Microscopy Artifacts: What They Are and How to Avoid Them

Every microscopist eventually gets excited about a strange structure that turns out to be a bubble, a bit of fluff, or a smudge on the eyepiece. These false features are called artifacts. Learning to recognise them is one of the most useful skills you can develop, because it stops you from misreading your own slides.

What is an artifact in microscopy?

In microscopy, an artifact is a feature in the image that isn’t really part of the specimen as it exists naturally. It is caused by the way the specimen was collected, prepared, illuminated, viewed or recorded. An artifact can be something added (a bubble or a fibre), something lost (cells that washed away), something distorted (shrunken or folded tissue), or something created by the optics or the camera (halos, colour fringes, pixel patterns).

A visual artifact is the general term for this kind of false feature in any image or display: a compression block in a video, a halo around an over-sharpened edge, or a ghost image in a lens. In microscopy, many visual artifacts come from the optics or the digital camera rather than from the slide.

Both spellings, “artifact” and “artefact”, are correct. For the wider scientific meaning of the word, see what “artefact” means in the context of science.

Types of microscopy artifacts

Artifacts are usually grouped by where they come from:

Specimen collection and preparation: bubbles, contamination, stain deposits, folds, crushing, shrinkage, fixation changes.
Optical and illumination: dirt on lenses, diffraction halos, colour fringes, uneven lighting, immersion oil problems.
Movement: Brownian motion, drifting liquid, evaporation currents.
Digital imaging and processing: compression, noise, over-sharpening, stitching and stacking errors.
Instrument-specific: in fluorescence and electron microscopy, problems such as autofluorescence, sample charging and beam damage. These are mostly relevant to lab work.

Specimen preparation artifacts

Air bubbles

Bubbles are the most common artifact in wet mounts. They are perfectly round (or smoothly rounded where squashed), with a thick, dark rim and a bright centre. The rim changes thickness as you focus up and down. Real cells are rarely that perfectly circular and almost never have such a heavy black outline. Our guide to making a wet mount slide without air bubbles explains how to prevent them.

Dust, fibres and contamination

Textile fibres, hairs, skin flakes, pollen and starch grains from gloves or hands find their way onto almost every slide. Common giveaways:

Fibres are long, uniform in width, and often twisted (cotton) or perfectly smooth and glassy (synthetics). They often lie across everything else.
Starch grains from food or glove powder are oval or polygonal with a central point. Under crossed polarizers they show a dark cross.
Pollen has a distinct, regular shape and often a patterned surface.

In histology, pieces of tissue from another specimen can be carried onto a slide. These are called floaters or carry-over contamination.

Stain deposits and crystals

Dried or old stain can form needle-like crystals, sparkling granules or dark specks that look a bit like bacteria or granules. These often lie on a different focal plane from the cells, sometimes on top of the coverslip. Filtering stains, using fresh solution, and not letting the mount dry out all help. Our methylene blue staining guide includes troubleshooting for this.

Folds, tears, crushing and shrinkage

These are the classic artifacts in tissue sections. They’re worth knowing if you look at prepared histology slides:

Folds and wrinkles show up as darker bands where the section doubled over and the stain is twice as dense.
Knife marks and chatter show as straight parallel scratches or a “venetian blind” pattern from a damaged or vibrating blade.
Tears and holes have sharp, empty edges with no cell lining.
Shrinkage spaces are clear gaps between tissues that should touch, caused during fixation and processing.
Crush artifacts appear as smeared, stretched or distorted nuclei where tissue was squeezed during sampling.
Fixative pigment, a fine brown-black granular deposit, can form with some fixatives under some conditions.

In home wet mounts, pressing on the coverslip, or using too little water, flattens and bursts delicate cells.

Optical artifacts

Dirt on lenses. Dust or smudges on the eyepiece, objective, camera sensor or condenser show up as dark specks, blurry blobs or a general haze. Specks on the eyepiece rotate with it; specks on the camera stay fixed whatever slide you use.
Halos and diffraction rings. Tiny objects and sharp edges can show bright or dark fringes, especially when the condenser diaphragm is closed far down. Phase contrast microscopes add characteristic bright halos around objects.
The Becke line. A bright line along the edge of a transparent particle moves in or out as you focus. It’s useful for comparing refractive indices, but it can be mistaken for a membrane or wall.
Colour fringes. Cheaper objectives can show coloured edges (chromatic aberration), especially near the edge of the field.
Oil problems. Oil smeared onto a dry 40x objective gives a hazy, low-contrast image. Bubbles in immersion oil cause similar haze at 100x.
Uneven illumination. A badly aligned or poorly focused condenser can give a bright spot, dark corners or a shadow.

Movement that isn’t life

Beginners often think every moving speck is alive. Two common causes of non-living movement:

Brownian motion. Very small particles, including dead bacteria, stain granules and fat droplets, jiggle constantly because water molecules bump into them. The motion is random and stays in roughly the same place.
Currents and drift. As a wet mount evaporates or the coverslip settles, everything flows in the same direction together.

Living swimmers usually move with purpose. They travel in straight or curving paths, change direction, and move independently of their neighbours.

Digital imaging artifacts

Microscope cameras and phone cameras add artifacts of their own:

Compression blocks and smeared detail from heavily compressed JPEGs or video.
Noise, a speckled grain in dim images or at high ISO, which can look like fine granules.
Over-sharpening halos, bright outlines around edges that weren’t there in the eyepiece.
Moiré and colour fringes when fine regular patterns interact with the camera’s pixel grid.
Stacking and stitching errors. Focus stacking software can create “ghost” outlines, smears or invented detail where objects moved between frames. Stitched panoramas can show seams or repeated sections.

Keep an unprocessed original of any image you want to rely on, and compare it with what you see through the eyepiece. Our guide to turning your phone into a microscope covers phone photography through an eyepiece.

Where is that speck? A quick diagnostic routine

When you see something odd, test where it is:

Rotate the eyepiece. If the speck rotates, it’s in the eyepiece. Clean it.
Move the slide. If the speck stays put while the specimen moves, it’s in the optics (objective, condenser, camera), not on the slide.
Switch objectives. If the speck appears only with one objective, that objective needs cleaning.
Focus up and down. If the speck comes into focus at a different level from your cells, it may be on top of the coverslip or under the slide.
Make a blank. Mount a drop of the same water or stain with no specimen. If the “organism” is still there, it came from your reagents.

How to avoid artifacts

Clean slides and coverslips before use, and handle them by the edges.
Use fresh, filtered water and stains; cap bottles to keep dust out.
Lower coverslips from one edge to avoid trapping bubbles.
Don’t press on coverslips over live specimens.
Keep wet mounts from drying out; make fresh ones often.
Keep lenses clean with lens tissue and an appropriate cleaner, and keep the dust caps on when not in use.
Align the condenser and set the diaphragm properly instead of using it as a brightness control.
Compare suspicious features with a blank control slide.
Treat software processing cautiously and keep originals.

Artifacts aren’t a sign that you’re doing badly. Every slide has some. The goal is to recognise them so that what you report, draw or photograph is really there. For a broader look at specimens worth exploring, see what everyday things look like under a microscope.