The Short Answer
Several kinds of microscope can give you a 3D image, but they do it in very different ways:
- Stereo (dissecting) microscopes give a live 3D view straight to your eyes, at low magnification.
- Confocal, light-sheet and multiphoton microscopes build a 3D volume of a specimen by recording it one thin optical slice at a time.
- Scanning electron microscopes (SEMs) produce images that look strongly 3D. True 3D data comes from tilting the sample to make stereo pairs, or from slicing it layer by layer (FIB-SEM and serial block-face SEM).
- Transmission electron microscopes (TEMs) can reconstruct 3D structure by electron tomography: imaging a thin sample at many tilt angles and combining the images by computer.
- Atomic force microscopes, optical profilers and many digital microscopes produce 3D height maps of a surface.
- X-ray microscopes and micro-CT scanners reconstruct the inside of a sample in 3D without cutting it.
An ordinary compound microscope, the kind used in schools, gives a flat image. It can still be used to collect 3D information if you photograph it at a series of focus positions.
Two Meanings of “3D”
When people ask which microscopes make 3D images, they usually mean one of two things:
- An image that looks three-dimensional. You perceive depth, either because each eye gets a slightly different view (stereo) or because shading and a large depth of field give strong visual depth cues (as in an SEM picture of an insect).
- An image that contains 3D data. The instrument records information along all three axes, so a computer can rotate the specimen, slice it virtually or measure heights and volumes.
A stereo microscope gives you the first kind. A confocal microscope gives you the second. Some methods, such as SEM stereo pairs, can give both.
Stereo Microscopes: 3D You See Directly
A stereo microscope has two separate optical paths angled at the specimen, one for each eye. Your brain fuses the two views into a single 3D image, just as it does with ordinary vision. This is the simplest and most affordable way to see microscopic objects in depth, and the image is live, so you can move the specimen or work on it while you watch.
The trade-off is magnification. Most stereo microscopes work somewhere between about 7x and 45x, so they show insects, seeds, minerals and circuit boards rather than cells. Our stereo vs compound microscope comparison covers this in detail.
It is worth repeating a common point of confusion: two eyepieces alone do not make a 3D image. A binocular compound microscope sends the same image to both eyes, so the view is still flat (see stereo and binocular microscope parts).
Optical Sectioning Microscopes
These instruments are the workhorses of 3D biological imaging. They capture a stack of thin, in-focus slices through a specimen (a “z-stack”), and software assembles the stack into a 3D volume. Most rely on fluorescence, so the specimen is usually labelled with fluorescent dyes or proteins.
Confocal microscopes
A confocal microscope focuses laser light to a point and places a pinhole in front of the detector. The pinhole blocks light from above and below the focal plane, so each image is a clean optical section rather than a blur of in-focus and out-of-focus detail. Moving the focus step by step through the sample builds up the stack. Confocal microscopy is widely used for cells, tissues and small organisms.
Light-sheet microscopes
A light-sheet microscope illuminates the specimen from the side with a thin sheet of light and views it from above at right angles. Only the plane being imaged is lit, which reduces light damage to living samples and allows fast imaging of whole embryos, small organisms and cleared tissue.
Multiphoton microscopes
Multiphoton (often two-photon) microscopes use pulsed infrared lasers so that fluorescence is excited only at the focal point. Infrared light scatters less than visible light, so these microscopes can image deeper into tissue than a standard confocal, which is why they are used for imaging inside living tissue.
Widefield microscopes with deconvolution
A conventional fluorescence microscope can also collect a z-stack. Each slice contains blur from neighbouring planes, but deconvolution software can computationally reassign much of that out-of-focus light, giving a usable 3D result from simpler hardware.
Electron Microscopes
Why SEM images look 3D
An SEM scans a focused electron beam across the surface of a sample and records the electrons given off. Surfaces tilted towards the detector appear brighter, edges glow, and the depth of field is very large compared with a light microscope at similar magnification. The result is a picture with strong shading and almost everything in focus, which reads to the eye as three-dimensional. Strictly, though, a single SEM image is a 2D picture of a surface. It does not contain height measurements.
Stereo pairs from a tilted SEM
If the sample is imaged twice, tilted by a few degrees between shots, the two images form a stereo pair. Viewed with a stereo viewer or combined into a red-cyan anaglyph, they give genuine depth. Software can also compare the two images to calculate a height map of the surface.
Slice-and-view: FIB-SEM and serial block-face SEM
To see inside a sample in 3D, the SEM can be combined with a way of removing material. In FIB-SEM, a focused ion beam mills away a very thin layer, the SEM images the freshly exposed face, and the cycle repeats hundreds or thousands of times. In serial block-face SEM, a tiny diamond knife inside the microscope shaves the surface of a resin-embedded block between images. Either way, the stack of images is reconstructed into a 3D volume. Both methods destroy the sample as they go.
Electron tomography in the TEM
A TEM sends electrons through a very thin sample, so each image is a projection, rather like an X-ray. In electron tomography, the sample is tilted through a range of angles and imaged at each step. A computer combines the projections into a 3D reconstruction, in a similar way to a medical CT scan. Cryo-electron tomography applies this to frozen, hydrated samples and is used to study structures inside cells. See our article on the principle of SEM and TEM for how the two electron microscopes differ.
Surface Height Maps: Digital Microscopes, Profilers and AFM
Several instruments measure surface topography rather than internal structure:
- Digital microscopes with motorised focus can capture images at many focus heights and combine them. The result is both an all-in-focus image and a 3D height map, which is useful in industrial inspection.
- Optical profilers, including white-light interferometers and laser scanning confocal profilers, measure surface height very precisely and are common in engineering and materials labs.
- Atomic force microscopes (AFMs) drag or tap a very sharp tip across a surface and record its height point by point. They can map surfaces at extremely fine scales, though they only measure the surface and only over small areas.
These are sometimes described as “2.5D”, because each point on the surface has a single height value. They cannot show overhangs or interiors.
X-ray Microscopy and Micro-CT
X-rays pass through many materials that light cannot, so X-ray microscopes and micro-CT scanners can rotate a sample, collect projection images and reconstruct its interior in 3D without cutting it. They are used for fossils, insects, rocks, bone, electronic components and manufactured parts. Resolution is generally coarser than electron microscopy, but the sample usually survives intact.
Comparison Table
| Method | Kind of 3D | Typical samples | Destroys sample? |
|---|---|---|---|
| Stereo microscope | Live 3D view to the eyes | Insects, plants, minerals, electronics | No |
| Confocal | 3D volume from optical sections | Fluorescently labelled cells and tissue | Not usually, though strong light can damage live samples |
| Light-sheet | 3D volume, fast and gentle | Embryos, small organisms, cleared tissue | No |
| Multiphoton | 3D volume, deeper in tissue | Living tissue | No |
| SEM stereo pair | Stereo view or height map | Surfaces of solid samples | Preparation may alter it |
| FIB-SEM / serial block-face | 3D volume at very fine scale | Cells, tissue, materials | Yes |
| TEM tomography | 3D reconstruction from tilt series | Thin sections, cells, macromolecular structures | Preparation is destructive |
| AFM / optical profiler | Surface height map | Flat surfaces, coatings, materials | No |
| Micro-CT / X-ray microscopy | 3D interior volume | Fossils, bone, rocks, parts | No |
3D Imaging for Amateurs
Most of the instruments above live in research facilities, but hobbyists can get real depth into their images with ordinary equipment:
- Use a stereo microscope. It is the only way to see in 3D directly at the eyepieces. A trinocular head lets you attach a camera, although the camera port sees only one of the two optical paths, so the photo is flat. Our binocular vs trinocular guide explains the camera port.
- Focus stacking. Take a series of photos through a compound or stereo microscope, moving the focus slightly between each, then combine them with stacking software. You get an image with far more in focus than any single shot, and some programs can also produce a rough depth map or a rotating 3D view.
- Stereo pairs. Photograph a specimen, tilt it or shift the camera slightly, and photograph it again. Viewed side by side or as an anaglyph, the pair shows depth. This works well with insects, crystals and small fossils.
- Z-series through a compound microscope. Recording a series of images at different focus positions through a thick specimen, such as a water flea or a section of moss, shows its structure layer by layer, even without confocal optics.
If you are capturing images for any of these methods, our guide to digital imaging and image analysis for microscopy covers cameras, adapters and software. For background on what a captured image actually represents, see what is a microscope image.