Diet composition - A critical variable in fluorescence imaging for preclinical cancer research

Background: Fluorescence based imaging is a cornerstone of preclinical cancer research, enabling noninvasive, real time assessment of tumor growth, metastasis, and therapeutic response. However, dietary ingredients can impact image quality and data integrity. Grain-based chow diets contain nonnutritive components such as phytochemicals and potential toxins including endotoxins, mycotoxins, and heavy metals, that vary by batch and influence animal phenotype, metabolism, and imaging outcomes. Dyes (FD&C Blue #1, Yellow #5, Red #40) commonly added to diets for visual differentiation may introduce background fluorescence, while alfalfa and other chlorophyll-rich dietary ingredients fluoresce strongly in the red and near infrared (NIR) range, overlapping with wavelengths used by many NIR fluorescent cancer imaging probes. The effect of different dietary ingredients on fluorescence imaging quality was evaluated in this study. Methods: Direct fluorescence imaging measurements of diet pellets were acquired on the IVIS TM Spectrum 2 using excitation wavelengths from 640 to 750 nm. For in vivo studies, 8 wk old male C57BL/6J and NU/J mice were initially fed alfalfa containing chow for a week, then transitioned to either purified diets (with or without dyes) or chows (with or without alfalfa) for two weeks. Multiple excitation wavelengths were used with appropriate excitation/emission filter sets to capture the range of diet- and dye-related signal from 500-750 nm with most analysis focused on the 640-750 nm range. Datasets were analyzed using Living Image TM 4.8.3 system software to quantify gastrointestinal fluorescence, using spectral unmixing algorithms to separate the signal from chlorophyll/dye and a relevant NIR fluorescent probe. Results: In vitro , both alfalfa (640 nm and 675 nm) and incorporated dyes (640 nm) contributed to strong fluorescence signal. However, only the alfalfa containing diets produced strong background signal at 640 and 675 nm in vivo , sufficient to obscure tumor and inflammation probe signals. Alfalfa free and purified diets, with or without added dyes, cleared the background chlorophyll signal within 1-2 weeks, improving signal to noise ratios and image clarity. Data were similar in both NU/J and C57BL/6J strains. Conclusions: Diet composition strongly influences fluorescence imaging. Alfalfa derived chlorophyll in chows introduces substantial NIR autofluorescence, which is minimized in alfalfa-free diets. Although dyes in purified diets fluoresce strongly in vitro , they likely degrade upon consumption contributing to minimal in vivo signal. Standardizing diet offers a simple, cost effective approach to improve image quality, reproducibility, and translational relevance in preclinical cancer imaging.