Your mouse model is ready, the reporter is still undecided, and the imaging system your group buys will run its studies for years. Bioluminescence and fluorescence imaging both carry a light signal from inside a living animal to a cooled camera. Both let you image the same animal again and again. Together they are the most common method of preclinical in vivo imaging. They suit different questions, though, and your choice of reporter shapes the system you need.
To track live cell populations, follow tumour growth or measure gene expression over time, bioluminescence is usually the more sensitive choice, because the animal itself gives off almost no light. To follow where a labelled compound, antibody or nanoparticle goes, or to image several labels in one animal, fluorescence gives you more freedom. Every Spectral Instruments Imaging system, sold in Israel by Merkel Technologies, images both, and some models add X-ray.
Why bioluminescence imaging is so sensitive
In bioluminescence imaging, a luciferase enzyme expressed by your cells converts its substrate, luciferin, in the presence of oxygen. The reaction emits light. Nothing has to be shone on the animal, so the background is close to zero. Spectral Instruments Imaging notes that this lets you detect very small numbers of viable cells, even in deep tissue.
The cost is in the preparation. Most luciferase reporters have to be genetically encoded and expressed inside the cells, which makes labelling slower and more laborious than with a chemical fluorophore. The signal also follows the substrate, rising and falling after each injection. Reliable data comes from consistent dosing and from imaging at the same point of the kinetic curve every time.
Colour matters here too. Firefly luciferase, whose substrate is inexpensive and distributes well in vivo, peaks at 612 nm at 37 °C. A significant part of its light falls above 650 nm, where tissue lets more of it through. Newer pairs such as AkaLuc and its substrate move the peak to 650 nm, at a higher substrate cost.
Where fluorescence imaging fits better
Fluorescence imaging needs excitation light, and the tissue answers with some light of its own. The background is therefore higher than in bioluminescence. In exchange, you have no substrate to time. A chemical fluorophore can label a drug candidate, an antibody or a nanoparticle as easily as a cell. That makes fluorescence the stronger choice for biodistribution studies, nanoparticle tracking and multiplexed imaging with several fluorophores.
Wavelength decides how deep you can see. Red light, especially above 650 nm, penetrates tissue better than blue or green. For deep targets, choose near-infrared fluorophores, and for several labels in one animal, choose fluorophores whose spectra barely overlap. Systems also differ in how many excitation and emission combinations they offer. The Lago carries 14 LED excitation wavelengths and 20 emission filters, so a new probe rarely means changing filters.
What a planar optical image can and cannot claim
An optical image reports where a reporter signal is and how strong it is. It is a planar view through the animal, so the same number of cells reads brighter near the skin than deep in an organ. Your cleanest comparisons are therefore within one animal over time. Absolute calibration, which Spectral Instruments Imaging sets at the factory, keeps those numbers comparable from one session to the next.
When the signal has to sit on anatomy, an X-ray image taken in the same position places it on the skeleton. The Lago X and Ami HTX include X-ray, and Lago and Ami units can be upgraded to it on site. For a quantitative measurement tied to anatomy, use tomographic imaging such as PET combined with MRI, as in our post on PET-MRI. At the other end of the scale, intravital microscopy shows what individual cells do at a single site.
How many mice per session
Throughput sets the size of the system. The Lago and Lago X image up to ten mice at once over a 25 by 25 cm field. The manufacturer builds them for imaging cores and CROs. The benchtop Ami HT and HTX take five mice over 25 by 17 cm. The Kino, also benchtop, images three mice over 13 by 13 cm and is aimed at small groups on a tight budget.
Count the animals you need at each time point. Say your study images 30 mice at a fixed time after injection. That is three sessions on a Lago and ten on a Kino, and each session needs its own injections on schedule.
Keeping a longitudinal study consistent
Imaging the same animals over time reduces the number of animals a study needs and removes variability between subjects, as long as every session runs the same way. Spectral Instruments Imaging recommends uniform anaesthesia and positioning. It also recommends injecting the substrate or probe at the same time and by the same route for every animal. Handling systems such as its DecaFlow position up to 20 mice on trays you can swap quickly, which keeps larger cohorts moving through the system.
Trying your reporter before you choose a system
The quickest way to settle bioluminescence against fluorescence is to image your own model. Merkel Technologies can arrange a demonstration on your samples. We support installation, training and service in Israel, alongside the other preclinical imaging systems we offer. Contact us to book the demonstration. No cost and no obligation for a first consultation.