# Microscope Scale Bar Calibrator

Calculate microscope object scale, field of view, diffraction limits, Nyquist sampling, and download a transparent SVG scale bar.

> Canonical page: https://elysiatools.com/en/tools/microscope-scale-bar-calibrator

- **Category:** Science & Education

- **Keywords:** microscope scale bar, microns per pixel, numerical aperture, field of view, Nyquist sampling

## Overview

Uses camera pixel pitch, binning, objective and relay magnification to calculate object-space microns per pixel. Optical resolution estimates use Abbe and Rayleigh criteria. The reported optical magnification is not screen viewing magnification.

## Inputs

- **Image width (px)** (number)
- **Image height (px)** (number)
- **Camera pixel size (µm)** (number)
- **Objective magnification** (number)
- **Relay magnification** (number)
- **Camera binning** (number)
- **Emission wavelength (nm)** (number)
- **Numerical aperture (NA)** (number)
- **Scale bar length (µm, 0 = auto)** (number)
- **Scale bar color** (color)
- **Scale bar position** (select)
- **Scale bar thickness (px)** (number)
- **Scale bar margin (px)** (number)
- **Show label** (checkbox)

## When to use

- When preparing microscopy images for scientific publication and you need to overlay an accurate, high-quality scale bar.
- When verifying if your camera sensor and optical setup satisfy the Nyquist sampling theorem for a specific emission wavelength.
- When calculating the physical field of view (FOV) and object-space pixel size of a new microscope configuration.

## How it works

- Enter your camera's physical pixel size, binning factor, objective magnification, and any relay lens magnification to determine the object-space microns per pixel.
- Input the light wavelength and numerical aperture (NA) to compute the theoretical diffraction limits using the Abbe and Rayleigh criteria.
- Specify your desired scale bar length, color, thickness, and position to generate a custom SVG overlay.
- Download the transparent SVG scale bar to overlay directly onto your acquired microscopy images.

## Use cases

- Calibrating a 40x objective setup with a sCMOS camera to generate a 20 µm scale bar for fluorescence imaging.
- Assessing if a 100x oil-immersion objective setup meets Nyquist sampling criteria at 488 nm excitation.
- Determining the total field of view (FOV) in microns for a large-format sensor on a stereo microscope.

## Frequently asked questions

### How is the object-space pixel size calculated?

It is calculated by dividing the physical camera pixel size (multiplied by the binning factor) by the total optical magnification (objective magnification multiplied by relay magnification).

### What is the difference between optical magnification and screen magnification?

Optical magnification is the physical magnification of the lenses, whereas screen magnification depends on the display size and resolution when viewing the image.

### How does the tool estimate optical resolution?

It uses the emission wavelength and numerical aperture (NA) to calculate the diffraction limits based on the Abbe and Rayleigh criteria.

### What is the Nyquist sampling check?

It evaluates whether your pixel size is small enough (at least twice as small as the optical resolution limit) to resolve the finest details without aliasing.

### Can I customize the appearance of the scale bar?

Yes, you can adjust the scale bar length, color, thickness, margin, position, and toggle the text label.

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