Online Ratio Tab

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.

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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.

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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.

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Image Ratio Type 2

The formula calculates the normalized ratio of the difference between two weighted channel intensities.

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Image Ratio Type 3

The formula calculates the ratio between the weighted difference and weighted sum of two channel intensities

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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:

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None

No background correction is performed.

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Constant

Allows a user defined numeric value to be entered for each channel in the appropriate spin box.

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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.

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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.