An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types.

Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.

Changes in Optical Properties of Plasmonic Nanoparticles in Cellular Environments are Modulated by Nanoparticle PEGylation and Serum Conditions.

When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments.

Continuously tunable nucleic acid hybridization probes.

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Implementation of a medical intensive care unit acute-care nurse practitioner service.

Demands for critical care services are increasing, but the supply of qualified physicians is not. Moreover, there are mounting national expectations for continuous on-site, senior providers and for adherence to quality and safety practices. In teaching institutions, manpower shortages are exacerbated by shrinking trainee duty hours, and there is a growing desire to recoup the revenue lost when a non-credentialed provider delivers a service. Increasingly, hospitalists and acute-care nurse practitioners (ACNPs) are meeting these needs. This article describes the development of an ACNP service in a university hospital medical intensive care unit (ICU) designed to improve the range and quality of services and faculty staffing when the ICU expanded from 22 to 34 beds without adding physicians. Eight ACNPs were hired and, over 9 months, received didactic, procedural, simulation center, and supervised patient care training. Progressive workload and graded responsibility were used to transition to a 24-hour, in-house, resident-independent, attending-supervised service, which now admits just under half of all patients (3.4 ± 1.3 patients/day), cares for approximately one-fourth of the unit's critically ill patients (6.0 ± 1.4 patients/day), and responds to medical rapid response team calls daily (1.5 ± 1.7 calls/day). Over the first 5 months of operation, work output in all categories continued to increase, with ACNPs documenting an average of 11.1 ± 2.7 activities per day (all data mean ± standard deviation). Acute-care nurse practitioners also provide 40% of the daily resident core lectures and a monthly staff nurse conference. Insufficient data exist at this time, however, to report accurate billing or collection results. Specific areas discussed within this article include service structure, hiring and training, implementation, scheduling, supervision, problems encountered, productivity, monitoring, and future plans.