Abstract
To understand the pathogenic processes for infectious bacteria, appropriate research tools are required for replicating and characterizing infections. Fluorescence and bioluminescence imaging have primarily been used to image infections in animal models, but optical scattering in tissue significantly limits imaging depth and resolution. Photoacoustic imaging, which has improved depth-to-resolution ratio compared to conventional optical imaging, could be useful for visualizing melA-expressing bacteria since melA is a bacterial tyrosinase homologue which produces melanin. Escherichia coli-expressing melA was visibly dark in liquid culture. When melA-expressing bacteria in tubes were imaged with a VisualSonics Vevo LAZR system, the signal-to-noise ratio of a 9×dilution sample was 55, suggesting that ∼20 bacteria cells could be detected with our system. Multispectral (680, 700, 750, 800, 850, and 900 nm) analysis of the photoacoustic signal allowed unmixing of melA-expressing bacteria from blood. To compare photoacoustic reporter gene melA (using Vevo system) with luminescent and fluorescent reporter gene Nano-lantern (using Bruker Xtreme In-Vivo system), tubes of bacteria expressing melA or Nano-lantern were submerged 10 mm in 1% Intralipid, spaced between <1 and 20 mm apart from each other, and imaged with the appropriate imaging modality. Photoacoustic imaging could resolve the two tubes of melA-expressing bacteria even when the tubes were less than 1 mm from each other, while bioluminescence and fluorescence imaging could not resolve the two tubes of Nano-lantern-expressing bacteria even when the tubes were spaced 10 mm from each other. After injecting 100-μL of melA-expressing bacteria in the back flank of a chicken embryo, photoacoustic imaging allowed visualization of melA-expressing bacteria up to 10-mm deep into the embryo. Photoacoustic signal from melA could also be separated from deoxy- and oxy-hemoglobin signal observed within the embryo and chorioallantoic membrane. Our results suggest that melA is a useful photoacoustic reporter gene for visualizing bacteria, and further work incorporating photoacoustic reporters into infectious bacterial strains is warranted.
Highlights
Visualization of gene expression with reporter genes has greatly increased our understanding of mammalian and bacterial cells.[1,2,3,4] Reporter genes encode proteins that can be detected due to the optical properties of the proteins or the products formed by the proteins
The current study investigates the utility of melA as a reporter gene in E. coli
To determine the minimum number of melA-expressing bacteria that can be detected with the Vevo LAZR system, the concentration of bacteria was multiplied by the voxel volume (9.33 × 10−6 cm3) that was divided by the Peak signal-to-noise ratios (PSNRs) from the photoacoustic images
Summary
Visualization of gene expression with reporter genes has greatly increased our understanding of mammalian and bacterial cells.[1,2,3,4] Reporter genes encode proteins that can be detected due to the optical properties of the proteins or the products formed by the proteins. Cells expressing lacZ, which produces a dark-blue product in the presence X-gal, generates strong photoacoustic signals, allowing lacZ to be a photoacoustic reporter gene.[9,17] due to the aforementioned limitations of lacZ, more biologically compatible photoacoustic reporter genes would be desirable. When expressed in human MCF-7 breast cancer cells, tyrosinase caused cells to become visibly dark, which provided strong photoacoustic signal.[23] Several studies have validated tyrosinase as a photoacoustic reporter gene for imaging cells in animals.[20,21,22,24,25] Melanin’s absorption spectrum is substantially different from oxy- and deoxy-hemoglobin, allowing relatively high-resolution (
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