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Xanthines such as caffeine and theobromine are among the most consumed psychoactive stimulants in the world, either as natural components of coffee, tea and chocolate, or as added ingredients. The present study assessed if xanthines affect liver sinusoidal endothelial cells (LSEC). Cultured primary rat LSEC were challenged with xanthines at concentrations typically obtained from normal consumption of xanthine-containing beverages, food or medicines; and at higher concentrations below the in vitro toxic limit. The fenestrated morphology of LSEC were examined with scanning electron and structured illumination microscopy. All xanthine challenges had no toxic effects on LSEC ultrastructure as judged by LSEC fenestration morphology, or function as determined by endocytosis studies. All xanthines in high concentrations (150 μg/mL) increased fenestration frequency but at physiologically relevant concentrations, only theobromine (8 μg/mL) showed an effect. LSEC porosity was influenced only by high caffeine doses which also shifted the fenestration distribution towards smaller pores. Moreover, a dose-dependent increase in fenestration number was observed after caffeine treatment. If these compounds induce similar changes in vivo, age-related reduction of LSEC porosity can be reversed by oral treatment with theobromine or with other xanthines using targeted delivery., (© 2023. Springer Nature Limited.)

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample's refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup., (© 2023. The Author(s).)

The point spread function of a fixed fluorophore with its dipole axis colinear to the optical axis appears donut-shaped when seen through a microscope, and its light distribution in the pupil plane is radially polarized. Yet other techniques, such as photolithography, report that this same light distribution in the pupil plane appears as a solid spot. How can this same distribution lead to a spot in one case but a donut in the other? Here, we show how the tube lens of the system plays a critical role in determining this shape. Using a vectorial treatment of image formation, we simulate the relative contributions of both longitudinal and radial components to the image of a dipole emitter and thus show how the donut (typically reported for z-polarized single molecule fluorescence microscopy) transforms into a solid spot (as commonly reported for photolithography) as the numerical aperture of the tube lens increases. We find that the transition point occurs around 0.7 NA, which is significantly higher than used for most microscopy systems and lower than for common photolithography systems, thus resolving the seeming paradox of dipole shape., (© 2022 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

Fenestrations in liver sinusoidal endothelial cells (LSEC) are transcellular nanopores of 50-350 nm diameter that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of the liver. Liver diseases, ageing, and various substances such as nicotine or ethanol can negatively influence LSECs fenestrations and lead to defenestration. Over the years, the diameter of fenestrations remained the main challenge for imaging of LSEC in vitro . Several microscopy, or rather nanoscopy, approaches have been used to quantify fenestrations in LSEC to assess the effect of drugs and, and toxins in different biological models. All techniques have their limitations, and measurements of the "true" size of fenestrations are hampered because of this. In this study, we approach the comparison of different types of microscopy in a correlative manner. We combine scanning electron microscopy (SEM) with optical nanoscopy methods such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. In addition, we combined atomic force microscopy (AFM) with SEM and STED, all to better understand the previously reported differences between the reports of fenestration dimensions. We conclude that sample dehydration alters fenestration diameters. Finally, we propose the combination of AFM with conventional microscopy that allows for easy super-resolution observation of the cell dynamics with additional chemical information that can be traced back for the whole experiment. Overall, by pairing the various types of imaging techniques that provide topological 2D/3D/label-free/chemical information we get a deeper insight into both limitations and strengths of each type microscopy when applied to fenestration analysis., (© 2022 Karolina Szafranska et al., published by De Gruyter, Berlin/Boston.)

Background: United States (US) policies to mitigate the opioid epidemic focus on reducing access to prescription opioids to prevent overdoses. We examined the impact of state policies in Vermont (July 2017) and Maine (July 2016) on opioid overdoses and opioid-related adverse effects., Methods: Study population included patients 15 years and older in all-payer claims of Vermont (N = 597,683; Jan.2016-Dec.2018) and Maine (N = 1,370,960; Oct.2015-Dec.2017). We used interrupted time series analyses to assess the impact of opioid prescribing policies on monthly opioid overdose rate and opioid-related adverse effects rate. We used the International Classification of Disease-10-CM to identify overdoses (T40.0 × 1-T40.4 × 4, T40.601-T40.604, T40.691-T40.694) and adverse effects (T40.0 × 5, T40.2 × 5-T40.4 × 5, T40.605, T40.695)., Results: Immediately after the policy, the level of Vermont's opioid overdose rate increased by 34% (95% confidence interval, CI: 1.09, 1.65) while the level of opioid-related adverse effects rate decreased by 29% (95% CI: 0.58, 0.87). In Maine, there was no level change in opioid overdose rate, but the slope of the adverse effects rate after the policy decreased by 3.5% (95% CI: 0.94, 0.99). These results varied within age and rurality subgroups in both states., Conclusion: While the decrease in rate of adverse effects following the policy changes is promising, the increase in Vermont's opioid overdose rate may suggest there is an association between policy implementation and short-term risk to public health., Competing Interests: Declarations of Interest None., (Copyright © 2021. Published by Elsevier B.V.)

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell-cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell-cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy., (© 2021 Abudu et al.)

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Background: Care at verified trauma centers has improved survival and functional outcomes, yet determining the appropriate location of potential trauma centers is often driven by factors other than optimizing system-level patient care. Given the importance of transport time in trauma, we analyzed trauma transport patterns in a rural state lacking an organized trauma system and implemented a geographic information system to inform potential future trauma center locations., Study Design: Data were collected on trauma ground transport during a 3-year period (2014 through 2016) from the Statewide Incident Reporting Network database. Geographic information system mapping and location-allocation modeling of the best-fit facility for trauma center verification was computed using trauma transport patterns, population density, road network layout, and 60-minute emergency medical services transport time based on current transport protocols., Results: Location-allocation modeling identified 2 regional facilities positioned to become the next verified trauma centers. The proportion of the Vermont population without access to trauma center care within 60 minutes would be reduced from the current 29.68% to 5.81% if the identified facilities become verified centers., Conclusions: Through geospatial mapping and location-allocation modeling, we were able to identify gaps and suggest optimal trauma center locations to maximize population coverage in a rural state lacking a formal, organized trauma system. These findings could inform future decision-making for targeted capacity improvement and system design that emphasizes more equitable access to trauma center care in Vermont., (Copyright © 2020 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.

Red blood cells (RBCs) have the ability to undergo morphological deformations during microcirculation, such as changes in surface area, volume and sphericity. Optical waveguide trapping is suitable for trapping, propelling and deforming large cell populations along the length of the waveguide. Bright field microscopy employed with waveguide trapping does not provide quantitative information about structural changes. Here, we have combined quantitative phase microscopy and waveguide trapping techniques to study changes in RBC morphology during planar trapping and transportation. By using interference microscopy, time-lapsed interferometric images of trapped RBCs were recorded in real-time and subsequently utilized to reconstruct optical phase maps. Quantification of the phase differences before and after trapping enabled study of the mechanical effects during planar trapping. During planar trapping, a decrease in the maximum phase values, an increase in the surface area and a decrease in the volume and sphericity of RBCs were observed. QPM was used to analyze the phase values for two specific regions within RBCs: the annular rim and the central donut. The phase value of the annular rim decreases whereas it increases for the central donut during planar trapping. These changes correspond to a redistribution of cytosol inside the RBC during planar trapping and transportation.

Total internal reflection fluorescence (TIRF) microscopy benefits from high-sensitivity, low background noise, low photo-toxicity and high-contrast imaging of sub-cellular structures close to the membrane surface. Although, TIRF microscopy provides high-contrast imaging it does not provide quantitative information about morphological features of the biological cells. Here, we propose an integrated waveguide chip-based TIRF microscopy and label-free quantitative phase imaging (QPI). The evanescent field present on top of a waveguide surface is used to excite the fluorescence and an upright microscope is used to collect the signal. The upright microscope is converted into a Linnik-type interferometer to sequentially extract both the quantitative phase information and TIRF images of the cells. Waveguide chip-based TIRF microscopy benefits from decoupling of illumination and collection light path, large field of view imaging and pre-aligned configuration for multi-color TIRF imaging. The proposed multi-modal microscopy is used to study inflammation caused by lipopolysaccharide (LPS) on rat macrophages. The TIRF microscopy showed that LPS inflammatory molecule disrupts the cell membrane and causes cells to significantly expand across a substrate. While, QPI module quantified changes in the sub-cellular content of the LPS challenged macrophages, showing a net decrease in its maximum phase values.

Objective: Overdose mortality from illicit and prescription opioids has reached epidemic proportions in the United States, especially in rural areas. Naloxone is a safe and effective agent that has been shown to successfully reverse the effects of opioid overdose in the prehospital setting. The National EMS Scope of Practice Model currently only recommends advanced life support (ALS) providers to administer naloxone; however, some individual states have expanded this scope of practice to include intranasal (IN) administration of naloxone by basic life support (BLS) providers, including the Northern New England states. This study compares the effectiveness and appropriateness of naloxone administration between BLS and ALS providers., Methods: All Vermont, New Hampshire, and Maine EMS patient encounters between April 1, 2014 and December 31, 2016 where naloxone was administered were examined and 3,219 patients were identified. The proportion of successful reversals of opioid overdose, based on improvement in the Glasgow Coma Scale (GCS), respiratory rate (RR), and provider global assessment (GA) of response to medication was compared between BLS and ALS providers using a Chi-Squared statistic, Fisher's exact or Wilcoxon rank-sum test., Results: There was no significant difference in the percent improvement in GCS between BLS and ALS (64% and 64% P = 0.94). There was no significant difference in the percentage of improvement in RR between BLS and ALS (45% and 48% P = 0.43). There was a significant difference in the percentage of improvement of GA between BLS and ALS (80% and 67% P < 0.001). There was no significant difference in determining appropriate cases to administer naloxone where RR < 12 and GCS < 15 between BLS and ALS (42% and 43% P = 0.94)., Conclusions: BLS providers were as effective as ALS providers in improving patient outcome measures after naloxone administration and in identifying patients for whom administration of naloxone is appropriate. These findings support expanding the National EMS Scope of Practice Model to include BLS administration of intranasal naloxone for suspected opioid overdoses.

Background: Targeted temperature management (TTM) confers neurological and survival benefits for post-cardiac arrest patients with return of spontaneous circulation (ROSC) who remain comatose. Specialized equipment for induction of hypothermia is not available in the prehospital setting, and there are no reliable methods for emergency medical services personnel to initiate TTM. We hypothesized that the application of surface cooling elements to the neck will decrease brain temperature and act as initiators of TTM., Methods: Magnetic resonance (MR) spectroscopy was used to evaluate the effect of a carotid surface cooling element on brain temperature in healthy adults., Results: Six individuals completed this study. We measured a temperature drop of 0.69 ± 0.38 °C (95% CI) in the cortex of the brain following the application of the cooling element. Application of a room temperature element also caused a measurable decrease in brain temperature of 0.66 ± 0.41 °C (95% CI) which may be attributable to baroreceptor activation., Conclusion: The application of surface cooling elements to the neck decreased brain temperature and may serve as a method to initiate TTM in the prehospital setting.

Background: Emergency Medical Services (EMS) are often the first medical providers to begin resuscitation of out-of-hospital cardiac arrest (OHCA) victims. The universal Basic Life Support Termination of Resuscitation (BLS-TOR) rule is a validated clinical prediction tool used to identify patients in which continued resuscitation efforts are futile., Objective: The primary aim is to compare the rate of transport of OHCA cases before and after the implementation of a BLS-TOR protocol and to determine the compliance rate of EMS personnel with the new protocol in a largely volunteer, rural system., Methods: A retrospective cohort study was conducted using the statewide EMS electronic patient care report system. Cases were identified by searching for any incident that had a primary impression of "cardiac arrest" or a primary symptom of "cardiorespiratory arrest" or "death." Data were collected from the two years prior to and following implementation of the BLS-TOR rule from January 1, 2012 through March 31, 2016., Results: There were 702 OHCA cases were identified, with 329 cases meeting inclusion criteria. The transport rate was 91.1% in the pre-intervention group compared with 69.4% in the post-intervention group (χ2=24.8; p<0.001). EMS compliance rate with the BLS-TOR rule was 66.7%. Of the 265 patients transported during the study, 87 patients met (post-intervention group; n=22) or retrospectively met (pre-intervention group; n=65) the BLS-TOR requirements for field termination of resuscitation. None of these patients survived to hospital discharge., Conclusion: Rural EMS systems may benefit from implementation and utilization of the universal BLS-TOR rule., (Published by Elsevier B.V.)

Idiopathic non-cirrhotic portal hypertension (INCPH) is portal hypertension (PHT) without cirrhosis and other identifiable causes. Esophageal and gastric varices are seen in INCPH which are mostly asymptomatic. We present a rare case of symptomatic isolated gastric varices (IGV) in the setting of INCPH. We report a case of a 60-year-old man who presented with an acute onset of hematemesis and no identifiable history. Upon further evaluation, he was found to have non-bleeding dilated gastric varices on esophagogastroduodenoscopy (EGD) and PHT without cirrhosis. Our patient is unique because he has symptomatic IGV and INCPH., Competing Interests: The authors have declared that no competing interests exist.

Objective: Intraosseous (IO) access is increasingly being used as an alternative to peripheral intravenous access, which is often difficult or impossible to establish in critically ill patients in the prehospital setting. Until recently, only Paramedics performed adult IO access. In 2014, Vermont Emergency Medical Services (EMS) expanded the Advanced Emergency Medical Technicians (AEMTs) scope of practice to include IO access in adult patients. This study compares successful IO access in adults performed by AEMTs compared to Paramedics in the prehospital setting., Methods: All Vermont EMS patient encounters between January 1, 2013 and November 30, 2015 were examined, and 543 adult patients with a documented IO access insertion attempt were identified. The proportion of successful IO insertions was compared between AEMTs and Paramedics using a Chi-Squared statistic and a non-inferiority test., Results: There was no significant difference in the percentage of successful IO access between AEMTs and Paramedics [95.2% and 95.6%, respectively; P = 0.84]. The confidence interval around this 0.4% difference (95% confidence interval = -4.2, 3.2) was within a pre-specified delta of ±10% indicating non-inferiority of AEMTs compared to Paramedics., Conclusions: This study's finding that successful IO access was not different among AEMTs and Paramedics lends evidence in support of expanding the scope of practice of AEMTs to include establishing IO access in adults.

Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy. Individual cells can be oriented arbitrarily but preferably either horizontally or vertically relative to the microscope's image plane, enabling access to sample sections that are impossible to achieve with conventional sample preparation and immobilization. This opens up new opportunities to super-resolve the nanoscale organization of chromosomal DNA in individual bacterial cells.

Purpose: A pharmacist's role in helping Vermont health officials standardize pharmacotherapy-related protocols used by emergency medical services (EMS) personnel across the state is described., Summary: Pharmacists with expertise in emergency medicine (EM) or critical care are ideally positioned to provide guidance on optimizing and standardizing medication-use aspects of state and local EMS protocols. In 2012, the medical director of the EMS division of the Vermont Department of Health requested that an EM pharmacist at a Burlington academic medical center review draft EMS protocols designed to replace the existing patchwork of local protocols with statewide standards of care; among the 92 draft protocols reviewed, 62 pertained to medication use. The pharmacist provided a wide range of suggestions on 33 protocols, including (1) evidence-based recommendations on use of vasopressor agents for septic shock, (2) recommendations to optimize medication ordering and preparation in the prehospital setting, (3) recommendations on prehospital management of pediatric shock and appropriate use of chemical restraints, and (4) recommendations to promote use of smart infusion pumps by EMS personnel. All of the pharmacist's suggestions were incorporated into the final protocols, which took effect in March 2014. The protocols have helped standardize care for patients receiving EMS services throughout Vermont while reducing the potential for medication errors., Conclusion: An EM pharmacist participated in the review and development of statewide EMS treatment protocols that focused on choice of medication therapy, dosage, administration, and identification and minimization of potential risks of medication errors., (Copyright © 2015 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.