BenchMasteryMolecular biology techniques & virtual lab

In vivo imaging

Luciferase-expressing cells imaged in an anaesthetised animal after a luciferin injection, in a light-tight camera such as an IVIS. Highly sensitive with almost no background, but semi-quantitative: light falls steeply with depth and depends on substrate timing.

photon fluxSimulator readyCore simulatorEngine: Microscopy & imaging
Choose a sub-technique

Our ruleSimulations prepare you for the bench but don't replace hands-on practice.

Sub-technique you chose

Bioluminescence (IVIS)

Luciferase-expressing cells imaged in an anaesthetised animal after a luciferin injection, in a light-tight camera such as an IVIS. Highly sensitive with almost no background, but semi-quantitative: light falls steeply with depth and depends on substrate timing.

How it differs from the standard method

  • Image at a fixed time after luciferin, from a kinetic series.
  • Report total flux in a region, background subtracted.
  • Keep the brightest pixel within the camera's range.
In this lesson · about 2 min read
  1. Seeing inside an animal without opening it
  2. What the numbers depend on
  3. Anaesthesia, welfare and the 3Rs
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Seeing inside an animal without opening it

In vivo imaging follows the same animal over days or weeks: a tumour's growth, the spread of labelled cells, the remodelling of bone around an implant. Each technique trades depth, resolution and specificity differently.

BLI

Bioluminescence

Luciferase cells plus injected luciferin emit light; almost no background, so very sensitive, but blurred and depth-dependent.

FLI

Fluorescence

Excite a reporter and collect its emission; works best in the near infrared, where tissue absorbs and autofluoresces least.

µCT

Micro-CT

Three-dimensional X-ray images with voxels down to ~10 µm; quantitative for bone, at the price of a radiation dose.

2What the numbers depend on

Bioluminescence depends on substrate kinetics: after an intraperitoneal luciferin injection the signal rises over minutes, peaks and decays, so every mouse must be imaged at the same time after injection, found from a kinetic series. It also falls with depth, dark fur and a cold animal.

3Anaesthesia, welfare and the 3Rs

Animals are imaged under inhalational anaesthesia on a warmed stage with eye lubricant, and recovered in a warmed cage under observation; long sessions need monitoring of breathing and temperature. Imaging needs institutional ethical approval and trained staff like any other procedure.

Key terms

Practise these as flashcards →
Total flux

Photons per second from a region of interest: the exposure-independent unit for bioluminescence.

Luciferin kinetics

The rise and fall of the bioluminescent signal after substrate injection; imaging is timed to its plateau or peak.

Saturation

Pixels at the camera's maximum count, where signal no longer rises with light.

Optical window

Roughly 650–900 nm, where haemoglobin and water absorb least and tissue is most transparent.

Autofluorescence

Fluorescence from the tissue or diet itself, such as chlorophyll in chow, that competes with the label.

Partial-volume effect

Blurring of a structure over neighbouring voxels when it is not much larger than the voxel.

BV/TV and Tb.Th

Bone volume fraction and trabecular thickness, the standard micro-CT measures of trabecular bone.

When to use it

  • Following tumour growth or metastasis in the same animals over time with luciferase-expressing cells.
  • Tracking a fluorescently labelled probe or cell population near the surface or in the near infrared.
  • Measuring bone architecture or implant integration longitudinally.

Compared with alternatives

Disease & implant models →

Callipers measure subcutaneous tumours directly; imaging adds deep or metastatic disease and implants hidden from view.

Calcium imaging →

For activity of individual cells in a window or a slice, microscope-based imaging gives cellular resolution that whole-body imaging cannot.

Fluorescent proteins & dyes →

How to choose a reporter's spectrum and brightness, which decides what can be seen through tissue.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Luciferase-expressing cells imaged in an anaesthetised animal after a luciferin injection, in a light-tight camera such as an IVIS. Highly sensitive with almost no background, but semi-quantitative: light falls steeply with depth and depends on substrate timing.

  • Image at a fixed time after luciferin, from a kinetic series.
  • Report total flux in a region, background subtracted.
  • Keep the brightest pixel within the camera's range.