CHAPTER 1 - SYSTEM OPERATIONZEISS Center Screen Area / Image Containers - Display and Image Analysis LSM 880472 000000-2071-464 10/2014 V_01Histogram Threshold:When the Threshold tick box is selected, the controls to set a lower and upper threshold for thehistogram calculation are displayed (Fig. 667). The threshold can be set for each channel (channelselection pull down menu).6.11 Colocalization ViewThe Colocalization function permits interactiveanalysis of two channels of an image by computinga scatter diagram (co-localization).The settings of the Dimensions, Display, Playerand Graphics view options control blocks apply.The additional view-specific Colocalization ViewOption control block is shown in Fig. 668.Any changes done with this View Option controlblock are effective immediately.The Image Display in the Colocalization View shows 3 panels: the scatter diagram, the data table and thepseudo-colored image display (see Fig. 669 and Fig. 670).Colocalization is defined by the presence of two or more different molecules at the samelocation in a specimen. However, in the context of digital imaging, the term colocalization refersto colors emitted by fluorescent molecules detected by the same pixel in the image. It isimportant to be aware of the fact that colocalization cannot be analyzed for fluorophores withsimilar emission spectra. Accurate colocalization analysis is only possible if the fluorescenceemission spectra are well separated between fluorophores and the correct filter sets (or spectraldetection bands) are used for data acquisition. If spectral bleed-through artifacts are presentbecause of spectral overlap between the fluorophore emission spectra, or due to the use ofincorrect filter sets, colocalization measurements will be meaningless. To avoid this, thefluorophores must be carefully chosen and matched to the excitation laser lines to obtain themaximum excitation efficiency while still maintaining a useful degree of separation betweenemission wavelengths. The choice of fluorophores is crucial for colocalization analysis.Fig. 668 Colocalization View Option controlblock
