How to calculate physical units from pixel values?

This article deals with questions about the resolution of an image and about how to get the physical quantities of your investigated sample from analyzed data in pixel format.

Image Resolution

Image resolution is the level of detail and clarity in digital images determined by several parameters. The topic of this article will be centered on sensor size, pixel size, and magnification. Although there are also other parameters on which the final resolution depends, those three are the most accessible.

A microscope is any system that magnifies small objects and makes them visible or detectable on a macroscopic scale. In optical digital microscopes, magnification is achieved by projecting the object's image onto an optical sensor through a system of lenses.

Magnification

Magnification is perhaps the most clear factor in microscopy. It dictates how much an object is enlarged when viewed through the microscope. Higher magnification levels allow you to see finer details and reveal the hidden intricacies of your specimen. However, increasing magnification decreases the field of view, meaning you zoom in on a smaller portion of your sample.

Sensor Size

You can think of the sensor size of your digital microscope as the “window“ through which you observe the microscopic world. Larger sensors can capture more light and detail, which leads to higher image quality. A bigger sensor size allows for better light sensitivity and reduces noise, especially in low-light conditions.

Pixel Size / Pixel Density

Pixel density refers to how closely packed the pixels are on the sensor. It is determined by the number of pixels (megapixels) divided by the sensor's physical size. Higher pixel densities provide greater resolution, which means that smaller details can be captured. In microscopy, resolution is vital because it defines the smallest distance at which two separate objects can be distinguished.

Resolution

Resolution in digital microscopy is the product of these three factors. A high magnification level zooms in on your specimen, while a larger sensor captures more detail, and higher pixel density ensures that tiny features are faithfully reproduced in the digital image.

To maximize resolution, balance these elements carefully. If you need to capture fine details, opt for a higher magnification. However, be aware that this may limit your field of view. A larger sensor size can enhance overall image quality, while higher pixel density ensures that each detail is noticed.

To aid the user when translating pixel values to physical values, a measurement on a digital microscope usually also delivers the length comparison value (as in µm/Px).

Translating values

If you want to translate pixel values to actual physical measurements, you need the length comparison factor, which we will refer to as “R” in length per pixel (µm/Px, mm/Px, etc.).

Next, you need to take a closer look at the quality of your output parameter, especially at which unit you want to translate:

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