Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Ultrasound Doppler as an Imaging Modality for Selection of Murine 4T1 Breast Tumors for Combination Radiofrequency Hyperthermia and Chemotherapy

Noninvasive radiofrequency-induced (RF) hyperthermia has been shown to increase the perfusion of chemotherapeutics and nanomaterials through cancer tissue in ectopic and orthotopic murine tumor models. Additionally, mild hyperthermia (37°C-45°C) has previously shown a synergistic anticancer effect when used with standard-of-care chemotherapeutics such as gemcitabine and Abraxane. However, RF hyperthermia treatment schedules remain unoptimized, and the mechanisms of action of hyperthermia and how they change when treating various tumor phenotypes are poorly understood. Therefore, pretreatment screening of tumor phenotypes to identify key tumors that are predicted to respond more favorably to hyperthermia will provide useful mechanistic data and may improve therapeutic outcomes. Herein, we identify key biophysical tumor characteristics in order to predict the outcome of combinational RF and chemotherapy treatment. We demonstrate that ultrasound imaging using Doppler mode can be utilized to predict the response of combinational RF and chemotherapeutic therapy in a murine 4T1 breast cancer model

Chemotherapy and radiofrequency-induced mild hyperthermia combined treatment of orthotopic pancreatic ductal adenocarcinoma xenografts

Patients with pancreatic ductal adenocarcinomas (PDAC) have one of the poorest survival rates of all cancers. The main reason for this is related to the unique tumor stroma and poor vascularization of PDAC. As a consequence, chemotherapeutic drugs, such as nab-paclitaxel and gemcitabine, cannot efficiently penetrate into the tumor tissue. Non-invasive radiofrequency (RF) mild hyperthermia treatment was proposed as a synergistic therapy to enhance drug uptake into the tumor by increasing tumor vascular inflow and perfusion, thus, increasing the effect of chemotherapy. RF-induced hyperthermia is a safer and non-invasive technique of tumor heating compared to conventional contact heating procedures. In this study, we investigated the short- and long-term effects (~20 days and 65 days, respectively) of combination chemotherapy and RF hyperthermia in an orthotopic PDAC model in mice. The benefit of nab-paclitaxel and gemcitabine treatment was confirmed in mice; however, the effect of treatment was statistically insignificant in comparison to saline treated mice during long-term observation. The benefit of RF was minimal in the short-term and completely insignificant during long-term observation

Optimizing non-invasive radiofrequency hyperthermia treatment for improving drug delivery in 4T1 mouse breast cancer model

Interactions of high-frequency radio waves (RF) with biological tissues are currently being investigated as a therapeutic platform for non-invasive cancer hyperthermia therapy. RF delivers thermal energy into tissues, which increases intra-tumoral drug perfusion and blood-flow. Herein, we describe an optical-based method to optimize the short-term treatment schedules of drug and hyperthermia administration in a 4T1 breast cancer model via RF, with the aim of maximizing drug localization and homogenous distribution within the tumor microenvironment. This method, based on the analysis of fluorescent dyes localized into the tumor, is more time, cost and resource efficient, when compared to current analytical methods for tumor-targeting drug analysis such as HPLC and LC-MS. Alexa-Albumin 647 nm fluorphore was chosen as a surrogate for nab-paclitaxel based on its similar molecular weight and albumin driven pharmacokinetics. We found that RF hyperthermia induced a 30-40% increase in Alexa-Albumin into the tumor micro-environment 24 h after treatment when compared to non-heat treated mice. Additionally, we showed that the RF method of delivering hyperthermia to tumors was more localized and uniform across the tumor mass when compared to other methods of heating. Lastly, we provided insight into some of the factors that influence the delivery of RF hyperthermia to tumors

A New Imaging Platform for Visualizing Biological Effects of Non-Invasive Radiofrequency Electric-Field Cancer Hyperthermia

Herein, we present a novel imaging platform to study the biological effects of non-invasive radiofrequency (RF) electric field cancer hyperthermia. This system allows for real-time in vivointravital microscopy (IVM) imaging of radiofrequency-induced biological alterations such as changes in vessel structure and drug perfusion. Our results indicate that the IVM system is able to handle exposure to high-power electric-fields without inducing significant hardware damage or imaging artifacts. Furthermore, short durations of low-power (< 200 W) radiofrequency exposure increased transport and perfusion of fluorescent tracers into the tumors at temperatures below 41°C. Vessel deformations and blood coagulation were seen for tumor temperatures around 44°C. These results highlight the use of our integrated IVM-RF imaging platform as a powerful new tool to visualize the dynamics and interplay between radiofrequency energy and biological tissues, organs, and tumors

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

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

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

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