Fluorescing molecules create a "Symphony of Photons in Time". The software package "SymPhoTime" helps you to read the score of such complex works of nature. It was first designed for use with the MicroTime 200 confocal microscope and is now available as a stand-alone version. Users of laser scanning microscopes upgraded to FLIM or FCS capability will love this versitile and comprehensive software package. The software is divided into a dedicated point and 2D / 3D measurement software. The functionality is described in the following.

Workspace concept

As both data acquisition and analysis is easy with the software, the amount of data to be managed can be large. The software provides a multidude of intuitive tools to master this task. Data is organized in a hierarchic workspace concept, which keeps track of the relation between saved analysis results and raw data. All actions taken either for acquisition or analysis are documented in a log file. All files, raw data as well as analysis results, include a comment tag; in addition text files can be added to the workspace, providing links to raw data files or results files. A full text and keyword search will locate files even in the most extensive workspaces.

TTTR File Format

The SymPhoTime software makes use of a very flexible way of raw data acquisition and storage, the so-called TTTR mode of the TimeHarp 200 TCSPC board and the PicoHarp 300 TCSPC module. Each single photon is recorded with its global arrival time and the 'microscopic' delay time with respect to the corresponding laser pulse. While the microscopic delay time is evaluated in lifetime related analyses, the global arrival time can be used to form a fluorescence intensity time trace, making all related analyses possible, like FCS, on / off analysis etc. In addition this global arrival time can be synchronized with external trigger pulses, for example the line or frame clock of LSMs, which is used to extract imaging information.

Data Acquisition

The SymPhoTime software allows to directly image the average fluorescence lifetime already during the scanning process ("online FLIM"). An intensity time trace or FCS curve can be displayed during point measurements ("online time traces" and "online FCS"). Multiple points can be pre-defined before the start of the measurement. Driving a Physik Instrumente E-710 piezo scanner controller or a large area scanner (cm range) it features an iteratively zoomable prescan function as well as point positioning and selection of regions of interest for image acquisition with high precision. The learning function of the piezo scanner controller allows enhanced bidirectional scanning (artefact free).

Data Analysis

All data analysis can separate up to four detector signals out of a single data file. The analyses can be performed applying time gates, i.e. filtering the photon stream according to their temporal position within the decay process. There are two major branches of the analysis methods: time trace based and imaging. Lifetime fitting can either be done using iterative reconvolution, taking into account the influence of the instrument response function (IRF), or as a tailfit, neglecting this influence. Decay models up to four exponential components can be applied. A Maximum Likelihood Estimator (MLE) method can be used to account for regions with low signal intensity.

Time Trace Analysis ('Point')

Sorting the photon events of a TTTR file into subsequent, equally spaced and equally sized time bins forms an intensity time trace (or MCS trace). Based on this first step a variety of analysis methods becomes available: FCS and FLCS calculation, total correlation, FCS fitting to 4 different established models, intensity gated TCSPC histogramming, BIFL or time trace TCSPC fitting including lifetime histogramming, on / off time histogramming, burst size histogramming, PCH usable for FIDA analysis, intensity FRET utilizing two-detector measurements, pulsed-interleaved FRET (PIE-FRET) with bleedthough and direct excitation correction. All results derived in these analyses can be exported as ASCII files, allowing for custom data processing by third party software and opening up almost unlimited scientific possibilities.

Image Analysis (2D / 3D)

Synchronizing the photon events of a TTTR file with a scanning process allows formation of a fluorescence image. Intensity gated imaging can be used to reduce stray light contributions, separation of different detector signals allows intensity FRET investigations, or extract polarization information. Applying a multiexponential fit to the image pixel by pixel reveals additional lifetime contrast (FLIM). Elaborate false coloring methods allow to maximize the amount of information presented in the display of the results, which can be exported directly as Windows™ bitmaps for publication or as ASCII files for subsequent data analysis. In addition an image calculator allows arithmetical operations on images on a pixel-by-pixel basis and facilitates even more complex analyses as for example FRET or calculating directional derivatives. Focussing on arbitrary sections of an image is possible selecting regions of interest for analysis. Low intensitiy images can be enhanced by a pixel binning. Analysed image data is represented by floating point values; saturation effects due to standard image formats as bitmaps can be excluded. Detailed lifetime histogramming features allow visualization of the significance of exponential components in the image as well as assessment of their spatial correlation.

Scritping interface

The SymPhoTime User Programming Script LANGuage (STUPSLANG) is intended to allow users to implement analysis techniques which are not part of the standard SymPhoTime environment. Its approach is to supply a limited amount of simple analysis steps, which may be freely combined to allow more complex analyses.

The software is available in three different packages in order to meet the different needs of the individual users:

• SPT 1A "SymPhoTime" : for analysis of 1-D measurements (stationary beam / sample)
• SPT 1 "SymPhoTime" : for 1-D data acquisition and analysis (stationary beam / sample)
• SPT 2 "SymPhoTime" : for 2-D and 3-D data acquisition and analysis
• SPT 1+2 "SymPhoTime" : complete version

The main features of the four versions are shown in the following table:

	SPT 1A	SPT 1	SPT 2	SPT 1+2
Hardware features with PicoHarp 300 / TimeHarp 200
Import of TTTR data files
Direct data acquisition in TTTR mode
Control of Physik Instrumente scanner (E-710) and large area scanner
Support for Laser Scanning Microscopes (LSM) and generic scanners via external trigger signals
Router support (separation of up to four detector signals)
Multi point measurements
Data analysis features
Time gating for all methods
Multi-channel scaling analysis - Lifetime and intensity time traces
TCSPC-lifetime histogramming and fitting to exponential decay functions up to 4th order incl. tail fitting, reconvolution analysis and maximum likelihood estimation
Burst integrated fluorescence lifetime (BIFL) analysis
On / Off time histogramming and analysis
Burst size histogramming
Fluorescence Correlation Spectroscopy (FCS) incl. Pulsed-Interleaved Excitation (PIE) treatment, (auto- or cross correlation) & FCS fitting
Fluorescence Lifetime Correlation Spectroscopy (FLCS)
FCS calculation and time trace display during the measurement ("online time traces", "online-FCS")
Total correlation (requires PicoHarp T2 mode)
Förster Resonance Energy Transfer (FRET) incl. Pulsed-Interleaved Excitation (PIE) treatment, corrections for direct excitation and bleedthough
Photon Counting Histogramming (PCH)
Generic ASCII export filter
Fluorescence Lifetime Imaging (FLIM) incl. online-visualisation of FLIM data during the measurement, arbitrary regions of interest for analysis, maximum likelihood estimation and bitmap export
Scripting ("STUPSLANG")

Current software version: 4.721

Owners of previous versions receive a free update. Simply contact us via e-mail.