimental conditions and fluorophores.

The non-descanned configuration (NDD) is used in systems with a multi-photon laser and enables deep-tissue FLIM. In this case one or two detectors (PMTs or Hybrid PMTs) are connected to the LSM via a large core liquid light guide. The light guide is either mounted to a suited NDD port of the microscope or, alternatively, a specially developed dove-tail adapter collects the fluorescence directly above the objective. The latter approach features a very high collection efficiency due the light collection very near to objective and the high fluorescence transmission through the liquid light guide. In case of the Olympus FluoView FV1000/1200 MPE system it is also possible to use up to four internal NDD standard PMT detectors of the LSM for FLIM measurements.

Multichannel detection unit
To offer greatest flexibility and multiple applications in one system, the multichannel detection unit allows for parallel confocal, polarization, and NDD measurements with up to four detection channels for multi color FLIM, FRET, deep tissue FLIM imaging, auto- and crosscorrelation (FCS, FLCS, FCCS, FLCCS) as well as anisotropy studies. Several detection configurations are available:


Schematic overview about possible detection options for an upright microscope

  1. Dual-channel confocal detection for FLIM, FRET, auto- and crosscorrelation (FCS, FLCS, FCCS, FLCCS)
  2. Four-channel standard confocal detection for FLIM, FRET, auto- and crosscorrelation (FCS, FLCS, FCCS, FLCCS)
  3. Dual channel polarization resolved confocal detection for anisotropy imaging
  4. Dual channel NDD set-up for deep tissue FLIM and FRET
  5. Parallel dual channel set-up enabling confocal and non-descanned detection for dual color FLIM, FRET, deep tissue FLIM imaging, auto- and crosscorrelation (FCS, FLCS, FCCS, FLCCS)
  6. Parallel four channel set-up enabling confocal as well as non-descanned detection and optionally polarization measurements for multi color FLIM, FRET, deep tissue FLIM imaging, auto- and crosscorrelation (FCS, FLCS, FCCS, FLCCS), and anisotropy.

All options are also available for inverse microscope setups.

more...

 

Intuitive software

SymPhoTime64 - FLIM-FRET analysisThe 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.
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.
Our interactive user forum as well as our regularly held SymPhoTime training days offer outstanding support for new and advanced users.

Course on time-resolved microscopy

Logo TRMic coursePicoQuant annually holds the European short course on "Time-resolved Microscopy and Correlation Spectroscopy". The course is intended for individuals wishing an in-depth introduction to the principles of time-resolved fluorescence microscopy and its applications to the Life Sciences. This 3-day event consists of lectures as well as instrumentation and software hands-on training. For details see the course website.

 


Supported LSMs
 

ZEISS Microscopy

LSM 880
LSM 780
LSM 710

 


TCS SP8
TCS SP5

 


A1
C1si, C1
C2+, C2


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

 

Detailed specifications are included in the datasheet.

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)
  • New: 560 nm picosecond pulsed excitation with the LDH-D-TA-560

Supported LSMs
  • Nikon: A1, C2+, C2, C1si, C1
  • Leica: TCS SP8, TCS SP5
  • Olympus: FluoView FV3000, FVMPE-RS, FluoView FV1200 (MPE), FluoView FV1000 (MPE)
  • Zeiss: 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

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: 405 - 900 nm (FV3000) and 375 - 900 nm (FV1200 / FV1000)
  • Simultaneous usage of pulsed lasers from PicoQuant and CW lasers from Olympus, 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 FV3000, FluoView FV1000 (MPE) / FV1200 (MPE), and FVMPE-RS.


  • 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

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


  • 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)
  • Parallel setup for confocal and NDD imaging as well as polarization measurements
  • Data analysis using the powerful SymPhoTime 64 software

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


  • 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
  • Spectral FLIM and anisotropy measurements
  • Attachment of PicoQuant detectors to a special exit port of the LSM
  • Usage of up to four internal NDD detectors for deep-tissue FLIM
  • Seamless integration of FLIM and FCS data acquisition using the Leica LASAF software
  • Data analysis using the powerful SymPhoTime 64 software

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

Generalized polarization plot of Laurdan stained, fixed BAEC cellFLIM 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:

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 following documents are available for download:

Presentations (as PDF files)


Related technical and application notes:



Latest 10 publications referencing LSM Upgrade Kit, SymPhoTime

The following list is an extract of 10 recent publications from our bibliography that either bear reference or are releated to this product in some way. Do you miss your publication? If yes, we will be happy to include it in our bibliography. Please send an e-mail to info@picoquant.com containing the appropriate citation. Thank you very much in advance for your kind co-operation.