Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
ZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880
472 000000-2071-464 10/2014 V_01
Histogram Threshold:
When the Threshold tick box is selected, the controls to set a lower and upper threshold for the
histogram calculation are displayed (Fig. 667). The threshold can be set for each channel (channel
selection pull down menu).
6.11 Colocalization View
The
Colocalization
function permits interactive
analysis of two channels of an image by computing
a scatter diagram (co-localization).
The settings of the
Dimensions
,
Display
,
Player
and Graphics view options control blocks apply.
The additional view-specific
Colocalization View
Option control block is shown in Fig. 668.
Any changes done with this View Option control
block are effective immediately.
The Image Display in the Colocalization View shows 3 panels: the scatter diagram, the data table and the
pseudo-colored image display (see Fig. 669 and Fig. 670).
Colocalization is defined by the presence of two or more different molecules at the same
location in a specimen. However, in the context of digital imaging, the term colocalization refers
to colors emitted by fluorescent molecules detected by the same pixel in the image. It is
important to be aware of the fact that colocalization cannot be analyzed for fluorophores with
similar emission spectra. Accurate colocalization analysis is only possible if the fluorescence
emission spectra are well separated between fluorophores and the correct filter sets (or spectral
detection bands) are used for data acquisition. If spectral bleed-through artifacts are present
because of spectral overlap between the fluorophore emission spectra, or due to the use of
incorrect filter sets, colocalization measurements will be meaningless. To avoid this, the
fluorophores must be carefully chosen and matched to the excitation laser lines to obtain the
maximum excitation efficiency while still maintaining a useful degree of separation between
emission wavelengths. The choice of fluorophores is crucial for colocalization analysis.
Fig. 668 Colocalization View Option control
block