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How To Quantify Intensity

Pixel values are generally relative values, where a brighter pixel shows a stronger signal, which is sufficient if images are to be printed or shared without any further post-processing or analysis. However, pixel values can be automatically calibrated to real signal values (i.e. quantified to physical values and units) if the acquisition hardware and software are designed for this purpose. Quantified signal values in images bring a number of benefits in microscopy

  • extraction of sample physical material parameters from images
  • direct comparison of signal levels between imagesexperiments andor samplesamples

Prerequisites

For intensity calibration/quantification to work

  • metadata much contain the necessary hardware values as tags
  • scan controller hardware has to be calibrated the particular signal/channel
  • images must be saved without compression, which means 16-bit depth

To verify that a file is recorded in 16-bit depth, if the file is already open in microDIP, press the File Information button in the Quick Access Toolbar, as shown in the screenshot below.

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This will open the native Microsoft Windows File Property windows, where select the Details tab and look for the 'Bit depth' property. 

Intensity Calibration

point electronic GmbH scan controllers are fully calibrated in the factory, therefore images acquired with either REVOLON or DISS6 scan controllers contain pixel intensities (pixel values) that can be calculated, displayed and exported as physical values in the microscope. Typical examples include signal level (V) at the input of the scan controller, or signal current (pA) at the input of a detector or Electrical Analysis (EA) system. These pixel values are calculated on the fly from the raw (ADC) pixel values and a combination of tag values and formulas in the metadata.

Similar with pixel size calibration information, which is shown in microscopy with a Scale Bar, pixel intensity calibration information is shown with an Intensity Bar. This shows the relationship between physical signal values in the microscope to image view grayscale (or colour) on screen. For example, in the screenshot below we have an intensity bar that informs that

  • -9.58 nA in the signal is displayed as black on the display
  • 18.24 nA in the signal is displayed as white on the display
  • there is a constant gradient (change) from black to white on the display

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Intensity bar can be displayed horizontally, as above, or vertically, as below. Vertical intensity bar is particularly useful if the image view on display uses a colour palette with many colour segments.

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If datatype is colour, then the Intensity Bar cannot be show.

Formulas

The basis for offline quantitative measurements is metadata information, which contains

  • hardware parameters and values (metadata tags) used for signal amplification at the time of the image acquisition
  • formulas that give the relationship between these metadata tags and physical signal values

Formulas are therefore essential to quantification, as they enable on-the-fly calculation of physical signal values from the raw 16-bit image values. A typical formula is shown below,

=((INPUT(MICS2)-mics:OutputOffset2)/mics:Gain2-mics:InputOffset2)*1e3

where MICS2 is the signal/channel that this formula applies to (and the selected signal in view), mics:OutputOffset2, mics:Gain2 and mics:InputOffset2 are hardware parameters used for amplification, and 1e3 is a coefficient for V/nA transformation.

On new file open, microDIP will default to RAW (ADC) pixel values and parse formulas found in the image metadata. To select a formula from metadata, use the selection menu in the formula bar, which will then allow a selection of units (e.g. nA, pA and so on).

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microDIP also keeps a library of formulas, which can be accessed by pressing the Library button and then selecting which XML collection of formulas to retrieve.

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If datatype is colour, 8-bit or Index8, then Formula bar cannot be shown.

Older microDIP version used a Value toolbar in the ribbon, with the same functionality as described here.

Calculated Pixel Values

Intensity (calculated value) for each pixel can be seen in the Status Bar, which displays coordinates (X and Y) from the top left corner of the image and pixel intensity for the pixel under the mouse cursor.

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Histogram and line profile plots will also use the calculated pixel intensities, including selection of the minimum and maximum range, as shown in the screenshots below. 

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Image export, as well as plot exports, will write calculated pixel values in the new CSV files.

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