Imaging cervical cytology with scanning near-field optical microscopy (SNOM) coupled with an IR-FEL

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (?0.2 \ensuremathμm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 \ensuremathμm to 30 \ensuremathμm due to the optical diffraction limit

Tracking the Impact of Excisional Cervical Treatment on the Cervix using Biospectroscopy

Local excisional treatment for cervical intra-epithelial neoplasia (CIN) is linked to significant adverse sequelae including preterm birth, with cone depth and radicality of treatment correlating to the frequency and severity of adverse events. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy can detect underlying cervical disease more accurately than conventional cytology. The chemical profile of cells pre- and post-treatment may differ as a result of altered biochemical processes due to excision, or treatment of the disease. Since pre-treatment cervical length varies amongst women, the percentage of cervix excised may correlate more accurately to risk than absolute dimensions. We show that treatment for CIN significantly alters the biochemistry of the cervix, compared with women who have not had treatment; this is due to the removal of cervical tissue rather than the removal of the disease. However, the spectra do not seem to correlate to the cone depth or proportion of cervical length excised. Future research should aim to explore the impact of treatment in a larger cohort

Infrared vibrational spectroscopy: a rapid and novel diagnostic and monitoring tool for cystinuria

Cystinuria is the commonest inherited cause of nephrolithiasis (~1% in adults; ~6% in children) and is the result of impaired cystine reabsorption in the renal proximal tubule. Cystine is poorly soluble in urine with a solubility of ~1 mM and can readily form microcrystals that lead to cystine stone formation, especially at low urine pH. Diagnosis of cystinuria is made typically by ion-exchange chromatography (IEC) detection and quantitation, which is slow, laboursome and costly. More rapid and frequent monitoring of urinary cystine concentration would significantly improve the diagnosis and clinical management of cystinuria. We used attenuated total reflection - Fourier transform infrared spectroscopy (ATR-FTIR) to detect and quantitate insoluble cystine in 22 cystinuric and 5 healthy control urine samples. Creatinine concentration was also determined by ATR-FTIR to adjust for urinary concentration/dilution. Urine was centrifuged, the insoluble fraction re-suspended in 5 μL water and dried on the ATR prism. Cystine was quantitated using its 1296 cm−1 absorption band and levels matched with parallel measurements made using IEC. ATR-FTIR afforded a rapid and inexpensive method of detecting and quantitating insoluble urinary cystine. This proof-of-concept study provides a basis for developing a high-throughput, cost-effective diagnostic method for cystinuria, and for point-of-care clinical monitoring

Surface-enhanced Raman spectroscopy of the endothelial cell membrane

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al

Subtle effects of environmental stress observed in the early life stages of the Common frog, Rana temporaria

Worldwide amphibian populations are declining due to habitat loss, disease and pollution. Vulnerability to environmental contaminants such as pesticides will be dependent on the species, the sensitivity of the ontogenic life stage and hence the timing of exposure and the exposure pathway. Herein we investigated the biochemical tissue ‘fingerprint’ in spawn and early-stage tadpoles of the Common frog, Rana temporaria, using attenuated total reflection- Fourier-transform infrared (ATR-FTIR) spectroscopy with the objective of observing differences in the biochemical constituents of the respective amphibian tissues due to varying water quality in urban and agricultural ponds. Our results demonstrate that levels of stress (marked by biochemical constituents such as glycogen that are involved in compensatory metabolic mechanisms) can be observed in tadpoles present in the pond most impacted by pollution (nutrients and pesticides), but large annual variability masked any inter-site differences in the frog spawn. ATR-FTIR spectroscopy is capable of detecting differences in tadpoles that are present in selected ponds with different levels of environmental perturbation and thus serves as a rapid and cost effective tool in assessing stress-related effects of pollution in a vulnerable class of organism

Safety and efficacy of Favipiravir in moderate to severe SARS-CoV-2 pneumonia

Background: We examined the safety and efficacy of a treatment protocol containing Favipiravir for the treatment of SARS-CoV-2. Methods: We did a multicenter randomized open-labeled clinical trial on moderate to severe cases infections of SARS-CoV-2. Patients with typical ground glass appearance on chest computerized tomography scan (CT scan) and oxygen saturation (SpO2) of less than 93 were enrolled. They were randomly allocated into Favipiravir (1.6 gr loading, 1.8 gr daily) and Lopinavir/Ritonavir (800/200 mg daily) treatment regimens in addition to standard care. In-hospital mortality, ICU admission, intubation, time to clinical recovery, changes in daily SpO2 after 5 min discontinuation of supplemental oxygen, and length of hospital stay were quantified and compared in the two groups. Results: 380 patients were randomly allocated into Favipiravir (1 9 3) and Lopinavir/Ritonavir (1 8 7) groups in 13 centers. The number of deaths, intubations, and ICU admissions were not significantly different (26, 27, 31 and 21, 17, 25 respectively). Mean hospital stay was also not different (7.9 days SD = 6 in the Favipiravir and 8.1 SD = 6.5 days in Lopinavir/Ritonavir groups) (p = 0.61). Time to clinical recovery in the Favipiravir group was similar to Lopinavir/Ritonavir group (HR = 0.94, 95% CI 0.75 � 1.17) and likewise the changes in the daily SpO2 after discontinuation of supplemental oxygen (p = 0.46) Conclusion: Adding Favipiravir to the treatment protocol did not reduce the number of ICU admissions or intubations or In-hospital mortality compared to Lopinavir/Ritonavir regimen. It also did not shorten time to clinical recovery and length of hospital stay. © 2021 Elsevier B.V