2.1.

Creation of E. coli with Inducible Expression of Nano-lantern and melA

The melA and Nano-lantern genes were individually inserted into the pBAD/His B plasmid using standard restriction enzyme digestion and ligation reactions. Briefly, cDNAs for melA (kindly provided by Dr. Guillermo Gosset²⁹) and Nano-lantern (Addgene, plasmid #51970²⁸) were PCR-amplified using forward and reverse primers containing XhoI and HindIII restriction enzyme cleavage sequences, respectively. PCR products and pBAD/His B plasmid were digested with XhoI and HindIII restriction enzymes, and reporter genes were inserted within the plasmid by ligation. Reporter gene expression was inducible through the araBAD promoter in the presence of l-arabinose. The pBAD/His B plasmid was modified with an A2056C mutation in the pBR322 origin, which enhanced plasmid replication at least 2-fold.

To generate the bacterial cells used for imaging, E. coli strain ElectroMAX™ DH10B™ (Invitrogen) was transformed by electroporation with pBAD/His B expression vectors containing melA or Nano-lantern. Individual bacterial colonies were used to inoculate 4 mL cultures that were grown overnight (37°C and 225 rpm) before being diluted into 50 mL of lysogeny broth (LB) medium containing $0.1\mathrm{mg}/\mathrm{mL}$ ampicillin. These cultures were grown in 50 mL baffled shake flasks (37°C and 225 rpm) with an optical density of 0.6 to 0.7, induced with 0.02% (w/v) l-arabinose, and cultured for 48 h at 30°C before cells were collected by centrifugation. MelA-producing cultures were supplemented with $200\mu \mathrm{g}/\mathrm{mL}$ tyrosine and $20\mu \mathrm{g}/\mathrm{mL}$ copper sulfate.

2.2.

Imaging melA-Expressing E. coli with Photoacoustic Imaging

In order to normalize bacterial concentrations in imaging tubes, bacteria were weighed and resuspended at specific weight to volume ratios. MelA-producing E. coli (1 mL) was centrifuged in preweighed tubes at $5000g$ for 5 min, and the supernatant was removed. Tubes with bacterial pellets were weighed again to determine bacterial weight, and bacterial pellets were resuspended at the desired concentration ($\mathrm{mg}/\mathrm{mL}$) in phosphate buffered saline (PBS). Absolute bacterial concentration (bacteria colony-forming $\text{units}/\mathrm{mL}$) was determined by serially diluting bacteria at $20\mathrm{mg}/\mathrm{mL}$ with LB medium, and dilutions were spread on plates with $100\mu \mathrm{g}/\mathrm{mL}$ ampicillin. Plates were incubated at 37°C overnight, and colonies were counted the following day.

2.3.

Imaging Nano-Lantern-Expressing E. Coli with Bioluminescence and Fluorescence Imaging

Escherichia coli-expressing Nano-lantern was resuspended in PBS at 0.5 to $10\mathrm{mg}/\mathrm{mL}$ and added to wells of a 12-well plate with $5\mu \mathrm{g}/\mathrm{mL}$ (final concentration) coelenterazine-h added to bacteria solutions as the substrate for luciferase activity. The plate was imaged with a Bruker In-Vivo Xtreme system with back-illuminated 4 mega-pixel camera (pixel dimensions $35\times 35\mu \mathrm{m}$). Imaging was also performed with tubes as described for melA-expressing bacteria earlier. Nano-lantern-expressing bacteria were resuspended at $20\mathrm{mg}/\mathrm{mL}$, and immediately before injecting bacteria into PFA tubing (1.06-mm inner diameter, 1.4-mm outer diameter), $5\mu \mathrm{g}/\mathrm{mL}$ coelenterazine-h was added to bacteria suspensions. Tubes with bacteria had 1 mL syringes and 18 gauge needles on both ends, which sealed the tubes. Tubes were placed in a tube-holding apparatus as described earlier that was placed in a clear plastic container containing 1% Intralipid^®. Bacteria in tubes were imaged with a Bruker In-Vivo Xtreme system using 10-s exposure for bioluminescence and 2-s exposure for fluorescence (480-nm excitation and 535-nm emission). Tubes were imaged with 5, 10, 15, and 20 mm spacing between each other with a 10-mm depth in Intralipid^® from the bottom-facing camera.

2.4.

Signal Analysis and Statistics

Photoacoustic signals from images were quantified using a MATLAB script that analyzed exported data from the Vevo LAZR system. Bioluminescence and fluorescence signals were quantified using Bruker Molecular Imaging software (v 7.1.3.20550) from the In-Vivo Xtreme system using regions of interest created on the centers of the tubes (signal from bacteria) and regions of interest created near the outer corners of the images (noise). Peak signal-to-noise ratios (PSNRs) were calculated as peak signal from bacteria/standard deviation of noise. 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 ($9.33\times {10}^{-6}{\mathrm{cm}}^3$) that was divided by the PSNR from the photoacoustic images. For comparing photoacoustic/fluorescence/bioluminescence imaging modalities for resolving bacteria in optically scattering medium, intensity plots from tubes were acquired in ImageJ using the rectangle selection tool, which was at least twenty pixels along the tubes. Full-width at half-maximum values were acquired from intensity plots using the fwhm MATLAB function available at MATLAB Central.³¹

3.1.

Photoacoustic Imaging of Phantoms Containing E. Coli Expressing melA

In the absence of induced melA expression with l-arabinose, E. coli transformed with pBAD/His B plasmid containing melA had a normal light yellow color. Escherichia coli cultured with l-arabinose induced melA expression, which caused the bacteria to become dark [Fig. 1(a)], with much of the produced melanin secreted into the growth medium since the medium was still dark after centrifugation of bacteria. The effect of melA on bacteria proliferation was assessed by enumerating colonies from bacterial cultures with or without melA induction. Induction of melA in E. coli had no effect on proliferation during the exponential growth phase when bacteria reached a near-maximum concentration at 6-h culture (data not shown). After bacteria concentration plateaued, melA expression caused fewer colony-forming units in culture compared to bacteria without melA induction. This delayed toxicity may be due to the high expression levels caused by the araBAD promoter. A previous study by Jathoul et al.²⁰ demonstrated that tyrosinase expression in 10 different human cell lines had minimal effect on cell growth, suggesting that tyrosinase enzymes such as melA may have minimal cell toxicity when expressed at moderate levels.

Fig. 1

Photoacoustic imaging of Escherichia coli-expressing melA. (a) Imaging setup of tubes containing phosphate buffered saline (PBS) with or without serially diluted bacteria expressing melA. (b) Top-down view of the three-dimensional photoacoustic image of tubes shown in (a). Photoacoustic and ultrasound signals are shown in yellow and gray scale, respectively. Scale bar represents 5-mm distance.

The sensitivity of imaging melA-expressing bacteria with photoacoustics was determined by imaging tubes containing 1-, 3-, 9-, and 27-fold serial dilutions of melA-expressing bacteria, which corresponded to approximately $9{\times 10}^8$, $3{\times 10}^8$, $1{\times 10}^8$, and $3.3{\times 10}^7\text{bacteria}/\mathrm{mL}$, respectively. Tubes with bacteria were submerged into a water bath and imaged with the Vevo LAZR system using the 21 MHz LZ250 transducer. Photoacoustic signal was observed along some regions of the tube containing PBS that likely corresponded to air bubbles and/or low levels of signal from the top of the plastic tube. Due to this background signal, the only photoacoustic signals analyzed were those within the tubes. Photoacoustic signal was detected in tubes with up to 9-fold diluted bacteria solutions with a PSNR of 55 [Fig. 1(b)]. Given that the 9-fold diluted sample had approximately $1{\times 10}^8\text{bacteria}/\mathrm{mL}$, the estimated minimum number of bacteria detectable with our system was $\sim 20\text{bacteria}/\text{voxel}$. The estimated minimum number of bacteria detectable with our photoacoustic system (and all photoacoustic systems) will depend on a variety of factors including the concentration of bacteria and the physical volume of the bacterial sample that will affect the frequency of emitted photoacoustic waves.

To determine if the photoacoustic signal from melA can be separated from that of blood using multispectral unmixing, imaging experiments were performed with tubes containing blood, PBS, or melA-expressing E. coli in PBS. The photoacoustic spectrum of melA-expressing bacteria was similar to that of melanin, which allowed spectral unmixing of the melA signal from that of blood [Fig. 2(a)]. All tubes contained at least a few regions with photoacoustic signals corresponding to melA or blood suggesting that the unmixing algorithm within the Vevo software was not perfect. However, the vast majority of photoacoustic signal classification was appropriate for blood and melA-expressing bacteria. To determine if melA-expressing bacteria can be visualized in an environment with optical scattering similar to tissue, melA-expressing bacteria were pelleted in $200\mu \mathrm{L}$ PCR tubes, placed underneath $\sim 10\mathrm{mm}$ of chicken tissue, and imaged with the Vevo LAZR system. When imaged underneath chicken tissue, the melA-specific photoacoustic signal was detectable from melA-expressing bacteria [Fig. 2(b)]. Very little melA-specific photoacoustic signal was detectable from bacteria without melA induction [Fig. 2(b)]. Surprisingly, the photoacoustic signal was not detectable from the tube containing chicken embryo blood. The 10 mm of chicken tissue may have absorbed/scattered much of the lower wavelengths of light that blood preferentially absorbs. Compared to blood, melA produced relatively greater levels of photoacoustic signal at longer wavelengths which can penetrate tissue with higher efficiency, possibly explaining the greater photoacoustic signal from the tube of melA-expressing bacteria compared to the tube of blood.

Fig. 2

Photoacoustic imaging of blood and E. coli-expressing melA. (a) Tubes containing PBS, melA-expressing bacteria ($20\mathrm{mg}/\mathrm{mL}$), and chicken embryo blood were imaged with photoacoustics at multiple wavelengths. Shown is a top-down view of the three-dimensional image of the tubes. melA and oxy-hemoglobin-specific photoacoustic signals were unmixed with Vevo software and are shown in yellow and red, respectively, over a gray scale ultrasound image. Scale bar represents 3-mm distance. (b) Bacterial pellets with or without melA induction as well as chicken embryo blood were imaged in $200\mu L$ tubes under approximately 10 mm of chicken tissue. Tube positions are marked by the white circles.

3.2.

Comparing Fluorescence/Bioluminescence/Photoacoustic Imaging with E. Coli Expressing Nano-Lantern or melA

To illustrate the imaging resolution disadvantages of fluorescence and bioluminescence imaging in an optical scattering medium, we created E. coli transformed with pBAD/His B plasmid containing Nano-lantern, which is a very bright chimera of a yellow fluorescent protein and Renilla luciferase.²⁸ To verify that bacteria expressing Nano-lantern produce fluorescence and bioluminescence, bacteria expressing Nano-lantern were diluted at 0.5 to $10\mathrm{mg}/\mathrm{mL}$ in a 12-well plate and imaged with a Bruker In-Vivo Xtreme system (Fig. 3). In the absence of an optically scattering medium, fluorescence and bioluminescence signals from bacteria were very strong with PSNR $>400$ at $10\mathrm{mg}/\mathrm{mL}$ bacteria. Although $0.5\mathrm{mg}/\mathrm{mL}$ bacteria expressing Nano-lantern provided relatively little fluorescence and bioluminescence signals, longer exposure times for fluorescence and bioluminescence imaging would theoretically increase the PSNR for these dilutions, allowing smaller amounts of bacteria to be detected. The results suggest that in the absence of an optically scattering medium, bacteria expressing Nano-lantern can be imaged with greater sensitivity with bioluminescence and fluorescence imaging compared to bacteria expressing melA imaged with photoacoustics.

Fig. 3

(a) Fluorescence and (b) bioluminescence imaging of E. coli-expressing Nano-lantern. Bacteria were diluted at different concentrations, aliquoted into wells of a 12-well plate, and imaged with a Bruker In-Vivo Xtreme system. Only the first row of wells contained bacteria.

However, when two tubes containing bacteria expressing Nano-lantern were spaced between 5 and 20 mm apart at a depth of 10 mm in 1% Intralipid^® (to mimic optical scattering in tissue), the fluorescence and bioluminescence signals from the two tubes significantly overlapped even when tubes were separated by 15 mm (Fig. 4). The two tubes could only be visually resolved when they were separated by 20 mm which is nearly 20-fold the inner diameter of the tubes. A similar experiment was performed but using tubes containing bacteria expressing melA, which were submerged 10 mm in 1% Intralipid^® and imaged using photoacoustics. The tubes could be resolved even when the tubes were less than 1 mm from each other (data not shown). Intensity plots from individual bacteria-containing tubes provided full-width at half-maximum values of 26.5, 19.5, and 0.499 mm when tubes were imaged with fluorescence, bioluminescence, and photoacoustics, respectively (Fig. 5), demonstrating the advantage of photoacoustics over fluorescence and bioluminescence for imaging in optically scattering environments at relatively high depths ($\sim 10\mathrm{mm}$).

Fig. 4

Comparison of Nano-lantern and melA as reporter genes for fluorescence/bioluminescence and photoacoustic imaging, respectively. Two tubes of bacteria expressing Nano-lantern or melA were spaced between 5 and 20 mm from each other and were imaged as indicated. Tubes were submerged 10-mm deep in 1% Intralipid^® to mimic tissue optical scattering. Scale bars correspond to 5-mm lengths.

Fig. 5

Intensity plots of images of single tubes containing melA or Nano-lantern-expressing bacteria, which were submerged 10-mm deep in 1% Intralipid^® and imaged with photoacoustics or fluorescence/bioluminescence, respectively.

3.3.

In Vivo Photoacoustic Imaging of melA-Expressing E. Coli Injected in a Chicken Embryo

Although chicken tissue has significant optical scattering, it lacks much of the blood normally found in healthy tissue, which can influence local fluence levels and background photoacoustic signal. To determine the in vivo utility of melA as a bacterial reporter gene for photoacoustic imaging, experiments with live animals are required. The chicken embryo model was used due to the model’s relative ease-of-use, rapid growth, and low cost.^30,32–34 melA-expressing bacteria (approximately $100\mu \mathrm{L}$ of $20\mathrm{mg}/\mathrm{mL}$ E. coli) was injected in the back flank of a chicken embryo, and the injection site was imaged with photoacoustics. melA-specific photoacoustic signal was detected at the bolus injection site that was unmixed from that of oxy- and deoxy-hemoglobin, all of which were laid over an ultrasound image detailing the anatomical structures within the embryo (Fig. 6). Some photoacoustic signal corresponding to melA can be seen outside the injection site, which may be due to embryo movement during imaging with different laser wavelengths that can cause signal misclassification. The presence of bubbles in the ultrasound coupling gel typically generated a melA signal below the bubble in the embryo as seen on the right side of Fig. 6. melA-specific signal could be detected almost 20-mm deep into tissue with a lateral resolution less than $200\mu \mathrm{m}$. As demonstrated in Fig. 4, such resolution at that depth is not achievable with fluorescence and bioluminescence due to optical scattering, which limits the resolution of purely optical imaging modalities.

Fig. 6

In vivo photoacoustic imaging of melA-expressing bacteria. (a) Representative image of a chicken embryo, which was the model used for in vivo imaging. (b) Multispectral three-dimensional photoacoustic and ultrasound image of a $100\mu \mathrm{L}$ bolus injection of melA-expressing bacteria in the back flank of a chicken embryo. Yellow, red, and blue represent the melA, oxy-hemoglobin, and deoxy-hemoglobin photoacoustic signal, respectively. Ultrasound is shown in gray scale. Green scale bar represents 5-mm distance.

Although it is relatively easy to compare resolution differences between photoacoustic, fluorescence, and bioluminescence imaging, it is more complicated to compare sensitivity differences between these imaging modalities. Optical scattering of fluorescent and bioluminescent signals complicates the reconstruction of the signals in three dimensions. As such, fluorescent and bioluminescent signals generated from Nano-lantern-expressing cells created two-dimensional images with signal intensities that cannot be directly compared to the photoacoustic signals from the three-dimensional images of melA-expressing bacteria. Additionally, the sensitivity of optical imaging modalities is dependent on exposure time, which further complicates comparisons of detection sensitivity of bacteria expressing melA and Nano-lantern. Longer exposure times provide greater sensitivity for optical imaging but may be impractical, especially due to the time-dependent depletion of luciferase substrate needed for bioluminescence. Based on our results, fluorescence and bioluminescence imaging can provide very high sensitivity for detecting bacteria expressing Nano-lantern when the bacteria are near the CCD camera with minimal optical scattering between the bacteria and camera. At these superficial depths, melA-expressing bacteria will also allow high-resolution photoacoustic images with the lateral resolution dependent on the ultrasound transducer frequency. Photoacoustic image sensitivity is greatest when imaging at superficial depths since laser fluence is highest at the tissue surface where there is minimal laser energy loss due to light scattering. As imaging depth increases, photoacoustic signal will decrease with decreasing laser fluence although resolution remains relatively unaffected due to low acoustic scattering in tissue. For high-resolution images of bacteria found relatively deep in tissue, melA may be a more appropriate reporter gene to be visualized with photoacoustics.

For in vivo experiments, melanin secreted from melA-expressing cells will provide a photoacoustic signal thus, in order to determine the location of cells, melA expression should be induced relatively soon before imaging. Alternatively, the continuous secretion of melanin from bacteria may be useful to track the migration paths of bacteria in animals using photoacoustic imaging.

melA is a tyrosinase homologue and as such requires oxygen for activity. Most common fluorescent proteins (e.g., green fluorescent protein and mCherry) and luciferases also require oxygen, thus anaerobic bacteria will require other reporter genes such as flavin binding proteins and SNAP-f fluorescent proteins to track gene expression and/or visualize anaerobic bacteria.^35–39

The current study demonstrates that melA can be used as a photoacoustic reporter gene for imaging E. coli. Expression of melA in E. coli causes significant melanin production causing the bacteria to become highly optically absorbing, which causes the bacteria to provide high contrast in photoacoustic imaging. When imaging in optically scattering environments (e.g., tissue), melA provides superior spatial resolution compared to fluorescent and luciferase reporter genes, since resolution in photoacoustic imaging is not affected by optical scattering. Future studies should involve tracking pathogenic bacteria with melA for precise assessment of the locations of infectious colonies in animals. Whole-animal tomographic photoacoustic imaging systems could be used to allow visualization of bacteria throughout entire animals.⁴⁰ Such experiments would allow noninvasive visualization of infection processes of pathogenic bacteria in entire live animals at unprecedented resolutions.