All ACAS experiment-sets require the user to enter the pixel per µm resolution of the uploaded images (i.e. the number of pixels for each µm of distance in the image).

This is a critical measurement and is used in two ways:

a) the ACAS software reads the number of pixels in the image, but it cannot know how many pixels a typical cell will occupy without this distance factor. This allows the algorithms to optimize the detection of structures.

b) the ACAS software reports its findings in terms of µm lengths and areas. Without this factor, it would report in pixels and leave the final conversions to µm to the researcher.

Resolutions lower than 0.4 pix/µm typically do not provide enough visual information for successful analysis. The typical range we see is 0.5 to 2.0 pix/µm.

Advanced Guide to Pixel Resolution

Advanced Guide to Pixel Resolution, provided by ibidi

Please note the ibidi technical note uses µm/pix whereas ACAS uses pix/µm.

So to get the units required by ACAS, simply  invert the result ( 1 divided by ibidi-number).

The Simple Calculation

The (pixel / µm) resolution is calculated by :

microscopy image resolution formula 1

A common mistake is to invert the calculation. If your microscope software provides resolution in µm/pixel, then you can simply apply 1/x to get the resolution in pixels/µm.

Your microscope imaging software should provide the image width in pixels as well as the image width in microns. If not, refer to the advanced guide linked at the bottom.

As an example, the following image has a resolution of 1.24, which is given by: 2048 pixels divided by 1648 um.

microscopy sample image

Automated Resolution Detection

The ACAS software analyzes the image to detect round-cells in an effort to estimate the image resolution.

However, the ACAS automated resolution detection is not accurate and only used to detect gross errors in the input (for example, the objective zoom may not have been considered in the calculation, or the units were inverted).

If the provided resolution is significantly different from the detected resolution, the user will see a warning in the final report in the Settings Tab and will be asked to validate the provided resolution. If the user is confident in the resolution, the warning can be ignored.