Specifications

Fluorescence Lifetime Imaging 33
the FLIM image. Moreover, many microscopes send a frame clock pulse some pixels before
the start of the useful part of the line. These pixels can be excluded from the recording by de-
fining a ‘Left Boarder’ larger than zero. Settings typical for FLIM with the Zeiss LSM 510 and
LSM 710 microscopes [7] are shown in Fig. 50, right.
Fig. 50: Scan synchronisation parameters. Left: FLIM image has same pixel numbers as microscope scan. Right:
Pixels and lines are binned by a factor of two, and the recording starts 11 pixels after the line clock.
The recommended main panel configuration for DPC-230 FLIM with the bh DCS-120 scan-
ner [4] is shown in Fig. 51. A time-gated image is displayed on the left, the scanner control
panel is kept open on the right.
Fig. 51: Recommended main panel configuration for FLIM. Gated Intensity image and scan parameters of
DCS-120 system displayed.
A measurement in the FLIM modes of the DPC module delivers the photon distribution over
the coordinates of the scan and the time within the fluorescence decay. The data can be con-
sidered an array of pixels, each containing a large number of time channels spread over the
fluorescence decay. To obtain fluorescence lifetimes the decay curves in the individual pixels
must be fitted with an appropriate model. The SPCM software has the SPCImage [4] FLIM
data analysis integrated. A lifetime image obtained by fitting a triple-exponential model to the
data is shown in Fig. 52.