FEASIBILITY STUDY ON REDUCING PIXEL RESOLUTION OF RAW HDR IMAGES FOR CALIBRATION OF SKY LUMINANCE MEASUREMENT

In the past, measuring the luminance distribution of the sky and the sun was done with either a sky scanner or a luminance meter. The conventional measurement methods are time consuming at low measurement resolution (with a maximum of 145 data points on the entire upper hemisphere), thus, cannot capture real-time changes typically seen with natural daylight. To solve this problem, a camera-array-based measurement technology was recently introduced by the University of Kansas lighting research laboratory to capture the spatial and temporal luminance distributions of the celestial hemisphere. This technique uses high dynamic range (HDR) photogrammetry for luminance mapping of the sky and the sun simultaneously. With two cameras mounted next to one another on a Sky Measurement Tripod Head developed in the lighting research laboratory, the sky and the sun are measured, respectively, by each camera. However, one issue that still remains with this type of data collection is the storage and treatment of big data embedded in the HDR images generated in the field. Each HDR image has a file size of approximately 40-50 MB, while the retrieved 18 million luminance data in text file format could have a file size of approximately 400-500 MB. Given at least hourly measurements for real-time sky conditions from sunrise to sunset, it is very tedious to deal with such large amounts of data that challenge the speed and storage capacity of current computation facilities. To solve this problem, the present research study was aimed to explore the feasibility of reducing pixel resolutions in the laboratory of raw HDR images taken in the field, in the hopes of speeding up the data treatment process of the sky and the sun luminance measurement while still maintaining an adequate degree of accuracy. An experiment was carried out at the Clinton State Park in Lawrence, KS at 1:30 pm on October 4th, 2015 to evaluate the null hypothesis that reducing the pixel resolution of the HDR images in the laboratory would not compromise the overall value of the obtained data. Two Canon digital cameras EOS Rebel T2i fitted with Sigma 4.5mm F2.8 EX DC HSM Circular Fisheye lenses were mounted side by side on a custom designed Sky Measurement Tripod Head to take measurements of the celestial sky using the HDR photography. One camera was mounted without a neutral density filter and was used to capture the luminance distribution of the sky while the other camera was equipped with a neutral density filter of 1/1000 and used to capture the luminance of the sun and its corona. The luminance data embedded in each of the two HDR images were later extracted in Radiance and outputted to Microsoft Access and Excel for the follow-up data treatment. It was discovered that the amount of data obtained from the cameras was very large and nearly impossible to handle in Microsoft Access or Excel due to their limited computation capacity of 18 million rows of data. This study then reduced such big data during the data extraction process in the laboratory by lowering the pixel resolutions of the raw HDR images obtained in the field. The size of the HDR images was reduced from 18 million data points to merely 270,500 data points. The reduced datasets were then treated using Excel spreadsheets containing pre-developed equations. Calibration Factor (CF) values were calculated by comparing the actual horizontal illuminance measured using an illuminance meter to the calculated illuminance from the sky and sun luminance data embedded in the synthesized HDR image. In theory, the CF ratio should be close to 1.0 indicating the robust data collection and treatment process was carried out with minimal error. In the present study, the CF value obtained during the laboratory data treatment was close to 0.05, indicating the dataset was improperly manipulated during the reduction process of pixel resolutions. Photometric calibrations using such a CF value (0.05) would lead to extraction of only 5% of the true luminance distributions of the sky and the sun. As a result, it is deemed inappropriate to reduce the pixel resolution of raw HDR images in the laboratory after the field measurement, since such a reduction found in this study is associated with a loss of useful data for luminance mapping of the sky and the sun. Further research to be conducted in the Lighting Research Lab will evaluate two possible ways to solve this problem. The first solution is to capture the HDR images with lower pixel resolutions by directly adjusting the camera settings in the field, which is not the optimal solution but recommended given the otherwise resulting big data and the limitations of current computing facilities. The second method is to conduct the data treatment in a more powerful computing software such as Matlab without reduction of the original 18 million pixels embedded in the HDR images

New advances in MR-compatible bioartificial liver

MR-compatible bioartificial liver (BAL) studies have been performed for 30 years and are reviewed. There are two types of study: (i) metabolism and drug studies using multinuclear MRS; primarily short-term (< 8 h) studies; (ii) the use of multinuclear MRS and MRI to noninvasively define the features and functions of BAL systems for long-term liver tissue engineering. In the latter, these systems often undergo not only modification of the perfusion system, but also the construction of MR radiofrequency probes around the bioreactor. We present novel MR-compatible BALs and the use of multinuclear MRS (13C, 19F, 31P) for the noninvasive monitoring of their growth, metabolism and viability, as well as 1H MRI methods for the determination of flow profiles, diffusion, cell distribution, quality assurance and bioreactor integrity. Finally, a simple flexible coil design and circuit, and life support system, are described that can make almost any BAL MR-compatible

Effect of Oxygen Concentration on Viability and Metabolism in a Fluidized-Bed Bioartificial Liver Using 31 P and 13 C NMR Spectroscopy

Many oxygen mass-transfer modeling studies have been performed for various bioartificial liver (BAL) encapsulation types; yet, to our knowledge, there is no experimental study that directly and noninvasively measures viability and metabolism as a function of time and oxygen concentration. We report the effect of oxygen concentration on viability and metabolism in a fluidized-bed NMR-compatible BAL using in vivo 31P and 13C NMR spectroscopy, respectively, by monitoring nucleotide triphosphate (NTP) and 13C-labeled nutrient metabolites, respectively. Fluidized-bed bioreactors eliminate the potential channeling that occurs with packed-bed bioreactors and serve as an ideal experimental model for homogeneous oxygen distribution. Hepatocytes were electrostatically encapsulated in alginate (avg. diameter, 500 μm; 3.5×107 cells/mL) and perfused at 3 mL/min in a 9-cm (inner diameter) cylindrical glass NMR tube. Four oxygen treatments were tested and validated by an in-line oxygen electrode: (1) 95:5 oxygen:carbon dioxide (carbogen), (2) 75:20:5 nitrogen:oxygen:carbon dioxide, (3) 60:35:5 nitrogen:oxygen:carbon dioxide, and (4) 45:50:5 nitrogen:oxygen:carbon dioxide. With 20% oxygen, β-NTP steadily decreased until it was no longer detected at 11 h. The 35%, 50%, and 95% oxygen treatments resulted in steady β-NTP levels throughout the 28-h experimental period. For the 50% and 95% oxygen treatment, a 13C NMR time course (∼5 h) revealed 2-13C-glycine and 2-13C-glucose to be incorporated into [2-13C-glycyl]glutathione (GSH) and 2-13C-lactate, respectively, with 95% having a lower rate of lactate formation. 31P and 13C NMR spectroscopy is a noninvasive method for determining viability and metabolic rates. Modifying tissue-engineered devices to be NMR compatible is a relatively easy and inexpensive process depending on the bioreactor shape

Hyperpolarized 13 C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism

The purpose of this study was to combine a three-dimensional NMR-compatible bioreactor with hyperpolarized 13C NMR spectroscopy in order to probe cellular metabolism in real time. JM1 (immortalized rat hepatoma) cells were cultured in a three-dimensional NMR-compatible fluidized bioreactor. 31P spectra were acquired before and after each injection of hyperpolarized [1-13C] pyruvate and subsequent 13C spectroscopy at 11.7 T. 1H and two-dimensional 1H-1H-total correlation spectroscopy spectra were acquired from extracts of cells grown in uniformly labeled 13C-glucose, on a 16.4 T, to determine 13C fractional enrichment and distribution of 13C label. JM1 cells were found to have a high rate of aerobic glycolysis in both two-dimensional culture and in the bioreactor, with 85% of the 13C label from uniformly labeled 13C-glucose being present as either lactate or alanine after 23 h. Flux measurements of pyruvate through lactate dehydrogenase and alanine aminotransferase in the bioreactor system were 12.18 ± 0.49 nmols/sec/108 cells and 2.39 ± 0.30 nmols/sec/108 cells, respectively, were reproducible in the same bioreactor, and were not significantly different over the course of 2 days. Although this preliminary study involved immortalized cells, this combination of technologies can be extended to the real-time metabolic exploration of primary benign and cancerous cells and tissues prior to and after therapy

Metabolic assessment of a novel chronic myelogenous leukemic cell line and an imatinib resistant subline by 1H NMR spectroscopy

The goal of this study was to examine metabolic differences between a novel chronic myelogenous leukemic (CML) cell line, MyL, and a sub-clone, MyL-R, which displays enhanced resistance to the targeted Bcr-Abl tyrosine kinase inhibitor imatinib. 1H nuclear magnetic resonance (NMR) spectroscopy was carried out on cell extracts and conditioned media from each cell type. Both principal component analysis (PCA) and specific metabolite identification and quantification were used to examine metabolic differences between the cell types. MyL cells showed enhanced glucose removal from the media compared to MyL-R cells with significant differences in production rates of the glycolytic end-products, lactate and alanine. Interestingly, the total intracellular creatine pool (creatine + phosphocreatine) was significantly elevated in MyL-R compared to MyL cells. We further demonstrated that the MyL-R cells converted the creatine to phosphocreatine using non-invasive monitoring of perfused alginate-encapsulated MyL-R and MyL cells by in vivo 31P NMR spectroscopy and subsequent HPLC analysis of extracts. Our data demonstrated a clear difference in the metabolite profiles of drug-resistant and sensitive cells, with the biggest difference being an elevation of creatine metabolites in the imatinib-resistant MyL-R cells

An NMR-compatible bioartificial liver for metabolomic investigation of drug action

NMR-compatible bioartificial liver (BAL) studies have been performed for thirty years and still have not been maintained beyond 8hrs. This doctoral work describes the engineering efforts in creating a long-term NMR-compatible BAL. Four general types of BALs have been reported: suspension, microcarrier, membrane, and entrapment. Reasons and efforts toward establishing a fluidized-bed entrapment bioreactor, which maintains hepatocytes entrapped in alginate for 30 hrs, and likely for long-term, are described. The electrostatically-encapsulated cells generate 1.5 mls of 500 μm diameter spherical encapsulates, containing about 10,000 cells each, in about 5 minutes. These encapsultates containing entrapped cells are then incolulated into a 10 mm glass NMR tube and are percholate in the bottom of the glass tube forming the fluidized-bed. To demonstrate the power of the NMR-compatible BAL in toxicity studies using in vivo 31P and 13C NMR spectroscopy, a rat hepatoma cell line, JM1, was used. The encapsulated cells were maintained overnite (16hrs) with 3-13C-cysteine and u-13C-glucose replaced in the perfused media, and production rates for glutathione, the body’s primary antioxidant, and lactate, an anaerobic glycolytic end-product common in cancer, were determined. The next day (16-20 hrs) when [3-13C-cysteinyl]glutathione was at 13C isotopic steady-state and the JM1 cells were at metabolic steady-state, the effects of two doses of bromobimane, a glutathione depleting agent, and three different doses of acetaminophen on the in vivo 31P and 13C NMR spectra were determined. The application of this time series data to toxicodyanamics and toxicokintetics is discussed. This is the first study demonstrating with 1 minute temporal resolution, the non-steady-state real-time toxicokinetics of glutathione. Once the NMR-compatible BAL was demonstrated with a relatively easy liver cell-type to culture, a cell line (i.e., JM1), the fluidized-bed bioreactor was established with primary rat hepatocytes. Liver is exquisitely sensitive to oxygen tension and ranges from 8% to 3% across its capillary-bed, yet all previous NMR-compatible BAL studies have all gasified the perfusion media with 95% oxygen. Therefore, the effect of four oxygen concentrations (20%, 35%, 55%, and 95%) on viability was monitored by in vivo 31P NMR. Only the 35% and 55% oxygen treatments maintained hepatocytes viability for 28 hours and likely beyond with no change in β-nucleotide triphosphate levels. Analysis of the in vivo 13C NMR data for the 55% oxygen treatment revealed synthetic rates for lactate and glutathione demonstrating differentiated functions were present and quantifying the function. This is the first demonstration of any primary hepatocyte culture being beyond 8 hrs in a NMR-compatible BAL

Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells

Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3&prime;-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately &lt; 2 min

Sphingosine kinase activity is not required for tumor cell viability.

Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology