Advanced microscopy

Why it matters

Ordinary light microscopes show cells and tissues, but many lab questions need more. Where in the cell is this protein? Is this bacterium alive? What does a virus or a crack in a metal grain boundary look like at 10 nanometres? Fluorescence and electron microscopy answer those questions, and they are central to biochemistry, cell biology, clinical microbiology and materials science. You may operate these instruments as a technician or depend on core facilities that do.

The resolution limit of light

Magnification makes an image bigger. ResolutionThe smallest distance at which two points still look separate in an image. is the smallest distance at which two points still look separate. Magnifying beyond the resolution limit only makes a blur bigger.

For a light microscope, the Abbe limit is approximately:

d = wavelength / (2 x NA)

where NA is the numerical aperture of the objective. With green light (about 500 nm) and a good oil-immersion objective (NA 1.4), d is about 500 / 2.8 = about 180 nm. Most viruses and all individual proteins are smaller than that.

Fluorescence microscopy

A fluorophoreA molecule that absorbs light of one wavelength and emits light of a longer wavelength. absorbs light of one wavelength and emits light of a longer wavelength. The difference is the Stokes shift. A fluorescence microscope uses this:

1. A bright light source (LED, mercury or xenon lamp, or a laser) passes through an excitation filter that selects the wavelength the fluorophore absorbs.
2. A dichroic mirrorA mirror that reflects excitation light onto the sample while letting longer-wavelength emitted light pass to the detector. reflects that light onto the sample.
3. The fluorophore emits longer-wavelength light, which passes through the dichroic mirror and an emission filter to the camera or eyepiece.
4. Only the labelled structures glow against a dark background.

The excitation filter, dichroic and emission filter are often mounted together as a filter cube matched to a fluorophore.

Common labels:

• DAPI and Hoechst bind DNA and stain nuclei blue.
• Green fluorescent protein (GFP) can be genetically fused to a protein so living cells show where it goes.
• Antibodies tagged with fluorophores (immunofluorescence) label specific proteins in fixed cells or tissue.

Confocal and beyond

A widefield fluorescence image includes out-of-focus glow from above and below the focal plane. A confocal microscopeA microscope that scans a laser point and uses a pinhole to block out-of-focus light, giving sharp optical sections. scans a laser point and uses a pinhole to block out-of-focus light, giving sharp optical sections that can be stacked into 3D images. Super-resolution methods (such as STED, PALM and STORM) use clever tricks with fluorophores to get below the Abbe limit, to tens of nanometres.

Limits

• PhotobleachingThe fading of fluorophores with light exposure.: fluorophores fade with exposure. Keep light exposure short and image consistently.
• Phototoxicity: strong light damages living cells.
• Autofluorescence: some tissues glow on their own; controls without the label reveal it.
• You only see what you labelled.

Electron microscopy

Electrons have a far shorter wavelength than visible light, so electron microscopes resolve down to the nanometre scale or below. The beam must travel in a vacuum, so samples must be dry or frozen and stable in vacuum.

SEM instruments often add energy-dispersive X-ray spectroscopy (EDS), which identifies elements in a spot on the sample. That makes SEM a workhorse in materials science for fracture surfaces, coatings and particles.

Cryo-electron microscopyElectron microscopy of samples frozen so fast that water forms glass-like ice, preserving molecules near their natural state. (cryo-EM) freezes samples so fast that water forms glass-like ice instead of crystals, preserving molecules close to their natural state. It is now a major method for determining protein structures.

Choosing a method

Common mistakes

• Confusing magnification with resolution.
• Interpreting a fluorescence image without no-label or no-primary-antibody controls.
• Comparing images taken with different exposure settings.
• Forgetting that EM sample preparation (drying, staining, coating) can create artefacts.

Quick recap

• Light microscopes are limited to about 200 nm resolution.
• Fluorescence uses excitation and emission filters with labels such as DAPI, GFP and antibodies; confocal adds sharp optical sections.
• TEM looks through thin sections; SEM scans surfaces; both need vacuum and careful preparation.
• Pick the method by the question: living or fixed, inside or surface, molecules or materials.

These instruments are operated only after training by the facility that runs them.

Try it

This lesson is quiz-only. Open the Do tab to check your understanding.