Brightfield & phase contrast
Check cell morphology and confluence during routine culture.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
1Why living cells are hard to see
A cultured cell is almost transparent. It absorbs a fraction of a percent of the light passing through it, so in plain brightfield it is nearly invisible.
Absorption
Shows stained or pigmented samples well. Unstained cells only appear as faint outlines, and closing the condenser aperture to make them darker also blurs them.
Phase contrast
A ring in the condenser and a matching phase ring in the objective shift and dim the undiffracted light so it interferes with the light the cell scattered. Thick parts look dark on grey, ringed by a bright halo.
DIC
Polarised light is split into two beams a fraction of a micron apart. Where the cell's thickness changes, they recombine out of step, giving a shaded, relief-like image of gradients with no halo and a thin focal plane.
2How phase contrast works and fails
The condenser annulus lights the sample from a hollow cone, so all the light the sample does not scatter passes through one ring at the back of the objective. That ring carries a phase plate that advances it by a quarter wave and absorbs most of it.
3Using it to judge a culture
The everyday use is a look at every flask before it is fed or passaged: how confluent it is, whether the cells have their normal shape, and whether anything is growing that should not be. Confluence is the fraction of the growth surface covered by cells.
4DIC in brief
DIC uses a polariser, a Nomarski (Wollaston) prism under the condenser, a matching prism behind the objective and an analyser. The two prisms must match the objective, the illumination must be set up by Köhler alignment, and the sample must sit on glass: moulded plastic dishes are birefringent and scramble the polarisation, so DIC through plastic is flat and grey.
Key terms
Practise these as flashcards →The extra distance light effectively travels through a denser object, refractive-index difference × thickness; what phase contrast and DIC make visible.
The matched pair of rings in the objective and the condenser; the condenser setting must match the number engraved on the objective.
The bright rim around objects in phase contrast; an artefact of the method that enlarges apparent outlines.
Alignment that focuses the field diaphragm in the sample plane and centres it, giving even, glare-free light; the starting point for DIC.
In DIC, the deliberate phase offset between the two sheared beams that sets the background grey level and the relief direction.
The percentage of the growth surface covered by cells; most lines are passaged at 70–90% before they pile up or become contact-inhibited.
Sets the illumination cone. In brightfield, closing it trades resolution for contrast; in phase contrast it is opened fully so the annulus is not clipped.
When to use it
- Daily checks of live cultures in flasks and plates: confluence, morphology, rounding, detachment and contamination.
- Watching unlabelled live cells over time (division, migration, wound closure) without dyes or phototoxic fluorescence light.
- DIC when you need fine detail in a thin optical section, such as neurites, filopodia or the edges of embryos, on a glass-bottom dish.
- Brightfield for stained material: Gram stains, H&E sections, crystal-violet colonies, trypan-blue counts.
Compared with alternatives
Counts cells and the fraction alive in suspension; confluence only estimates coverage of a surface.
Labels specific molecules; use it when you need to know what a structure is, not just that it is there.
Builds on phase contrast or DIC with an environmental chamber to follow cells for hours or days.
Automated confluence and cell-shape measurement from phase images needs segmentation that copes with halos.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Differential interference contrast: polarised, sheared beams turn thickness gradients into a shadow-cast relief image with no halo and a thin depth of field. It needs glass vessels, matched prisms and Köhler illumination.
- Polariser, two Nomarski prisms and an analyser, all matched to the objective.
- Samples must be on glass: plastic is birefringent and destroys the contrast.
- Bias retardation is set by the user; a small bias gives the best relief.
- Higher resolution and thinner optical section than phase contrast, but more expensive optics.