Introduction
Why Use FLIM
Requirements to a FLIM Technique
Multi-Dimensional Time-Correlated Single Photon Counting
One-Photon FLIM versus Multi-Photon FLIM
The Leica FLIM Systems
External FLIM System Configuration
Internal SP FLIM System Configuration
SP5 Setup for FLIM measurement
Laser Setup for D FLIM
Laser Setup for MP FLIM
Setup of Imaging Parameters in LASAF Software
FLIM data acquisition using the external FLIM detector
Scanning a Sample
FLIM data acquisition using internal SP FLIM detectors
FLIM Data Acquisition Software
DCC-100 Software
SPCM Software
TCSPC System Parameters
Measurement Control Parameters
CFD, SYNC and TAC Parameters
Data Format and Page Control
Scan Parameters
Access of System Parameters from Main Panel
Display of Images in the SPCM Software
Online Display
Display Parameters
Window Parameters
Saving Setup and Measurement Data
Loading Setup and Measurement Data
Setup Files Delivered with the Leica SP5 FLIM System
Predefined Setups
FLIM Measurements
Steps of a FLIM Measurement
Details of FLIM Data Acquisition
Count Rates
Photobleaching
Acquisition Time of FLIM
Image Size
Data Analysis
Introduction
Analysing fluorescence lifetime images
Loading of FLIM Data
Hot Spot and Region of Interest Selection
Instrument Response Function
Fit Selection Parameters
Binning of Pixels in the Data Analysis
Model Selection
Calculation of the Lifetime Image
Display of Lifetime Images
Lifetime Parameter Histogram
Special Commands
Applications
Measurement of Local Environment Parameters
Fluorescence Resonance Energy Transfer (FRET)
Autofluorescence Microscopy of Tissue
References
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