Microscope Illumination: Lamps, Mirrors and Condensers

What illumination does in a microscope

Most specimens on a compound microscope are thin and nearly transparent. You see them because light passes through them from below, and the way that light is delivered has a large effect on what you see. Poor illumination can make a good objective look mediocre, while careful illumination gets the most from even a basic microscope.

Illumination affects three things:

• Brightness: enough light to see comfortably, without glare.
• Evenness: the whole field lit uniformly, without hot spots or dark edges.
• Resolution and contrast: the angle of the light cone reaching the specimen sets how much fine detail the objective can resolve and how much contrast transparent structures show.

That third point is the one most beginners miss, and it’s why the condenser and its diaphragm matter so much. For a list of every part involved, see which parts of the microscope are the illuminating parts.

Light sources: mirrors, tungsten, halogen and LED

For most amateurs, an LED illuminator is the practical choice. If you have a halogen microscope, a blue daylight filter can correct its yellow tint; see our guide to blue, green and neutral density filters.

Using a mirror: plane side vs concave side

Many older and simple microscopes have a mirror under the stage instead of a lamp. Its role is to catch light from a window or lamp and send it straight up through the stage opening. Mirrors are often double-sided:

• Plane (flat) side: use it when the microscope has a condenser. The condenser expects roughly parallel light and does the focusing itself.
• Concave (curved) side: use it when there is no condenser. The curved surface converges the light toward the specimen, acting as a simple condenser.

To aim the mirror, use the lowest-power objective, look through the eyepiece, and tilt the mirror until the field is evenly bright. A frosted desk lamp or an overcast sky gives more even light than a bare bulb.

Never point the mirror at direct sunlight. Sunlight concentrated through the optics can seriously harm your eyes.

We cover what the word means and the mirror’s role in more detail in which means mirror.

The condenser

The condenser sits under the stage and focuses light into a cone that passes through the specimen and into the objective. The common types are:

• None, or a simple disc diaphragm: found on toy and very basic microscopes. Workable at low power but limiting at 400x.
• Abbe condenser: the usual two-lens condenser on most lab and amateur microscopes. It’s often rated at NA 1.25, though it only reaches values above about 1.0 if a drop of immersion oil is placed between the condenser top and the slide. For most work it’s used dry.
• Achromatic or aplanatic-achromatic condensers: better-corrected designs used for higher-quality work and photography.

Why does the condenser matter? The resolving power of the system depends on the NA of both the objective and the illumination cone. A 40x objective with NA 0.65 needs a condenser able to fill a cone of similar angle to perform at its best. If the condenser is too weak or set too low, the objective underperforms.

For most work, the condenser should sit near the top of its travel, just below the slide. It’s focused properly as part of Köhler illumination below.

Diaphragms: disc, aperture iris and field diaphragm

Microscopes can have up to two adjustable diaphragms:

• The aperture diaphragm (iris diaphragm) in the condenser controls the angle of the light cone. Closing it increases contrast and depth of field but reduces resolution. It’s the control that most affects image quality; see how to set the iris diaphragm.
• The field diaphragm, on the light port in the base, controls the diameter of the illuminated area. It’s used to stop stray light and is essential for Köhler illumination.

Basic microscopes may have a disc diaphragm instead: a wheel with holes of several sizes under the stage. It does a rougher version of the aperture diaphragm’s job.

Critical vs Köhler illumination

There are two classic ways of setting up transmitted light:

• Critical (Nelsonian) illumination focuses an image of the light source directly onto the specimen. It’s simple and bright, but any unevenness in the source (a filament, or individual LED chips) can show up in the image.
• Köhler illumination focuses the light source away from the specimen and instead focuses the field diaphragm onto it. The result is an evenly lit field, with brightness, field size and light-cone angle all controlled separately. It’s the standard for good brightfield microscopy and photography. Our Köhler illumination walkthrough goes into more depth.

When should you use Köhler illumination? Whenever your microscope has a field diaphragm and a focusable condenser, and you care about image quality. On microscopes without these, the best you can do is raise the condenser close to the slide and set the aperture diaphragm.

Setting up Köhler illumination

1. Switch on the lamp, place a stained slide on the stage, and focus it with the 10x objective.
2. Close the field diaphragm most of the way. You should see a small, blurry polygon of light.
3. Raise or lower the condenser until the edges of that polygon are sharp.
4. Use the condenser centering screws to move the polygon to the center of the field.
5. Open the field diaphragm until its edges just disappear past the edge of the field of view.
6. Set the aperture diaphragm: remove an eyepiece and look down the tube at the bright disc of the objective’s back lens, then close the iris until it covers roughly 20 to 30 percent of that disc. Replace the eyepiece.
7. Adjust brightness with the lamp control (or a neutral density filter), not the diaphragms.

Repeat steps 2 to 6 when you change objectives. At least the field and aperture diaphragms usually need resetting.

Transmitted vs reflected light

Everything above concerns transmitted light: light passing up through a thin specimen. Compound microscopes for biology work this way.

Opaque specimens such as rocks, coins, circuit boards and insects need reflected (incident) light, shining down onto the surface. Stereo microscopes usually have a top light for this, and many have a bottom light too. Metallurgical compound microscopes send light down through the objective itself. For a fuller comparison, see transmitted vs reflected light microscopes. If you’re working with a stereo microscope, see our guide to stereo and binocular microscope parts.

Controlling brightness without ruining the image

The right way to change brightness is the lamp dimmer or a neutral density filter. Closing the aperture diaphragm or lowering the condenser to dim the image changes contrast and resolution at the same time, usually for the worse.

With halogen lamps, there’s an extra wrinkle: as you turn the dimmer down, the light becomes noticeably yellower. For photography with consistent color, many users run the lamp at a fixed setting and dim with neutral density filters. LEDs shift color much less when dimmed.

Common illumination problems and fixes

If you can’t see anything at all, start with our microscope troubleshooting guide. For lighting-specific problems:

With a little practice, setting up the lighting takes under a minute and makes a large difference to every slide you look at. For a broader look at how lighting fits with the rest of the instrument, see our guide to the parts of a compound microscope.