| dc.description.abstract |
<p>In vivo imaging is an important scientific tool for studying bio-molecular interactions,
but lack of preserved functionality during imaging restricts scientists’ abilities
to gain critical knowledge. Structure can be preserved while using high-resolution
optical imaging by utilizing window chambers in murine models1, yet the use of anesthesia
for immobilization is problematic. Anesthesia affects tissue oxygenation2, blood cell
velocities3, immunosuppression4, and allowable duration of imaging5–thus its usage
restricts in vivo bio-molecular imaging accuracy and duration. </p><p>Developing a
portable imaging system that attaches to murine dorsal window chambers enables imaging
without anesthesia, avoiding previous drawbacks of window chamber models. A raspberry
pi camera (RPI-CAM-V2, Raspberry Pi) was modified for microscopy and used alongside
3D printed panels for attaching the camera, optical filters, and LED light source
to murine window chambers. Multiple applications for the portable system were developed,
each requiring their own setup of filters and stimulating LEDs. The system is powered
by a Raspberry Pi 3 Model B single-board computer (RASPBERRYPI3-MODB-1GB, Raspberry
Pi), allowing for stream-lined data acquisition. </p><p>Imaging tissue oxygenation
was the first application developed for the portable system. Oxygen sensing boron
nanoparticles were injected into window chambers, while a UV LED was used to stimulate
fluorescent and phosphorescent signals. When stimulated by UV light, the boron nanoparticles
emit fluorescence and phosphorescence. Fluorescence is stable regardless of oxygenation,
while phosphorescence signal from the nanoparticles is quenched in the presence of
higher oxygenations. The ratio of fluorescence to phosphorescence was used to calculate
oxygen concentration maps of window chamber tissue. Tissue oxygenations in awake and
anesthetized mice inhaling varied oxygen concentrations were analyzed. In 5 awake
nude mice inhaling 20% O2, the median partial pressure of oxygen was measured as 49
mmHg within their window chambers. From a one-tailed t-test with a false positive
correction, 3 of the mice had significantly higher (p ≤ 0.05) tissue oxygenation while
anesthetized compared to the awake measurements.</p><p>Developing the portable systems
ability to image blood cells was another focus of this project. Blood cells were visible
with white LED exposure. A frame rate of 30 frames/second was adequate for tracking
cell motion while allowing for the highest resolution possible with the system. Blood
cell velocities in a mouse awake and anesthetized were analyzed, while also observing
change in blood cell velocities during sepsis that was induced by cecal ligation puncture
(CLP). Three days after CLP, the mean awake blood cell velocity was measured as 0.21
± 0.03 mm/s, while the mean anesthetized blood cell velocity was measured as 0.080
± 0.002 mm/s. Six days after CLP, the awake measurement had reduced to 0.019 ± 0.005
mm/s, while the anesthetized measurement was reduced to 0.031 ± 0.002 mm/s (91% decrease
in awake measurement, 61% decrease in anesthetized measurement). A two-way ANOVA on
the factors of anesthesia and time post-CLP performed on multiple vessel regions calculated
significance (p ≤ 0.05) for both of these factors on blood cell velocities within
the pilot mouse’s window chamber. </p><p>Noting the differences between data collected
on awake and anesthetized mice, our system has been validated as a tool for real-time
imaging of tissue without the observed effects of anesthesia. By avoiding anesthesia,
the developed device allows for continual data acquisition to increase from hours
to days. The system is generalizable, and while only two applications are presented
in this study, the system could be modified for imaging fluorescently labeled cells/proteins
for other bio-molecular interactions.</p>
|
|
