Live-cell & time-lapse
A heated, gassed and humidified chamber on the stage, holding a dish or plate at incubator conditions for hours to days of imaging. The difference between a movie of healthy cells and a movie of stressed ones.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Stage-top incubators
A heated, gassed and humidified chamber on the stage, holding a dish or plate at incubator conditions for hours to days of imaging. The difference between a movie of healthy cells and a movie of stressed ones.
How it differs from the standard method
- 37 °C with 5% CO₂ for bicarbonate media; HEPES alone buffers only for short runs.
- Humidification and a lid, or a gas-permeable lid, to stop evaporation.
- Let the system equilibrate before starting, and use hardware autofocus.
1Keeping cells alive on a microscope
A time-lapse turns a microscope into an incubator with a camera. Cells are filmed for hours to days to watch division, migration, differentiation or death as they happen, one cell at a time.
Light is a drug
Every frame costs the cells some photodamage, mostly through reactive oxygen made by excited fluorophores. The harm builds up with the total dose: frames × power × exposure. It shows first as slower mitosis and cell-cycle arrest, long before cells die.
Sample the event
The frame interval has to be short compared with what you time: four or more frames per mitosis, and short enough that a cell moves less than about half a nucleus between frames so it can be tracked.
Focus drifts
A dish and stage warming up settle by micrometres over the first hour, then creep. Hardware (reflection) autofocus finds the coverslip at each time point without extra light on the cells.
Imaged vs not imaged
Compare cells that were filmed with cells kept in the same incubator but not imaged, or imaged less often. If the two differ, the light is part of the experiment.
2Spending the light budget
There is a fixed amount of light a sample can take, and a time-lapse spends it across frames, channels, z-planes and focusing. The usual trade: fewer photons per frame (lower power, binning, a more sensitive camera, a brighter label) let you take more frames, and fewer frames or planes let you use more photons each.
3From movies to screens
High-content screening uses the same automation to image every well of 96- to 1536-well plates and extract numbers per cell: nuclei counts, intensities, shapes. The statistics that matter are those of the plate.
Key terms
Practise these as flashcards →A heated, gassed and humidified chamber on the microscope stage that holds cells at 37 °C and 5% CO₂ during imaging.
Damage to living cells caused by illumination, largely through reactive oxygen species; it grows with the total light dose.
Time between consecutive time points; it limits how precisely events can be timed and cells tracked.
Focus held by detecting the reflection of an infrared beam from the coverslip, without exposing the fluorophores.
Outer wells of a plate evaporate and warm differently, so cells there grow differently and read differently.
Assay-quality score from control wells: 1 − 3(σ₊ + σ₋)/|μ₊ − μ₋|; ≥ 0.5 separates hits well.
One camera image within a well; a well is usually sampled with several fields.
When to use it
- Following individual cells through division, migration, differentiation or death, where a snapshot of a population would average away the order of events.
- Measuring rates and durations (mitotic timing, migration speed, oscillations) that need the same cells observed repeatedly.
- Screening many conditions on plates with image-based readouts, where per-cell numbers matter more than single beautiful images.
Compared with alternatives
The usual optics for live-cell movies; fast and light-efficient for thin cultured cells.
Gives far lower light dose per plane for long 3D movies of embryos and organoids.
Population assays of growth and cell cycle without imaging; quicker when individual histories do not matter.
Measures many cells at one time point quickly; prefer it when you need distributions, not trajectories.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A heated, gassed and humidified chamber on the stage, holding a dish or plate at incubator conditions for hours to days of imaging. The difference between a movie of healthy cells and a movie of stressed ones.
- 37 °C with 5% CO₂ for bicarbonate media; HEPES alone buffers only for short runs.
- Humidification and a lid, or a gas-permeable lid, to stop evaporation.
- Let the system equilibrate before starting, and use hardware autofocus.