Zeiss Lightsheet Z.1 Operating Manual

Contents

CHAPTER 4 - SYSTEM OPERATIONLightsheet Z.1 Center Screen Area / Image Containers - Display and Image Analysis Carl Zeiss02/2013 000000-1790-528 225Histogram Threshold:When the Threshold tick box is selected, thecontrols to set a lower and upper threshold for thehistogram calculation are displayed (Fig. 337). Thethreshold can be set for each channel (channelselection pull down menu).4.10 Colocalization ViewThe Colocalization function permits interactiveanalysis of two channels of an image by computinga scatter diagram (co-localization).The settings of the Dimensions, Display, Player,and Graphics view options control blocks apply.The additional view-specific Colocalization ViewOption control block is shown in Fig. 338.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. 339 and Fig. 340).Colocalization is defined by the presence of two or more different molecules at the same locationin a specimen. However, in the context of digital imaging, the term colocalization refers to colorsemitted by fluorescent molecules detected by the same pixel in the image. It is important to beaware of the fact that colocalization cannot be analyzed for fluorophores with similar emissionspectra. Accurate colocalization analysis is only possible if the fluorescence emission spectra arewell separated between fluorophores and the correct filter sets are used for data acquisition. Ifspectral bleed-through artifacts are present because of spectral overlap between the fluorophoreemission spectra, or due to the use of incorrect filter sets, colocalization measurements will bemeaningless. To avoid this, the fluorophores must be carefully chosen and matched to theexcitation laser lines to obtain the maximum excitation efficiency while still maintaining a usefuldegree of separation between emission wavelengths. The choice of fluorophores is crucial forcolocalization analysis.Fig. 337 Histo View Option control block –ThresholdFig. 338 Colocalization View Option controlblock