NDD detection is now available for the Olympus FluoView FV1000MPE allowing deep tissue FLIM imaging

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

• Independence from fluorophore concentration
• Discrimination of fluorescence light against elastic and Raman scattering by temporal resolution
• Reduction of needed detectors - one detector can detect different lifetimes simultaneously
• Decay time as further dimension enhances the accuracy of analytical measurements

In order to upgrade a Laser Scanning Microscope, the LSM must

• be equipped with a fiber input port to couple the pulsed laser light to the microscope (except 2-photon excitation setups)
• be equipped with a fiber exit port to guide the emission to a photon counting detector (except NDD setups)
• provide synchronization signals from the laser scanner controller in order to synchronize the measurement with the movement of the scanning head

The final assembly, alignement and testing is a demanding task. Therefore an installation from PicoQuant is always recommended.

The individual components of the upgrade kit are:

Excitation 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 nm to 900 nm. The laser heads are integrated along with multiple optical components in one Laser Combining Unit (LCU) for easier handling and coupling into an optical fiber. As an alternative to pulsed diode lasers, especially for two photon excitation schemes, short pulse laser systems such as Titanium:Sapphire lasers can be integrated as well.

		TCSPC Data Acquisition The photon counting module PicoHarp 300 contains the complete timing electronics for Time-Correlated Single Photon Counting (TCSPC) with picosecond resolution. The versatile Time-Tagged Time-Resolved mode (TTTR) is used to study fluorescence dynamics and allows to synchronize the measurement with external events through special marker signals. These markers allow later the reconstruction of 2D or 3D images. If more than one detector is required a router solution allows to connect and record the signals of up to 4 detectors simultaneously.

Detector The standard detectors for the upgrade kit are Single Photon Avalanche Diodes (τ-SPAD or PDM SPAD) which feature a high temporal resolution and a very high detection efficiency, necessary e.g. for single molecule studies or Fluorescence Correlation Spectroscopy (FCS). As an alternative, Photomultiplier tubes (PMTs) can be used. As they have a lower detection efficiency than SPADs, they are not suited for FCS, but can still be used for FLIM. In case of the Olympus FluoView FV1000MPE system it is also possible to use the internal NDD PMT detectors for FLIM measurements. One or two detector channel set-ups are available with integrated filter holders that allow quick change of emission filters in order to adapt to different experimental conditions. The detector(s) are usually connected to the LSM via a suited optical fiber to an appropriate fiber exit port of the microscope (except NDD set-ups).

		Software The analysis software "SymPhoTime" is based on the powerful but generic TTTR data collection. Users can perform an unlimited number of analysis steps without losing track of the interdependence and origin of their measurement and analysis data. All derived data is maintained in the hierarchic workspace, including a log file, keeping track of all measurement and analysis steps. The analysis possibilities include intensity time traces, burst analysis, lifetime histogramming, Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Correlation Spectroscopy (FLCS), Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging (FLIM), to name only a few. The lifetime image as well as FCS auto- and crosscorrelation curves are already displayed during the data acquisition. Up to 4 lifetimes can be fitted for each pixel or selected regions of an the image. Mixtures of different dye molecules can thus be analysed. Reconvolution of the TCSPC histogram can be performed with a measured or fitted Instrument Response Function (IRF) in order to yield high accuracy even for very short lifetimes.

Supported LSMs:
		FluoView FV1000 FluoView FV1000MPE FluoView FV300		A1 C1si,C1		TCS SP5 TCS SP2		LSM 780 LSM 710 LSM 510
		Upgrades of other LSM types are also possible - please contact us for details.