More information

• For more information on Vevo F2 LAZR-X, go to Fujifilm Visualsonics.

Consider the eyes

• Following anaesthesia, ensure to protect the eyes of your animals from drying out by applying eye ointment with a clean cotton wool bud. Contact animal facility personnel for purchase of eye ointment.

Bioluminescence and fluorescence in short

• Bioluminescence imaging is based on endogenous production of light by expression of the enzyme luciferase upon reacting with the substrate luciferin in presence of oxygen and ATP. Since bioluminescent imaging does not require an excitation light source, the level of background (often caused by autofluorescence) is extremely low. Hence, bioluminescence imaging enables imaging of processes that produce minimal signal, e.g. lymphocyte trafficking or detection of small numbers of cancer cells.
• Fluorescence results from a process that occurs in molecules known as fluorophores. Typically, acquisition of a fluorophore signal relies on excitation by an external light source. The fluorophore absorbs the excitation light, reaching a higher energy state. By returning to its former state, it emits fluorescent light. Fluorescence imaging is widely used for e.g. gene expression and protein detection.

Bioluminescence or fluorescence?

• Absorption and scattering of light by water, lipids, ect. reduces tissue penetration depth of light, depending on the wavelength. These effects are more pronounced for fluorescence-based detection, where light is both required for excitation and is emitted. Using bioluminescence, it is possible to observe deep organs of experimental animals, e.g., liver, heart, or lungs, although signal intensity will vary depending on the amount of light emitted and organ depth.

• For imaging under bone structures such as the brain and spinal cord, consider choosing a bioluminescence setup rather than fluorescence as these areas are difficult to illuminate sufficiently.

• For deep penetration with fluorescence imaging, longer wavelength bands in the red range (650-900 nm) are recommended. This is due to the improved light transmittance afforded the by reduced scattering and absorption, as well as the higher signal-to-noise ratio owing to minimized interference from endogenous fluorophores (e.g. collagen and elastin which autofluoresce in the blue/green range)

• For studies quantifying tumor diameters, consider using 2D bioluminescence as opposed to 3D fluorescence. 3D fluorescence is ok for generating images but is rarely published like that.
• Bioluminescence and fluorescence imaging may also be combined in the same animal.

Which fluorophores are possible?

• Consult our Specifications section for installed filters and filter bandwiths.
• In case of multiple fluorophores, consult a spectra viewer to estimate the risk of cross-talk/bleed-through.

Inside the Newton

• Consider placing the rodent so the area of interest is facing upwards, i.e. towards the top of the instrument.
• In case of strong bioluminescence/fluorescence, consider using spacers between each animal. Spacers are located inside the door of the Newton.

Consider the eyes

• Following anaesthesia, ensure to protect the eyes of your animals from drying out by applying eye ointment with a clean cotton wool bud. Contact animal facility personnel for purchase of eye ointment

Did my experiment work at all?

• Obtain a quick image for swift evaluation by adjusting the binning settings.

Consider autofluorescence

For fluorescence-based studies, use a control mouse to investigate the degree of autofluorescence potentially masking fluorescence in the area of interest.

• Autofluorescence in abdominal region: Consider alfalfa-free chow for 4-7 days prior to imaging to reduce autofluorescence caused by phytochromes in the abdominal region. Consult quick-guide on site for info on wavelengths. Consider emptying the bladder prior to imaging. Contact stable personnel for guidance.
• Autofluorescence of fur: Consider removing the fur by shaving and/or depilatory cream and/or to work in the red spectrum. Fur autofluorescence may mask results particularly when detecting fluorescence in the blue-green spectrum. Consider the possibility of fur removal 24 h prior to imaging as mild skin inflammation caused by fur removal might affect distribution and/or activation of certain inflammation-targeted probes.
• Autofluorescence of tail: Consider covering the tail with e.g. black paper (located on site in drawer under Newton) prior to imaging.

Spectral unmixing

• Your fluorophores may have a significant overlap of excitation and emission spectra, also known as crosstalk or bleed-through. With SpectraViewer, you can determine overlap of your fluorophores. In case of overlap, you may have to consider spectral unmixing to be able to distinguish between the signals. Spectral unmixing can only be done on-site while acquiring the image.
• Read more about spectral unmixing
• If you need further spectal unmixing files, please contact the Phenotyping Core Facility for guidance.