Traditional methods for observing molecular events in living organisms involve either sacrificing animals at multiple time points or performing subjective measurements over time. Optical imaging provides several advantages over existing techniques, including improved sensitivity, higher throughput, and the capacity for rapid real-time data acquisition. Additionally, because optical imaging is non-invasive, it both reduces animal use and supports whole organism imaging, meaning it can be used for long-term monitoring of experimental cohorts or assessing disease progression. This article highlights some key factors to consider for optical in vivo imaging experiments and offers practical guidance to help researchers achieve reliable results.
Optical imaging studies are based on either bioluminescence or fluorescence, depending on the target(s) being investigated. Bioluminescent reporters such as luciferase have a high signal-to-noise (SNR) ratio since they typically represent the only light emitting source in a model animal, making them well-suited to single, cellular targets. In contrast, fluorescence has a relatively low SNR but collectively exhibits distinct excitation/emission wavelengths spanning the entire optical spectrum, allowing for multi-target imaging with multi-channel imaging ability. To facilitate the latter, a vast array of fluorescent reagents has been developed for labeling targets that include small molecules, antibodies, and cells.
The absorption and scattering of light by water, lipids, oxyhemoglobin, and deoxyhemoglobin reduces the tissue penetration depth in a wavelength-dependent manner. Consequently, these effects are more pronounced for fluorescence-based detection (where light is both emitted and required for excitation), making target depth a key consideration for experimental design. Using bioluminescence, it is possible to observe deep organs of experimental animals (e.g., liver, heart, or lungs), although the signal intensity will vary depending on the amount of light emitted and organ depth. For deep penetration with fluorescence imaging, longer wavelength bands of yellow/orange (550–700nm) and near-infrared range (700–900nm) are strongly recommended; this is due to the improved light transmittance afforded by reduced scattering and absorption, as well as the higher SNR owing to minimized interference from endogenous fluorophores (e.g., collagen and elastin). Whichever imaging modality is used, it is essential that the animal is oriented correctly to maximize experimental sensitivity.
Where optical imaging studies are based on fluorescence, it is important to note that unwanted background signal can come from sources other than endogenous fluorophores. For example, the most commonly used food for laboratory mice contains alfalfa, a plant that is rich in chlorophyll. This has been shown to produce intestinal autofluorescence, highlighting the value of implementing an alfalfa-free diet for at least a week prior to imaging.
Because the hair and skin absorb and scatter light, hairless, albino, or Hr mutant animal strains are often preferred for optical in vivo imaging studies. Where using these strains is impractical due to the genetic background or immunocompetency status of the model, the hair should be removed by shaving or using a depilatory cream; this is best done 24 hours prior to imaging as mild skin inflammation caused by hair removal can affect the biodistribution and/or activation of inflammation-targeted probes.
In any experiment using an optical imaging system, the best SNR is usually obtained by adjusting the binning and exposure times. For bioluminescence, this involves taking an initial image with moderate binning (4x4) and a short exposure time (5–10 seconds), which can then be increased incrementally (binning of 8x8, 16x16, and so forth, and exposure times of up to 60–600 seconds). During fluorescence-based experiments, the best SNR is usually achieved at lower binning (1x1 to 4x4) and shorter exposure times (1–10 seconds) compared to luminescence, and special care should be taken to avoid long exposure times, which increase background noise.
In addition to binning and exposure, saturation should also be considered. To put this into context, remember that the camera sensor has a pixel that receives light and stores signal information, and that the maximum amount of signal the pixel can store is fixed. This maximum signal amount is determined by the bit of pixel so, if the optical imaging system is fitted with a 16-bit pixel, the maximum signal strength will be 65535 (216 minus 1). If the signal exceeds the fixed maximum, the correct signal strength cannot be measured—a phenomenon known as saturation. Ways of preventing saturation include shortening the exposure time and lowering the binning.
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