Biology Reference
In-Depth Information
14.3.6.1
Evaluation procedure
Preprocessing includes steps from 1 to 7
1.
Data import to evaluation software must be achieved individually. In our case,
the Fluorolog data were imported in ASCII format, and the microscope images
and metadata were imported with self-written routines.
2.
Saturated regions of images were indicated and excluded from further
processing.
3.
Since acquisition devices work with unsigned integer, we introduced an
artificial offset while acquisition. Images were offset corrected either done by
selecting regions where fluorescence signal was absent or done by using dark
images recorded extra with no laser excitation. Values from dark regions were
further subtracted from the sample image.
4.
The inhomogeneous illumination correction was done according to the specific
excitation wavelengths. The inhomogeneous illumination was recorded by
fluorescent slides (Chroma Technology). The correction for inhomogeneous
illumination is essential for low-quality objectives and large field of views,
because the intensity at the peripheral regions of the images can drop to 80%.
The intensity drop usually shows an individual characteristic for the used
excitation wavelength and the specific emission channels.
5.
Since bleaching correction is hardly possible, we preferred to adjust and
optimize acquisition conditions to avoid it. This check is also recommended for
Fluorolog data.
6.
For pixel-based analysis, subpixel shift correction between two excitations for
Zeiss LSM and between emission channels caused by imperfect alignment of
the OptoSplit II and dichrotome in case of spinning disk and TIRF is necessary.
Data shift correction was performed by self-written routines based on image
registration and regional image transformation, allowing shifts with a low-order
polynomial spatial function.
7.
Gauss blurring (standard 0.4 pixel) was applied to soften the image view in order
to slightly reduce noise level that could be increased according to imaging
conditions.
Reference spectra analysis (steps 8-10)
8.
From reference images, several regions of interest were selected to define
reference spectra for unmixing in further FRET analysis.
9.
Reference spectra were determined from ROIs (microscopes) and reference
samples (Fluorolog) selected at each excitation. In addition, all acquisition
parameters were kept constant along with stored spectra.
10.
Spectral analysis was done and the characteristic spectra and relative
amplitudes were stored for further FRET calculation (for Fluorolog data
as well).
