Portable RF system retrofitted to the IVM.

<p>(A) The RF system integrated into the intravital microscope (IVM) for real-time imaging under RF exposure. (B) Mouse manipulation for imaging–an incision is made to expose and gently manipulate the 4T1 tumor for IVM imaging. (C) 4T1 tumor under IVM illumination with a x4 objective lens.</p

High-temperature vessel degradation.

<p>(A)–(D) Impact of RF exposure on vessel architecture at four different time-points: 0:22, 6:53, 16:18, and 20:31 minutes, respectively. The tumor temperatures and RF power at those time points are shown in the upper-middle and upper-right hand side sections, respectively. Figure (E) illustrates the change in temperature and power with respect to time. Vessel degradation can be seen for temperatures > 41°C. A complete breakdown of the vessel architecture can be seen for temperatures > 47°C.</p

Modulation of tumor temperature using RF exposure.

<p>(A) Thermal fiber optic probe placement. Probes #1–3 are positioned (i) under the skin but above the tumor; (ii) under the skin in between the tumor and the main body; and (iii) under the skin next to the intraperitoneal cavity. (B) Extracted thermal probe data. The recorded temperature of the probes was modulated by turning on and off the RF system (+RF and–RF). The system was turned off once the tumor temperature (probe #1) reached 45°C, 43°C, and 41°C, respectively, and was turned on when all probes had values in the range ~29–31°C. (C) The IR camera simultaneously measured the surface temperature of the points where the thermal probes were located.</p

Real-time RF-IVM imaging and post capture analysis.

<p>RF exposure shows transport of fluorescently bound albumin across the perfusion barrier into tumor region. Figure (A) and (B) depict the blue image channel (albumin) before and after (4.5 min) RF exposure. This data is shown superimposed with the tumor (red) channel in Figure (C) and (D). Figure (E) Control mouse (no RF) was imaged for 30 minutes on both channels. There is no transport of albumin into the tumor across the perfusion barrier. (F) Time lapsed images of the data shown in Figure (A) and (B). Figure (G) 4T1 tumor slices immunohistologically stained to the antibodies CD31 (green, vasculature endothelial cells), and albumin (red) for both RF (left image) and non-RF (right image) groups. Figure (H) depicts positive area fraction (PAF) of albumin accumulation in tumor slices. Finally, (I) is a quantitative video analysis of relative increase in albumin fluorescence (RAIF) in multiple 4T1 tumor surfaces exposed to RF under IVM (n = 4).</p

Thermal probe and IR camera calibration.

<p>(A) Three thermal probes were places in a quartz cuvette filled with phosphate buffered saline (PBS) and exposed to 200 W of RF. The IR camera captured the surface temperature of cursor points located next to the thermal probes for the RF exposure time 0 s—380 s (B and C, respectively). (D) Comparison of thermal probe and IR camera heating data.</p

Portable RF system setup and generated electric field.

<p>(A) Portable RF system consists of the transmitting unit (TX) and receiving head (RX) that generates a high-power electric field across the specimen (e.g. mouse). The system is driven by a variable power fixed RF amplifier (0–200 W, 13.56 MHz) that is cooled during operation by a water chiller. Heat production is monitored using an infrared (IR) camera or direct insertion of fiber optical probes. (B) Circuit representation of the portable RF system. (C) Setup for extracting electric-field intensities. An electric-field probe (EFP) is placed at specific points along the x- and z-axis in between the TX and RX heads and measures the voltage at each point for 20 W RF-power. (D) The electric field is derived from the voltage data and is plotted as an intensity contour plot.</p

Post-RF IVM analysis of images using template and masking algorithms.

<p>FITC-dextran was injected followed by 30 mins RF exposure. Albumin was then injected and imaged for 30 minutes without RF exposure. (<b>A</b>) The tumor area (i) is demarcated using a green line and allows FITC-dextran (ii) and albumin (iii) perfusion to be monitored. (<b>B</b>) These masks are applied to the full time-lapsed video for all channels. (<b>C</b>) Areas where both albumin and FITC-dextran overlap are processed to quantify the relative average intensity of albumin perfusion after 30 mins of RF exposure. (<b>D</b>) Relative tumor dye intensity (RTDI) versus time. The intensity of FITC-dextran gradually increases over the 30 min of RF exposure and continues for another 30 mins after the RF is turned off. The intensity of albumin increases once injected after the RF is turned off (t = 30 mins) and continues for 30 minutes. This suggests RF-mediated effects are prevalent even after RF exposure.</p