SymPhoTime 64

Fluorescence Lifetime Imaging and Correlation Software

  • Powerful 64bit TTTR data acquisition and analysis software
  • Point, 2D, and 3D data acquisition with online preview of FLIM, FCS, time-traces, or TCSPC histograms
  • FCS, FCCS, FLCS, PIE-FCS, coincidence correlation, total correlation
  • Fluorescence time trace analysis, single molecule burst analysis
  • Anisotropy
  • TCSPC lifetime fitting with advanced error treatment
  • User programming script language "STUPSLANG"

The SymPhoTime 64 software package is an integrated solution for data acquisition and analysis using PicoQuant's time-resolved confocal microscope MicroTime 200, MicroTime 100, LSM upgrade kits or TCSPC electronics. Its clearly structured layout and powerful analysis routines allows the user to focus on the results rather than on the data processing. The software is designed for a 64 bit operation system and features a graphical user interface (GUI), which guides the user through all necessary steps for an individual analysis or measurement process. Data dependencies are directly visible in the underlying workspace concept. An integrated scripting language ("STUPSLANG") even puts the user in a position to freely add new analysis procedures or customize existing ones.

Data acquisition with PicoQuant TCSPC modules

SymPhoTime 64 - easy-to-use data acquisition which preserves all photon timing informationThe SymPhoTime 64 is the dedicated data acquisition software for PicoQuant's time-resolved confocal microscopes MicroTime 200 and MicroTime 100 and LSM upgrade kits. It can of course also be used with custom set-ups based on PicoQuant TCSPC electronics. All data acquisition and analysis features of the SymPhoTime 64 are based on the unique time-tagging modes of the MultiHarp 150, TimeHarp 260, PicoHarp 300, PicoHarp 330 or HydraHarp 400. In these modes, photons on each detection channel are tagged with the absolute arrival time since the beginning of the measurement and, in certain cases, also along with the time difference to the last laser pulse. This scheme preserves all photon timing information and allows a large range of data interpretation ranging from simple TCSPC histograms to complex imaging and correlation analysis.

Adapted interfaces for imaging analysis

SymPhoTime 64 - screen shot showing the FLIM-FRET analysis moduleWith the SymPhoTime 64, analysis of (time-resolved) imaging measurements will be easier than ever before. The software provides special adapted interfaces for many standard analysis procedures ranging from Fluorescence Lifetime Imaging (FLIM) to Fluorescence Resonance Energy Transfer (FRET) and Anisotropy. Each interface only makes those procedures available that are directly required for the individual analysis. This ensures a steep learning curve as well as quick and correct analysis results.

Ultrafast software correlator

SymPhoTime 64 - screen shot of the FCS analysis module with ultrafast software correlatorThe SymPhoTime 64 also sets a new standard for analysis of fluorescence correlation spectroscopy measurements. The software provides a wide range of specially adapted correlation analysis procedures, which range from classical auto-correlation (FCS) and cross-correlation (FCCS) to lifetime based correlation analysis (FLCS) and total correlation. Even coincidence-correlation analysis is possible based on the unique time-tagging modes of the PicoQuant TCSPC modules. By exploiting the full power of a multi-core computer system, the SymPhoTime 64 is one of the fastest software correlators on the market.

Intensity time trace analysis

SymPhoTime 64 - screen shot of the time trace analysis moduleThe analysis of fluorescence intensity time traces is another core feature of the SymPhoTime 64. Fluorescence intensity time traces display the measured fluorescence dynamics and can be analysed in a variety of ways. Prominent examples are classical single molecule methods such as on/off histograms, burst size histograms or fluorescence lifetime traces.


STED super-resolution

SymPhoTime 64 - performing STED analysisThe SymPhoTime 64 supports the super-resolution STED add-on for the MicroTime 200. Different data acquisition modes including interleaved trains of excitation and STED laser pulses are integrated in the GUI. STED images as well as time-gating for gSTED are displayed online during data acquisition. Multiple image processing approaches such as gSTED and Pattern Matching are implemented for even further improvement of the resolution. STED-FCS is also available.

Different packages for different requirements

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

  • SPT64 1A "SymPhoTime 64": for analysis of 1-D measurements (stationary beam / sample)
  • SPT64 1 "SymPhoTime 64": for 1-D data acquisition and analysis (stationary beam / sample)
  • SPT64 2 "SymPhoTime 64": for 2-D and 3-D data acquisition and analysis
  • SPT64 1+2 "SymPhoTime 64": complete version
  • SPT64 1+2+3 "SymPhoTime 64": complete version for STED

Current software version: 2.8

The latest version supports the PicoHarp 330, ASCII ROI import/export as well as manual adjustment of laser position during point measurements.

Data Acquisition
Supported TCSPC modules HydraHarp 400, PicoHarp 300, PicoHarp 330, TimeHarp 260, MultiHarp 150, MultiHarp 160, TimeHarp 200 (Data import ONLY!)
Supported Configurations MicroTime 200 with 100 x 100 (x 100) µm or 10x7.5 cm piezo scanner or FLIMbee galvo scanner
MicroTime 100 Laser Scanning Microscopes (LSM)from Nikon, Olympus or Zeiss
Stand-alone TCSPC modules
Remote control via TCP/IP interface (software handshake with ZEN and NIS Elements)
Routing 1 to 8 detectors
Measurement modes Single point, multi-point, 2D imaging (XY, XZ, YZ), 3D imaging (XYZ), time lapse (XYT), oscilloscope mode for alignment purposes
Measurement previews FLIM, FCS, FLCS and FCCS, Time Trace, TCSPC histogram
Parallel calculation and display of up to 4 different previews
Automated Measurements Z–Stacks, Time Stacks,  Image Stitching, Multi-Point Measurements
Hardware control PDL 828 "Sepia II" laser driver
E-710, E-725, E-727 and wide range scanner controller (Physik Instrumente)
Shutter of MicroTime 200
Wide-field fluorescence camera IDS uEye USB3 in MicroTime 200
Data Analysis
General Features Time gating
TCSPC Binning
TCSPC Fitting (Multi–Exponential Decay (1 to 5 Exponentials), Least–Squares Fitting, MLE Fitting, IRF Reconvolution, Tailfit, Bootstrap error analysis)
Global analysis of TCSPC curve fitting
GUI Themes
Imaging FLIM, FLIM-FRET, Intensity FRET, Anisotropy Imaging, (Time-Gated) Fluorescence Intensity Imaging
Pattern Matching, Fast Pattern Matching
Adjustable color scale
Region-of-Interest (ROI)
Bin export for phasor analysis (via third party software Globals developed by the Laboratory for Fluorescence Dynamics)
Correlation FCS, FCCS, FLCS, PIE–FCS
FCS Fitting (Models: Diffusion Constants, Triplet state, Conformational, Protonation, Gaussian PSF, User-defined models via scripting, Bootstrap error analysis)
Global analysis
FCS calibration
Antibunching/Coincidence correlation, Total correlation
FRET PIE (Pulsed Interleaved Excitation)
Bleedthrough Correction
STED STED, gatedSTED, STED-FLIM, Pulsed Interleaved STED and Confocal, Resolution estimation
Fluorescence Intensity Traces Blinking (On/Off Histogramming), Count Rate Histogram (PCH), Burst Size Histogram, Intensity–Gated TCSPC, Fluorescence Lifetime Traces, Lifetime Histogram, BIFL (Burst Integrated Analysis
Steady–State Anisotropy Objective correction factors included
Export data formats BMP, ASCII, TIFF, BIN
User Scripting (STUPSLANG) User–Defined Analysis Procedures, Fitting Functions, Multi–Parameter Filtering
Control of external hardware via suited interface
Required PC
with SSE2 and EMT64 or AMD64 extension; recommended: quad–core or better
CPU clock 2.2 GHz (or better) quad-core CPU
RAM minimum 4 GB (suggested 16/32 GB)
Operating system Windows 10 x64
Disk space >= 100 MB (except data storage)
Display(s) Full HD Display
Protection Module (HASP) USB


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.

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

  SPT64 1A SPT64 1 SPT64 2 SPT64 1+2 SPT64 1+2+3
Data Acquisition Features
Direct data acquisition in TTTR mode
using MultiHarp 150/160, TimeHarp 260, PicoHarp 300, PicoHarp 330 or HydraHarp 400
Control of Physik Instrumente scanner    
Support for Laser Scanning Microscopes (LSM)
and generic scanners via external trigger signals
STED imaging, STED-FLIM, STED-FCS        
Router support (separation of up to eight detector signals)
Multi point measurements      
Data Analysis Features
Time gating for all methods
TCSPC-lifetime histogramming and fitting to exponential
decay functions up to 5th order incl. tail fitting, reconvolution
analysis, maximum likelihood estimation and bootstrap error analysis
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
Pattern Matching, Fast Pattern Matching    
Fluorescence Correlation Spectroscopy (FCS)
incl. Pulsed-Interleaved Excitation (PIE) treatment,
(auto- or cross correlation) & FCS fitting with bootstrap error analysis
Fluorescence Lifetime Correlation Spectroscopy (FLCS)  
FCS calculation and time trace display during the
measurement ("online time traces", "online-FCS")
Total correlation  
Förster Resonance Energy Transfer (FRET)
incl. Pulsed-Interleaved Excitation (PIE) treatment,
corrections for direct excitation and bleedthough
STED imaging, STED FLIM, gatedSTED, Pulsed Interleaved STED and Confocal, Resolution estimation        
Multi-channel scaling analysis – Lifetime and
intensity time traces
Burst integrated fluorescence lifetime (BIFL) analysis  
On/Off time histogramming and analysis  
Burst size histogramming  
Calculation of Photon Counting
Histogramm usable for PCH
Generic ASCII export filter  
Scripting ("STUPSLANG")

The following documents are available for download:

Presentations (as PDF files)

The following step-by-step tutorials for the SymPhoTime 64 are available at

  • Script registration
  • Focal width determination
  • Calibrate the confocal volume using the FCS calibration script
  • TCSPC fitting script
  • Lifetime fitting using the FLIM script
  • Lifetime separation using the FLCS script
  • Using the intensity time trace script
  • Caluclate and fit FCS traces with the FCS script
  • Calculate FCCS traces with the grouped FCS script
  • Using the FLCS script for spectral crosstalk removal via FLCS
  • ROI-fitting using the FLIM script
  • Visualizing dynamics using the multi frame FLIM script
  • Pattern matching
  • FLIM-FRET calculation for multi-exponential donors
  • FLIM-FRET calculation for single-exponential donors
  • Calculate ratiometric FRET images with the FRET image script
  • Single-pair FRET distributions using the FRET script
  • Single-pair FRET distributions using the PIE-FRET script
  • Using the anisotropy image script
  • Using the antibunching script

Latest 10 publications referencing MicroTime 200, LSM Upgrade Kit, MicroTime 100, 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 containing the appropriate citation. Thank you very much in advance for your kind co-operation.

How can we help you?