Confocal microscopy
A single focused laser spot is raster-scanned by galvanometer mirrors and the emission passes an adjustable pinhole to a photomultiplier or GaAsP detector. Flexible zoom, pinhole and spectral detection; slower and harsher on live cells.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Point scanning
A single focused laser spot is raster-scanned by galvanometer mirrors and the emission passes an adjustable pinhole to a photomultiplier or GaAsP detector. Flexible zoom, pinhole and spectral detection; slower and harsher on live cells.
How it differs from the standard method
- Adjustable pinhole (usually 1 AU) and pixel size set by zoom.
- Photon-counting detectors with excess noise; pixel dwell times of microseconds.
- Best for fixed, thick or multi-colour samples.
1Throwing away the blur
A widefield image of a thick sample is the sharp focal plane buried under light from every plane above and below it. A confocal microscope removes that light physically.
The pinhole
Sized in Airy units (the diameter of the focused spot). At 1 AU it passes about 84% of the in-focus light and gives a section of about 0.8 µm with a 1.4 NA lens; smaller is thinner but darker, larger is brighter but hazier.
Sampling in 3D
Pixels about 2.3 times smaller than the lateral resolution, and z-steps about 2.3 times smaller than the section. Coarser loses detail; finer only adds dose and time.
Every slice costs the whole sample
The excitation cone passes through the planes above and below, so each slice bleaches the whole stack. Oversampling, high laser power and repeated scans add up.
2Three ways to do it
A point-scanning confocal moves one spot with galvanometer mirrors and counts photons with a photomultiplier or GaAsP detector; the pinhole is adjustable and the zoom sets the pixel size, so it is the flexible choice for fixed samples and thick specimens. A spinning disk sweeps thousands of pinholes across the field at once and images them onto a camera: it is many times faster and gentler on living cells, but the pinholes are a fixed size and, in thick or dense samples, out-of-focus light leaks through neighbouring pinholes.
3What goes wrong
Opening the pinhole to get more signal turns a confocal back into a widefield microscope. Taking slices far apart turns the side view into a staircase.
Key terms
Practise these as flashcards →The thickness (axial FWHM) of the slab that contributes to a confocal image; set by NA, wavelength and pinhole size.
The diameter of the focused spot's central disc, 1.22 λ/NA; pinhole sizes are quoted in AU.
A series of optical sections taken at regular focus steps, viewed as slices, side views or 3D renderings.
Out-of-focus light from a thick sample passing through neighbouring pinholes on the disk.
Simultaneous absorption of two infrared photons; it scales with intensity squared, so it only happens at the focus.
A detector placed close to the objective that collects emitted light without sending it back through the scanner and pinhole, so scattered photons still count.
The distance over which light loses about two thirds of its unscattered photons; ~50–100 µm for visible light and ~150–200 µm near 920 nm in brain.
A difference between immersion medium and sample index that causes depth-dependent aberration and axial distortion.
When to use it
- Sharp optical sections and 3D reconstructions of samples thicker than a few microns: organoids, cysts, embryos, tissue sections.
- Colocalisation in thick specimens, where out-of-focus light would otherwise create false overlap.
- Live imaging of thicker or dimmer samples by spinning disk, when speed and low dose matter.
- Two-photon imaging hundreds of microns deep in scattering tissue, such as brain slices or intravital imaging.
Compared with alternatives
Faster and more photon-efficient for thin, flat cells, but with no optical sectioning.
Lights only the plane being imaged, so large, cleared or living samples get far less dose.
Confocal still sits at the ~200 nm diffraction limit; STED, SIM, SMLM or expansion go beyond it.
Two-photon microscopy through a cranial window is a core method for imaging in living animals.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A single focused laser spot is raster-scanned by galvanometer mirrors and the emission passes an adjustable pinhole to a photomultiplier or GaAsP detector. Flexible zoom, pinhole and spectral detection; slower and harsher on live cells.
- Adjustable pinhole (usually 1 AU) and pixel size set by zoom.
- Photon-counting detectors with excess noise; pixel dwell times of microseconds.
- Best for fixed, thick or multi-colour samples.