Saving and Printing the Results and Data. UVP DigiDoc-It

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You can also Update the results from the Table menu on the Area Density Results window.

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Save the Results

From the Area Density Results window > File menu you can select from multiple options if you need to save the analysis data:

Save Outlines: Lets you save the file

Load Outlines

Print the Results

From the Area Density Results window > File menu you can select from multiple options if you need to save the analysis data:

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Page Setup

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Print Preview

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Print

Export the Results

From the Area Density Results window > Data menu you can select from multiple options if you need to save the analysis data:

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Copy to Clipboard

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Export to CVS

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Export to Excel

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Intensity Calibration is the method of creating a mapping (and subsequently a curve) of input intensities to output intensities. Ideally, such a mapping would be linear. However, if you require finely tuned results, you may need to take into account, various errors introduced in the process

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ENSITY of lighting and imaging. These errors may change the mapping of original input intensity value to higher or lower than that. E.g. a pixel value of 100 might look slightly brighter, say 120. Or possibly the camera being used has a noise step of 40, which will make all values below 40 look as dark as 0.

One can use Area Density tool in LS Software to carry out analysis based on two different metrics of light intensity:

LS software lets you create curves for both types of calibrations.

Optical Density

Standard Optical Density (OD) is used when the sample of interest is imaged with transmitted light. (i.e. light going thru the sample, into the camera for imaging.) OD value of an area gives an idea of how much light can pass through that area. If the area belongs to a sample in question, it simply how much of sample might be present in that area. Higher OD means less light can get through, suggesting presence of higher quantity of sample.

Following Beer’s Law, the Optical Density of a given pixel P (say at position x,y) is calculated by LS software in the following way:

OD = - Log [ (P(x,y) – Black) / (Incident – Black) ], if P(x,y) < Black

= - Log [ 1/ (Incident - Black) ], otherwise

Where

White = value of brightest white pixel in the imaging environment

Black = value of darkest black pixel in the imaging environment

Total Optical Density of an area is simply the sum total of OD values of all pixels.

Grey Levels

Grey Level calculations are used when the sample is imaged using reflective light. Grey level of an area is simply the sum of grey levels of all pixels in the area.

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To Calibrate the Intensity

To add points on the Intensity Calibration Curve, click Add on the Points List

Collection window. Click OK. You can also add points by clicking the Edit List button on

Points List Collection window.

Select the Free Form curve for use on fluorescent/luminescent samples to produce results in Grey Levels.

Select the Std. Optical Density for colorimetric samples imaged using transmitted white light to produce results in Optical Density units.

The scroll-box Number of Samples lets you select the total samples you want displayed on the X-axis of the curve. By default, it is set to the highest value of the dynamic range of the image (8 bit=>256, 16bit =>65536).

To Calibrate Free Form Intensity Curve

Click on the Data Points tab in the Intensity Calibration Curve window. This window lets you manually add or edit each point on the curve (hence the name Free Form). For each intensity volume there is a calibrated volume value that can be assigned. You can also fit a proper curve (in the Graph tab > Curve Model).

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Changes are reflected on the curve after the window is closed.

Special Cases of Free Form Curves

Calibration Always Positive

A curve of the following type (left) has a significant section in the negative ‘Y’. If you may not want such a curve, click on Calibration Always Positive checkbox shown above in the Intensity Calibration Curve > Graph Options tab. Result is the curve on the right.

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Monotonic Calibration

A curve of the following type (left) can have two values of input (X) for some values of output (Y). In order to avoid that, check the box Monotonic Calibration from the

Intensity Calibration Curve > Graph Options tab to turn the slope of the curve all positive (or negative). Corrected curve looks like the one on the right below.

Saving and Loading calibration

In LS software, Intensity Calibration can be saved as well as loaded back when required. The calibration is stored in a .cal file. It stores information about all calibration curves created.

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