ZEISS Knowledge Base

In the Mean ROI view you can draw ROIs and measure their intensity profile after acquiring time series experiments. The intensity profiles are displayed as charts and can be exported to data tables.

The Physiology (Dynamics) module activates additional features to those of MeanROI for the offline analysis of physiology experiments, e.g. Ratio functions and ROI tracing.
In this view the Image area is always to the left, charting area always to the right. Depending on which Region layout you have selected in Layout tab, the MeanROI view can have a different appearance.

1	Image area Here you see the images for each channel of the time series and the ratio image (if the ratio calculation is enabled). The display of images can be adapted in the Layout tab.
2	Charting Area Here you see the charts for the values of all channels selected in the Layout tab as well as for the ratio calculation (if it is enabled on the Ratio tab). If a ROI is selected in the image area on the left, the corresponding plot line is highlighted (the plot line is thicker) in the charts. The lines in the merged channel image can either have the color of the ROI, or the color of the channel (option available on the Charts tab). Playhead (blue line) Indicates the current frame of the time series visible in the image panel(s). The position is synchronized with the displayed image frame number and vice versa and it can be moved via drag & drop. The current time point of the visible frame is displayed to the right of the playhead line in the same time unit as the x-axis of the chart.
3	Table Displays the values for all channels and regions at the different time points, as well as the temperature, focus (if present in the image metadata) and information about markers. If you deactivate a channel in the Visible Charts section of the Layout tab, the corresponding columns are hidden in the table. This table is synchronized with the image view and the charts. If you select a field in the table, the corresponding ROI is selected in the image view and the charts (playheads) are updated accordingly.
4	Time Line View This view is activated in the Layout tab. The chart supports similar functions as the other charts in MeanROI. Here you can limit the time range with the zoom functionality. The actions in this chart are synchronized with the others in the MeanROI view.
5	View Options Here you have your standard view options as well as specific options for MeanROI, for example for the Layout or the calculation of the Ratio.

Hover over the plot with the mouse (crosshair). A tool tip appears with details of the intensity value at this position, ROI ID #, Channel, and time point (in currently set time unit of x-axis). Note these values (intensity and time) are interpolated. You can visualize the time points alone a plot by activation of the Show Tick marks function on the Chart tab.

Charts Tab

Parameter		Description
All Chart Settings (X-/Y-Axis)		Note that a function is active when the button is highlighted in blue. The settings for X- and Y-Axis (only if Show All is activated) are the same, see description below. The Y- axis settings are always applied to the selected chart. The currently selected chart name (channel) is displayed above the Y-axis settings.
-	Auto	The scaling of the respective axis is automatic, allowing for an optimal, and appropriate adjusting display of the all values.
-	Fixed	The upper and lower limit of the axis can be defined using the min and max spin boxes.
X-Units
-	Auto	The units are selected automatically.
-	Fixed	You can select the desired unit for the x-axis from the dropdown list.
Show Tick Marks		Activated: Displays tick marks in the chart. You can set the Form and Size of the tick marks. The tick marks have to be set per chart.
Show Legend		Activated: Displays the chart legend.
Show Axis Captions		Activated: Displays captions of the axis.
Use Channel Color		Only visible if you display the merged channels chart. Activated: Displays the lines in the chart in the colors of the channel. If a channel has no color defined, the chart line is displayed in white. The colors are synchronized with the channel settings, i.e. they update accordingly if the channel colors are changed.

Ratio Tab

This view option is almost identical to the Online Ratio tab in the MeanROI Setup, see Online Ratio Tab. In fact when an experiment is finished the exact same values used for the display of the online ratio are transferred to the offline ratio tool of the MeanROI view and are stored with your image for later reference.
For offline ratio assessment the settings can be changed and are applied to the ratio image and the measured values. For large data sets (high resolution or many time points), a smooth playback of the ratio image can be achieved by clicking on the Cache ratio image button. This stores the ratio images temporarily to the computers memory for very fast playback or adjustments of the time slider. The following descriptions describe the differences on the Ratio tab:

Parameter	Description
Ft₀= Average intensity of frame	Only available if the Single wavelength method is selected. Defines the frames of the time series image from which the reference value Ft ₀ should be calculated. The numbers in the input fields refer to the frame number of the current experiment in the MeanROI view. The desired frame numbers can be entered with the buttons or directly by typing a number into the field. Typically, your experiment should include a baseline of 5-10 images before and/ or in between a simulation/ activation.
–	Update	Applies the changes of the frame made with the input fields.
Cache ratio image	Caches all the ratio images of the current time series with the given ratio calculation parameters. This is done to avoid image flickering when moving through the time series with the slider quickly.

Parameter

Description

Ft₀= Average intensity of frame

Only available if the Single wavelength method is selected.

Defines the frames of the time series image from which the reference value Ft ₀ should be calculated.
The numbers in the input fields refer to the frame number of the current experiment in the MeanROI view. The desired frame numbers can be entered with the buttons or directly by typing a number into the field. Typically, your experiment should include a baseline of 5-10 images before and/ or in between a simulation/ activation.

–

Update

Applies the changes of the frame made with the input fields.

Cache ratio image

Caches all the ratio images of the current time series with the given ratio calculation parameters. This is done to avoid image flickering when moving through the time series with the slider quickly.

Parameter		Description
Calculation Dropdown		Selects the ratiometric method you want to use. Single and Dual Wavelength dyes are supported with an additional three formulas for further adapted (online or offline) image ratio calculations. The ratio set-up will change in accordance with your selection.
-	Single Wavelength	Select the channel in the dropdown menu. The Ft₀ value is the averaged fluorescence from the specified number of image frames. The number of frames to average is defined in the spin box of the reference image set-up (see 10). The spin box at the far left is a multiplication factor.
-	Dual Wavelength	Select the channels in the dropdown list required to calculate the ratio values/image e.g. for Fura-2, a dual excitation dye, the numerator is the 340 nm image the denominator the 380 nm image. For dual emission dyes the function is identical. The spin box at the far right is a multiplication factor.
-	Image Ratio Type 2	The formula calculates the normalized ratio of the difference between two weighted channel intensities.
-	Image Ratio Type 3	The formula calculates the ratio between the weighted difference and weighted sum of two channel intensities
-	Image Ratio Type 4	The formula calculates the ratio between the intensity difference of two channels in relation to the intensity of one channel.
Background Correction		A background correction can be performed on a channel-by-channel basis. The selection of a background correction method modifies the ratio set-up formula accordingly. Note that the correction by ROI is only available if there are at least two ROI defined in the image! The following modes are available:
-	None	No background correction is performed.
-	Constant	Allows a user defined numeric value to be entered for each channel in the appropriate spin box.
-	ROI	Allows to select the background ROI defined in the Mean ROI view/setup. Note that for dual wavelength protocols the same ROI is used in each case, but its channel specific values are applied for the correction. For the ratio types Single Wavelength, Image Ratio Type 2, 3, and 4, no ROI background correction is available.
Ratio Clipping		Activated: Sets the factor for ratio clipping.
Color		Select the color (LUT) used to display the ratio image. Per default the Rainbow LUT is used as it allows intensity changes to be followed easily.
Enable Threshold		Activated: Allows the threshold values to be set for the ratio calculation.
-	Channel/Threshold	A threshold value can be applied in the form of a constant integer value for each channel individually. Thresholds help to reduce noise anomalies that are cause by pixel-to-pixel variations in areas between cells or near cell boarders during the ratio calculation. Enter the desired threshold value for each channel into the spin boxes provided. For more detailed information on how ZEN handles thresholds, see Basics of Calculation of Intensity and Ratio Values.

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Mean ROI View

Charts Tab

Ratio Tab

Online Ratio Tab

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