Brightfield Illumination: A Guide to the Most Widely Used Microscopy Technique

In microscopy, contrast means the difference in brightness or color that makes microscopic details stand out from the background.

Microscopy relies on good contrast so you can see

• shapes,
• edges,
• features clearly inside of transparent specimen like cells and tissues, and
• features on the surface of intransparent specimen like metal samples.

Contrast can be produced by

• the specimen’s own contrast (amplitude object).
• staining (e. g. with histological dyes for biological tissues or etching for metal surfaces), if the specimen has no own contrast (phase object).

If your microscopy sample doesn’t have enough contrast, it can blend into the background – even under a powerful microscope. In such cases, optical contrasting techniques are essential.

Optimizing contrast in microscopy is crucial to producing clear, sharp images, especially when looking at transparent samples.

Microscopy samples can be

• amplitude objects – absorb light (thus showing up well in brightfield microscopy), such as stained tissues,etched metal surfaces or specimen with own contrast.
• phase objects – don’t absorb much light and are mostly transparent (thus usually hard to see in brightfield microscopy), like living cells, unstained bacteria or untreated metal surfaces.

When studying transparent specimens with microscopy, you need optical contrasting techniques to make them visible.

Not every microscopy sample is visible with the same illumination or contrast method. Brightfield microscopy is excellent for stained or pigmented samples, but not as effective for clear, living cells under the microscope.

Some cells and structures shouldn’t be stained if you want to keep them alive during microscopic observation or, in the case of material samples, if you don’t want sensitive material surfaces to be treated with chemicals.

Other microscopy contrast techniques – like phase contrast, differential interference contrast (DIC), or darkfield microscopy – help you see clear, living, or delicate samples under the microscope.

Using the right contrast method in microscopy allows you to

• observe a wider variety of microscopic samples.
• keep microscopy specimens in their natural state.
• reveal microscopic specimen details that brightfield alone would miss.

Learning when and why to use each microscopy contrast method is a key skill for beginners exploring the microscopic world.

• Biological and medical labs (e.g. hematology, histology, pathology, etc.)
• Medical diagnostics (e.g., blood smear analysis)
• Microbiology (e.g., bacteria stained with Gram stain)
• Botany and plant sciences 

• Metallography
• Ceramics analysis
• Polymers
• Semiconductors  

Brightfield microscopy uses transmitted or reflected light to illuminate a specimen. The image is formed based on how much light is absorbed or reflected by the sample’s structures.

Transmitted light passes through the sample from below, ideal for transparent specimens. Reflected light illuminates the sample from above and is used for opaque materials with surface features.

Start with a 10x objective (or 20x if 10x is unavailable). Use well-stained samples, align Koehler Illumination, and adjust the condenser aperture for optimal contrast and resolution.