The short answer
Brightfield microscopy makes contrast from how much light the specimen absorbs. It’s ideal for stained slides and naturally colored specimens. Phase contrast microscopy makes contrast from how much the specimen delays light. It converts invisible phase shifts into visible differences in brightness, so it’s ideal for living, unstained, transparent cells.
Most phase contrast microscopes can also do brightfield. The real question is whether your specimens are mostly colored or mostly clear.
How brightfield creates an image
In brightfield, light from the lamp passes through the condenser, through the specimen, and into the objective. Parts of the specimen that absorb light look darker than the bright background around them. Stained nuclei, chloroplasts, pollen and insect parts all show up well, because they absorb light strongly.
The problem is transparent material. A living cheek cell or a bacterium absorbs very little light, so it transmits almost as much as the surrounding water. The result is a faint outline at best. You can boost edges by closing the condenser’s aperture diaphragm, but that costs resolution. Our article on what bright field imaging is covers the basics and how to get the most from it.
How phase contrast works
Phase contrast was developed by the Dutch physicist Frits Zernike in the 1930s, and it earned him the Nobel Prize in Physics in 1953. It relies on the fact that light passing through a cell is slowed slightly, because the cell’s refractive index is a little higher than that of water. The light comes out with a small phase shift that your eye can’t detect.
A phase contrast microscope adds two matched parts:
- A condenser annulus. This is a ring-shaped opening below the condenser, so the specimen is lit by a hollow cone of light.
- A phase plate in the objective. A ring inside the objective, sitting exactly where the image of the annulus falls, shifts the phase of the direct (undeviated) light and dims it.
Light scattered by the specimen mostly misses the phase ring, while the direct background light goes through it. When the two recombine in the image, they interfere. Small phase differences become differences in brightness. In the usual “positive” phase contrast, denser structures look darker than the background, and nuclei, vacuoles and membranes stand out clearly in cells that were nearly invisible a moment earlier.
The annulus has to match the phase ring in each objective. That’s why phase objectives carry markings such as Ph1, Ph2 or Ph3, and why the condenser usually has a turret with a matching annulus for each.
Side-by-side comparison
| Brightfield | Phase contrast | |
|---|---|---|
| What creates contrast | Light absorption (and color) | Phase shifts from refractive index differences |
| Best specimens | Stained sections and smears, pigmented organisms, pollen, hairs | Living cells, unstained bacteria, protozoa, cell cultures, cheek cells |
| Background | Bright | Usually medium gray (positive phase) |
| Special parts needed | None beyond a standard condenser | Phase objectives plus matched condenser annuli |
| Alignment | Basic Köhler setup | Annuli must be centered on the phase rings, usually with a centering telescope |
| Artifacts | Faint or invisible transparent detail | Bright halos and shade-off around edges |
| Thick specimens | Workable with focusing | Poor; halos and confusion increase with thickness |
| Cost | Lowest | Higher; phase objectives cost more than standard ones |
What the same specimen looks like in each
Scrape a little material from the inside of your cheek, spread it in a drop of water under a coverslip (a simple wet mount), and look at it both ways.
- Brightfield, aperture diaphragm open: pale, nearly invisible cell outlines. The nucleus may be just a hint.
- Brightfield, aperture diaphragm partly closed: the outlines become more visible, the image looks grainier, and fine detail softens.
- Phase contrast: clear cell borders, a distinct nucleus, granules in the cytoplasm, and often the folds of the cell membrane. A thin bright halo surrounds each cell.
Once the cells are stained with methylene blue, brightfield becomes the better view. Stained specimens often look odd in phase contrast, because absorption and phase effects mix together.
Disadvantages of phase contrast
Phase contrast is excellent at its job, but it isn’t perfect:
- Halo artifacts. Bright rings appear around edges, especially at large refractive index jumps such as cell edges in water. They can hide fine detail right at the boundary.
- Shade-off. Large, even areas of a specimen can fade toward the background brightness in their centers, which makes broad, flat structures look hollow.
- Thick specimens perform poorly. Phase contrast works best on thin layers, such as a single layer of cells. Thick tissue gives a confusing image.
- Matched optics. Each objective needs its own matching annulus, and both must be centered. Misaligned annuli give weak, uneven contrast.
- Slightly reduced brightfield performance. You can use phase objectives for brightfield work, but the phase ring slightly degrades the image compared with an equivalent plain objective.
- Cost. Phase objectives and a phase condenser add noticeably to the price of a microscope.
What is the light source in a phase contrast microscope?
The same kind of lamp you’d find in a brightfield microscope: usually an LED or a halogen bulb. Phase contrast doesn’t need any special light source. What makes it different is the annular ring in the condenser, which shapes the light into a hollow cone, and the phase plate in the objective. Many users also put a green filter in the light path, because phase objectives are often optimized for green light and it can sharpen contrast.
Which should you use?
Choose brightfield if you mainly look at:
- Prepared or stained slides
- Plant sections, pollen, insect parts and hairs
- Pigmented algae and other naturally colored specimens
Choose phase contrast if you mainly look at:
- Living protozoa, cells and bacteria without stains
- Cheek cells, blood cells in thin films and cell cultures
- Anything transparent and thin that you don’t want to kill or stain
Most serious hobbyists eventually want both. A microscope with phase objectives and a turret condenser can switch between them in seconds.
Adding phase contrast to a hobby microscope
Some student and hobby microscopes accept a phase contrast kit: a set of phase objectives plus a slider or turret condenser with matching annuli. Before buying one, check:
- Compatibility. Objectives must match your microscope’s optical standard (finite or infinity tube length, parfocal distance and thread). Mixing standards gives poor results.
- Centering. A good kit includes a centering telescope or Bertrand lens, so you can see and align the annulus with the phase ring.
- Which objectives. A 10x and a 40x phase objective cover most living-cell work. A 100x oil phase objective is useful for bacteria.
Where darkfield fits in
Darkfield is the other common way to see unstained specimens. It blocks the direct light completely, so specimens glow against black. It’s cheaper than phase contrast and gives very high contrast for small, thin and motile objects, but it reveals less internal detail. For a full comparison, see dark field microscopy vs phase contrast. For the bigger picture of why contrast matters as much as resolution, read our guide to microscope contrast.