IVIS Spectrum - Best practices

Imaging preparation

  • Reduce light absorption by tissue: Use hairless or albino animals to minimize melanin-associated photon absorption. If using hairy or pigmented strains, fully shave the imaging area before acquisition to reduce photon loss due to melanin and hair scattering. If using chemical depilation, rinse thoroughly to avoid skin irritation that could impact signal

  • Limit tissue autofluorescence: Feed animals an alfalfa-free (chlorophyll-free) diet for at least 1 week prior to imaging. This minimizes gastrointestinal autofluorescence

  • Standardize physiological conditions: Use consistent anesthesia protocols and a heated stage to prevent hypothermia, which can affect probe biodistribution and signal intensity

  • Ensure consistent animal positioning: Position animals in the same orientation across imaging sessions to allow accurate and reproducible ROI placement

Signal acquisition

  • Avoid auto-exposure for raw signal quantification:

    • When working with uncalibrated photon counts, disable auto-exposure

    • Keep all camera settings (exposure time, binning, f/stop) constant for comparability

  • Use radiance (bioluminescence) or radiance efficiency (fluorescence) for calibrated, cross-study comparisons:

    • Exposure-independent quantification: Radiance is normalized for exposure time, making it robust to minor variations in acquisition settings

    • Enables cross-animal and group comparisons: Because it accounts for imaging variables, radiance allows for reliable signal comparison between animals, groups, and imaging sessions, even if ROI sizes or exposures differ

    • Improves reproducibility in longitudinal studies: Ideal for tracking biological changes over time in the same subject, since it removes technical bias from repeated measurements

    • Essential for multi-center or multi-operator studies: Radiance allows for standardized data interpretation across labs using same systems, assuming proper calibration

Imaging optimization

  • Start with a pilot experiment: Define signal kinetics and substrate/probe timing, especially for bioluminescence imaging

  • Avoid imaging during circadian fluctuations: Image animals at consistent times of day to reduce variability in metabolism and gene expression

  • Allow imaging system warm-up: Let CCD cameras and optics stabilize thermally for consistent sensitivity and minimal signal drift

  • Log comprehensive metadata: Record animal ID, age, weight, reporter info, imaging settings, and experiment date/time.

Fluorescence-specific tips

  • Use far-red fluorophores: Prefer fluorophores emitting 650–900 nm for better tissue penetration and reduced autofluorescence

  • Apply spectral unmixing and background subtraction:

    • Use reporter-negative control animals to characterize tissue autofluorescence

    • Define background ROIs in non-targeted regions (contralateral tissue) for subtraction

    • Ensure consistent ROI placement and size across background and target areas

  • Ensure proper controls for multiplex imaging:

    • Include single-reporter control animals for each fluorophore to establish accurate spectral fingerprints and avoid cross-channel bleed-through

    • Use contralateral tissue or a background ROI within the same animal as internal reference region to estimate and subtract non-specific signal

    • Use reporter-negative animals imaged with all channels to define baseline autofluorescence across filters

Bioluminescence-specific tips

  • Use freshly prepared substrate

  • Store luciferin/coelenterazine at -80°C in the dark, and avoid repeated freeze-thaw cycles

  • Optimize substrate administration route: Choose IP or IV injection based on timing and biodistribution needs

  • Account for metabolic and physiological variability: Luciferase signal depends on ATP and oxygen availability, consider how disease state, perfusion, and tissue viability may affect interpretation

Data quantification

  • Define consistent ROIs: Use anatomical landmarks to apply identical ROI shape and placement across animals and time points

  • Use average or total signal appropriately:

    • Use total signal when ROIs vary in size (growing tumors)

    • Use average signal when ROIs are consistent in size/shape, especially in comparative studies

    • Always specify which metric is used to ensure interpretability

  • Never quantify signal from saturated ROIs:

    • Saturated signals are clipped, meaning they do not represent true photon flux, and should be excluded from quantitative analysis

    • To prevent saturation: Use a shorter exposure time, increase the f/stop to reduce the aperture and hence reduce the light and reduce binning

  • Always keep your signal well within the dynamic range of the detector

Image quality

In vivo optical imaging relies on detecting low-intensity light emitted from within biological tissues. Achieving high image quality requires optimizing the signal-to-noise ratio (SNR), spatial resolution, and light sensitivity of the imaging system. The key tunable parameters are outlined below:

  • Binning

    • It consists in combining adjacent pixels on the CCD or CMOS sensor into a single “superpixel”.

    • It increases SNr as more photons are collected per binned pixel.

    • It decreases spatial resolution as each superpixel covers a larger area.

    • It speeds up the data acquisition leading to faster scans with lower read noise.

    • Use 4×4 or 8×8 binning for very weak bioluminescent signals; use lower binning for high-detail fluorescence.

  • Exposure time

    • Longer exposures collect more photons, increasing signal strength and SNR.

    • It is especially critical in bioluminescence, where the signal is inherently weaker than in fluorescence.

    • It increases the risk of motion blur in live animals.

    • There is a potential of signal saturation if exposure is too long for bright fluorescent reporters.

  • Aperture opening of the lens (f/stop)

    • Low f/stop corresponds to wide aperture enabling the collection of more light.

    • High f/stop corresponds to narrow aperture collecting less light but offering a greater depth of field.

    • Lower f/stops increase light sensitivity and improve SNR but reduce depth of field and may blur regions slightly out of focus.

    • Higher f/stops improve sharpness across depth but require longer exposure.

    • Use low f/stop (f/1.2–f/2.8) for dim signals; increase if you need more DOF for large subjects.

    • Do not use f/1.2 for fluorescence as there is a risk of signal saturation.

  • Cooling temperature

    • Lower Camera temperature reduce thermal noise (dark current noise), improving SNR during long exposure.

    • It is crucial in bioluminescence imaging due to the long integration time and the weak signals.

    • Always ensure maximum cooling for low-light bioluminescence imaging.

  • Light source power

    • High excitation leads to stronger signals.

    • It also increases background autofluorescence, the risk of photobleaching or phototoxicity.

    • Use the minimum excitation power necessary to achieve acceptable SNR.

  • Background substraction and calibration

    • Dark current subtraction, flat-field correction, and background ROI calibration improve final image clarity and quantification accuracy.

    • This reduces artifacts and enhances perceived contrast and SNR.

  • Bioluminescence: long exposure, high binning, low f/stop, and deep sensor cooling to maximize SNR

  • Fluorescence: balance exposure, binning, and excitation power to avoid saturation while maintaining spatial clarity

*Imaging parameters affecting optical imaging quality*

Imaging parameters affecting optical imaging quality