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Microscope pixel size calculator: µm per pixel and ImageJ Set Scale

Q: What pixel size do I need for Nyquist sampling?

At most half the resolution, with the resolution r = 0.61 λ/ NA. For 520 nm emission and NA 0.75, r = 0.423 µm, so the pixel should be 0.211 µm or smaller; with a 6.5 µm camera pixel that takes about 31× total magnification.

Pixel size in the specimen = camera pixel size × binning ÷ total magnification: a 6.5 µm camera pixel behind a 20× objective covers 0.325 µm (3.077 pixels per µm). To measure it instead, photograph a stage micrometer and divide the known distance by its length in pixels; for an SEM or TEM image, divide the horizontal field width by the image width in pixels. The calculator gives µm per pixel, pixels per µm, the field of view, whether the pixel meets Nyquist sampling, and what to type into ImageJ’s Analyze › Set Scale.

What it does: Calculates microscope pixel size in µm from the camera pixel, magnification and binning, a stage micrometer or an SEM field width, with the values for ImageJ Set Scale.
Formula: $\begin{aligned} &\text{pixel size} = \text{camera pixel} \times \text{binning} \\ &\quad {}\div \text{total magnification} \\ &\text{or known distance} \div \text{pixels} \\ &\text{Nyquist: pixel} \le r \div 2, \\ &\quad r = 0.61\,\lambda \div \mathrm{NA} \end{aligned}$
Example: A 6.5 µm camera pixel behind a 20× objective without binning: 6.5 ÷ 20 = 0.325 µm per pixel (3.077 pixels per µm).

Last updated 8 November 2026

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At a glance

Microscope pixel size calculator at a glance
Formula	$\text{pixel size} = \text{camera pixel}$ ${}\times \text{binning}$ ${}\div (\text{objective}$ ${}\times \text{extra magnification})$
Or measured	$\text{known distance}$ ${}\div \text{its length in pixels}$ (stage micrometer, scale bar); $\text{field width}$ ${}\div \text{image width}$ (SEM, TEM)
Units	nm, µm or mm in; µm/px, nm/px and px/µm out
Nyquist	$\text{pixel} \le r / 2$ , with $r = 0.61\,\lambda / \mathrm{NA}$
ImageJ	Distance in pixels, Known distance, Pixel aspect ratio 1, Unit of length
Output	Pixel size, field of view, sampling check, Set Scale values, scale bar length in pixels
Runs in	Your web browser; nothing is uploaded
Price	Free, no account needed

What it does

How to use the microscope pixel size calculator

Choose the routeFrom the camera if you know its pixel size, from a calibration image if you photographed a stage micrometer or have a scale bar, or from the field width for an electron micrograph.
Enter the valuesCamera pixel (µm), objective and any extra magnification and the binning; or the known distance and its length in pixels; or the field width and the image width.
Read the pixel sizeRead µm per pixel and pixels per µm, and the field of view if you entered the sensor size.
Check the samplingAdd the objective’s NA and the emission wavelength to see whether the pixel is at most half the resolution (Nyquist).
Set the scaleType the four values shown into Analyze › Set Scale in ImageJ or Fiji, or into Image › Scale in LabCanvas Image Analysis, then measure in µm.

How it works

$\text{pixel size} = \frac{\text{camera pixel} \times \text{binning}}{\begin{array}{c} \text{objective} \\ \times\ \text{extra magnification} \end{array}}$ $\text{pixel size} = \frac{\text{known distance}}{\text{its length in pixels}}$ $\text{pixel size} = \frac{\text{field width}}{\text{image width in pixels}}$ $r = \frac{0.61\,\lambda}{\mathrm{NA}}; \quad \text{Nyquist: pixel} \le r / 2$

From the camera. The objective, with the tube lens and any relay optics, projects a magnified image onto the camera, so one sensor pixel covers its own size divided by the total magnification. The total magnification is the objective’s magnification multiplied by that of the other components between it and the camera (North 2006). Binning $n \times n$ adds $n \times n$ sensor pixels into one, so the image pixel is $n$ times larger and the field of view stays the same: with 6.45 µm photodiodes behind a 100× objective one pixel covers about 65 nm of the specimen, 129 nm with 2 × 2 binning and 258 nm with 4 × 4 (Waters 2009). The field of view is the sensor width in pixels times the sensor pixel divided by the magnification. Several scientific CMOS cameras have 6.5 µm pixels on a 2048 × 2048 sensor (for example the Hamamatsu ORCA-Flash4.0 V3 and the Teledyne Photometrics Prime BSI Express, by their makers’ specifications); take your camera’s value from its data sheet.

From a calibration image. The nominal magnification is not exact, so measure it: photograph a stage micrometer (a slide with a ruled scale of known spacing) with the same objective, camera adapter and binning as your images, as North (2006) recommends for finding the total magnification. Draw a straight line along as many divisions as fit, end to end on the marks, and divide the known distance by the line’s length in pixels. A longer line keeps the error small: one pixel of uncertainty on a 154-pixel line is 0.65 %. A scale bar burnt into an image works the same way, but only as well as the bar itself was drawn.

Electron micrographs. An SEM shows the horizontal field width (HFW), the width of the scanned area, in its data bar; the pixel size is the field width divided by the image width in pixels. Many SEM and TEM files also carry the pixel size in their metadata.

Set Scale in ImageJ. In ImageJ or Fiji, draw a straight line along the known distance and choose Analyze › Set Scale…: Distance in pixels is filled in from the line; type the Known distance, keep Pixel aspect ratio at 1 for square pixels, and type the Unit of length (ImageJ accepts “um” for µm). From a pixel size, type 1 as Distance in pixels and the pixel size as Known distance. Global applies the scale to every image instead of only the active one, so tick it only when they all share the pixel size. ImageJ and Fiji are independent open-source projects; LabCanvas is not affiliated with them.

Nyquist sampling. The objective resolves details down to $r = 0.61\,\lambda / \mathrm{NA}$ in the image plane, with $\lambda$ the emission wavelength and NA its numerical aperture (Thorn 2016). To record that detail there must be at least two camera pixels per resolvable element (Thorn 2016): the pixel should be at least two times smaller than the resolution limit (Waters 2009). The calculator calls a pixel no larger than $r / 2$ sampled at Nyquist and gives the total magnification that reaches it, $2 \times \text{camera pixel} \times \text{binning} \div r$ . Larger pixels, from binning or a lower magnification, trade resolution for signal, which is often worth it in low-light imaging (Waters 2009).

Sources: Waters JC. Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 2009;185:1135–1148, doi:10.1083/jcb.200903097. Thorn K. A quick guide to light microscopy in cell biology. Mol Biol Cell 2016;27:219–222, doi:10.1091/mbc.E15-02-0088. North AJ. Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. J Cell Biol 2006;172:9–18, doi:10.1083/jcb.200507103.

Worked examples: a camera, a micrometer and an SEM image

1. From the camera. A camera with 6.5 µm pixels and a 2048 × 2048 sensor, a 20× objective, no camera adapter (1×) and no binning:

$\text{Pixel size} = 6.5\ \upmu\mathrm{m} \times 1 \div 20$ $= \mathbf{0.325\ \upmu m}$ per pixel, or $1 \div 0.325 = 3.077$ pixels per µm.
$\text{Field of view} = 2048 \times 0.325\ \upmu\mathrm{m}$ $= \mathbf{665.6 \times 665.6\ \upmu m}$ .
In ImageJ’s Analyze › Set Scale: Distance in pixels 1, Known distance 0.325, Pixel aspect ratio 1, Unit of length um.
A 50 µm scale bar is $50 \div 0.325 = 153.8$ pixels long. Areas scale with the square: 1000 px² is $1000 \times 0.325^2 = 105.6\ \upmu\mathrm{m}^2$ .

Is that enough for the objective? With NA 0.75 and green emission at 520 nm, $r = 0.61 \times 0.520\ \upmu\mathrm{m} \div 0.75 = 0.423\ \upmu\mathrm{m}$ , so Nyquist needs pixels of 0.211 µm or less. At 0.325 µm there are only 1.30 pixels per resolution: the image is undersampled, and full resolution needs $2 \times 6.5 \div 0.423 = 30.7\times$ in total. A 1.5× magnification changer (30× in total, 0.217 µm per pixel) comes close at 1.95 pixels per resolution; a 60× NA 1.4 oil objective gives 0.108 µm per pixel against $r = 0.227\ \upmu\mathrm{m}$ , 2.09 pixels per resolution, which meets it. For counting cells or measuring objects many micrometres across, the 20× image is usually fine.

2. From a stage micrometer. With a 10× objective, ten 10 µm divisions (100 µm) measure 154 pixels: $100 \div 154 = \mathbf{0.6494\ \upmu m}$ per pixel (1.54 pixels per µm). The camera route predicts $6.5 \div 10 = 0.65\ \upmu\mathrm{m}$ per pixel; the measured value is 0.1 % smaller. Set Scale: Distance in pixels 154, Known distance 100, Pixel aspect ratio 1, Unit of length um.

3. From an SEM field width. A horizontal field width of 10.0 µm over an image 1024 pixels wide: $10\ \upmu\mathrm{m} \div 1024 = \mathbf{9.766\ nm}$ per pixel. Set Scale: Distance in pixels 1024, Known distance 10, Pixel aspect ratio 1, Unit of length um.

Frequently asked questions

How do I calculate the pixel size of my microscope camera?

Divide the camera’s pixel size by the total magnification and multiply by the binning: $\text{pixel size} = \frac{\text{camera pixel} \times \text{binning}}{\text{objective} \times \text{any extra magnification}}.$ A 6.5 µm camera pixel with a 20× objective and no binning gives 0.325 µm per pixel.

How do I convert pixels to micrometres?

Multiply a length in pixels by the pixel size in µm per pixel; for areas, multiply by the square of the pixel size. At 0.325 µm per pixel, 100 pixels is 32.5 µm and 1000 px² is 105.6 µm².

How do I set the scale in ImageJ?

Draw a straight line along a known distance (a stage micrometer or a scale bar), choose Analyze › Set Scale…, type the Known distance and the Unit of length (um for µm) and keep Pixel aspect ratio at 1; Distance in pixels comes from the line. If you know the pixel size, type 1 as Distance in pixels and the pixel size as Known distance.

Where do I get a calibration image for ImageJ?

It has to come from your own microscope: photograph a stage micrometer with the same objective, camera adapter and binning as the images you will measure. A calibration image from another microscope gives that microscope’s pixel size, not yours. Repeat it for each objective.

Does binning change the pixel size?

Yes. 2 × 2 binning combines four sensor pixels into one, so the pixel size doubles (0.325 µm becomes 0.65 µm at 20× with a 6.5 µm camera) while the field of view stays the same and the image has half as many pixels in each direction.

What pixel size do I need for Nyquist sampling?

At most half the resolution, with the resolution $r = 0.61\,\lambda / \mathrm{NA}$ . For 520 nm emission and NA 0.75, $r = 0.423\ \upmu\mathrm{m}$ , so the pixel should be 0.211 µm or smaller; with a 6.5 µm camera pixel that takes about 31× total magnification.

How do I find the pixel size of an SEM or TEM image?

Divide the horizontal field width shown in the data bar by the image width in pixels: 10 µm over 1024 pixels is 9.766 nm per pixel. Many SEM and TEM files also store the pixel size in their metadata, and a scale bar can be measured like a micrometer.

Why does ImageJ measure in pixels instead of µm?

The image has no scale: it was never set, or the file does not carry it, as with most JPEGs and pictures copied out of a figure. Set it again with Analyze › Set Scale before measuring; ImageJ keeps the scale in the TIFF files it saves.

Set the scale and measure in LabCanvas Image Analysis

Open your image in LabCanvas Image Analysis, set the scale under Image › Scale (draw along the micrometer or type the pixel size), add a scale bar and measure lengths, areas and particle sizes in µm. Image Analysis is free to use with a free LabCanvas account.

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