Detection of acquired radioresistance in breast cancer cell lines using Raman spectroscopy and machine learning.

Radioresistance-a living cell's response to, and development of resistance to ionising radiation-can lead to radiotherapy failure and/or tumour recurrence. We used Raman spectroscopy and machine learning to characterise biochemical changes that occur in acquired radioresistance for breast cancer cells. We were able to distinguish between wild-type and acquired radioresistant cells by changes in chemical composition using Raman spectroscopy and machine learning with 100% accuracy. In studying both hormone receptor positive and negative cells, we found similar changes in chemical composition that occur with the development of acquired radioresistance; these radioresistant cells contained less lipids and proteins compared to their parental counterparts. As well as characterising acquired radioresistance in vitro, this approach has the potential to be translated into a clinical setting, to look for Raman signals of radioresistance in tumours or biopsies; that would lead to tailored clinical treatments.

Label-free biomarkers of human embryonic stem cell differentiation to hepatocytes.

Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.

Label-free identification and characterization of living human primary and secondary tumour cells.

We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type ­ colorectal. Raman spectroscopy distinguished cells by their biochemical 'fingerprint' in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH2 bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity ­ secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis ­ to assign a tumour as primary or secondary.

Raman spectroscopy of brain and skin tissue in a minipig model of Huntington's disease.

We applied Raman spectroscopy to brain and skin tissues from a minipig model of Huntington's disease. Differences were observed between measured spectra of tissues with and without Huntington's disease, for both brain tissue and skin tissue. There are linked to changes in the chemical composition between tissue types. Using machine learning we correctly classified 96% of test spectra as diseased or wild type, indicating that the test would have a similar accuracy when used as a diagnostic tool for the disease. This suggests the technique has great potential in the rapid and accurate diagnosis of Huntington's and other neurodegenerative diseases in a clinical setting.

Nine golden codes: improving the accuracy of Helicopter Emergency Medical Services (HEMS) dispatch-a retrospective, multi-organisational study in the East of England.

BACKGROUND: Helicopter Emergency Medical Services (HEMS) are a limited and expensive resource, and should be intelligently tasked. HEMS dispatch was identified as a key research priority in 2011, with a call to identify a 'general set of criteria with the highest discriminating potential'. However, there have been no published data analyses in the past decade that specifically address this priority, and this priority has been reaffirmed in 2023. The objective of this study was to define the dispatch criteria available at the time of the initial emergency call with the greatest HEMS utility using a large, regional, multi-organizational dataset in the UK. METHODS: This retrospective observational study utilized dispatch data from a regional emergency medical service (EMS) and three HEMS organisations in the East of England, 2016-2019. In a logistic regression model, Advanced Medical Priority Dispatch System (AMPDS) codes with ≥ 50 HEMS dispatches in the study period were compared with the remainder to identify codes with high-levels of HEMS patient contact and HEMS-level intervention/drug/diagnostic (HLIDD). The primary outcome was to identify AMPDS codes with a > 10% HEMS dispatch rate of all EMS taskings that would result in 10-20 high-utility HEMS dispatches per 24-h period in the East of England. Data were analysed in R, and are reported as number (percentage); significance was p < 0.05. RESULTS: There were n = 25,491 HEMS dispatches (6400 per year), of which n = 23,030 (90.3%) had an associated AMPDS code. n = 13,778 (59.8%) of HEMS dispatches resulted in patient contact, and n = 8437 (36.6%) had an HLIDD. 43 AMPDS codes had significantly greater rates of patient contact and/or HLIDD compared to the reference group. In an exploratory analysis, a cut-off of ≥ 70% patient contact rate and/or ≥ 70% HLIDD (with a > 10% HEMS dispatch of all EMS taskings) resulted in 17 taskings per 24-h period. This definition derived nine AMPDS codes with high HEMS utility. CONCLUSION: We have identified nine 'golden' AMPDS codes, available at the time of initial emergency call, that are associated with high-levels of whole-system and HEMS utility in the East of England. We propose that UK EMS should consider immediate HEMS dispatch to these codes.

Optical spectroscopy for noninvasive monitoring of stem cell differentiation.

There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications.

Development of tip-enhanced optical spectroscopy for biological applications: a review.

Tip-enhanced optical spectroscopy is an approach that holds a good deal of promise for the nanoscale characterisation of matter. Tip-enhanced Raman spectroscopy (TERS) has been demonstrated on a variety of samples: inorganic, organic and biological. Imaging using TERS has been shown for carbon nanotubes due to their high scattering efficiency. There are a number of compelling motivations to consider alternative approaches for biological samples; most importantly, the potential for heat damage of biomolecules and long acquisition times. These issues may be addressed through the development of tip-enhanced coherent anti-Stokes Raman scattering microscopy.

Raman spectroscopy and related techniques in biomedicine.

In this review we describe label-free optical spectroscopy techniques which are able to non-invasively measure the (bio)chemistry in biological systems. Raman spectroscopy uses visible or near-infrared light to measure a spectrum of vibrational bonds in seconds. Coherent anti-Stokes Raman (CARS) microscopy and stimulated Raman loss (SRL) microscopy are orders of magnitude more efficient than Raman spectroscopy, and are able to acquire high quality chemically-specific images in seconds. We discuss the benefits and limitations of all techniques, with particular emphasis on applications in biomedicine--both in vivo (using fiber endoscopes) and in vitro (in optical microscopes).

Simulations of tip-enhanced optical microscopy reveal atomic resolution.

We have performed finite element electromagnetic analysis of 'apertureless' scanning near-field optical microscope tips. A variety of radii was considered, from 40 nm down to 1 nm, and the enhancement of optical scattering from the region beneath the probe apex was modelled in air and water. We observed sizeable spectral shifts when the radius decreases, together with a surprising increase in the amount of scattering from small tips. The lateral resolution is considered, which is shown to be always smaller than the tip radius, and for 1-nm-radius tips can allow atomic resolution imaging with sufficient optical enhancement. Gold, silver, copper and aluminium were modelled as tip materials.

Heating effects in tip-enhanced optical microscopy.

Finite element simulations of laser-induced heating in scanning probe microscopy are presented. The electromagnetic field is first simulated for a variety of tip and substrate materials, and for air and aqueous environments. This electromagnetic field, in the end of the tip and substrate under the tip, produces Joule heating. Using this Joule heat source, steady state thermal simulations are performed. As a result of the large enhancement of optical power by the tip-substrate cavity, predicted temperature rises can be over 3 orders of magnitude higher than the values predicted without a tip present, but the optical signal can be enhanced by over 10 orders. Gold tips and substrates are predicted to give the highest optical signal for a given temperature increase.

Simulations of atomic resolution tip-enhanced optical microscopy.

Optical techniques can access a wealth of information but traditionally their resolution has been restricted by the diffraction limit. Near-field techniques, which used nanoscale apertures or nanotip electric field enhancement, have succeeded in circumventing Abbe's law. We show that atomic resolution is theoretically achievable for tip enhanced optical microscopy. Using finite element analysis of the electromagnetic field around a small radius metallic scanning probe microscopy tip, we modeled various tip radii and materials, and an aqueous environment as well as ambient air. For a 1 nm gold tip we predict a strong red shift, and surprisingly high values for the enhancement of the intensity of scattered light - over 10(7). For this tip, we predict that 0.2 nm lateral resolution in optical imaging is achievable - good enough to resolve individual atomic bonds. The promise of optical data at these spatial scales offers great potential for nanometrology and nanotechnology applications.

Finite element simulations of tip-enhanced Raman and fluorescence spectroscopy.

Finite element electromagnetic simulations of scanning probe microscopy tips and substrates are presented. The enhancement of the scattered light intensity is found to be as high as 10(12) for a 20 nm radius gold tip, and tip-substrate separation of 1 nm. Molecular resolution imaging (< 1 nm) is achievable, even with a relatively large radius tip (20 nm). We also make predictions for imaging in aqueous environments, noting a sizable red shift of the spectral peaks. Finally, we discuss signal levels, and predict that high-speed Raman mapping should be possible with gold substrates and a small tip-substrate separation (< 4 nm).

A multicenter study of the family educational rights and privacy act and the standardized letter of recommendation: impact on emergency medicine residency applicant and faculty behaviors.

BACKGROUND: Residency applicants have the right to see letters of recommendation written on their behalf. It is not known whether applicants are affected by waiving this right. OBJECTIVES: Our multicenter study assessed how frequently residency applicants waived their FERPA rights to view their letters of recommendation, and whether this affected the ratings they were given by faculty. METHODS: We reviewed all ERAS-submitted letters of recommendation to 14 ACGME-accredited programs in 2006-2007. We collected ERAS ID, program name, FERPA declaration, standardized letter of recommendation (SLOR) use, and SLOR Global Assessment ranking. The percentage of applicants who waived their FERPA rights was determined. Chi-square tests of independence assessed whether applicants' decision to waive their FERPA rights was associated with their SLOR Global Assessment. RESULTS: We examined 1776 applications containing 6424 letters of recommendations. Of 2736 letters that specified a Global Assessment, 2550 (93%) applicants waived their FERPA rights, while 186 did not. Of the applicants who chose not to waive their rights, 45.6% received a ranking of Outstanding, 35.5% Excellent, 18.3% Very Good, and 1.6% Good. Of applicants who waived their FERPA rights, 35.1% received a ranking of Outstanding, 49.6% Excellent, 13.7% Very Good, and 1.6% Good. Applicants who did not waive their FERPA rights were more likely to receive an Outstanding Assessment (P  =  .003). CONCLUSIONS: The majority (93%) of residency applicants waived their FERPA rights. Those who did not waive their rights had a statistically higher chance of receiving an Outstanding Assessment than those who did.

CENP-A confers a reduction in height on octameric nucleosomes.

Nucleosomes with histone H3 replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes from human and Drosophila have been reported to have reduced heights as compared to canonical octameric H3 nucleosomes, thus suggesting a unique tetrameric hemisomal composition. We demonstrate that octameric CENP-A nucleosomes assembled in vitro exhibit reduced heights, indicating that they are physically distinct from H3 nucleosomes and negating the need to invoke the presence of hemisomes.

Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy.

Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 µm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy.

Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

Raman spectroscopy and CARS microscopy of stem cells and their derivatives.

The characterisation of stem cells is of vital importance to regenerative medicine. Failure to separate out all stem cells from differentiated cells before therapies can result in teratomas - tumours of multiple cell types. Typically, characterisation is performed in a destructive manner with fluorescent assays. A truly non-invasive method of characterisation would be a major breakthrough in stem cell-based therapies. Raman spectroscopy has revealed that DNA and RNA levels drop when a stem cell differentiates into other cell types, which we link to a change in the relative sizes of the nucleus and cytoplasm. We also used Raman spectroscopy to investigate the biochemistry within an early embryo, or blastocyst, which differs greatly from colonies of embryonic stem cells. Certain cell types that differentiate from stem cells can be identified by directly imaging the biochemistry with CARS microscopy; examples presented are hydroxyapatite - a precursor to bone, and lipids in adipocytes.