Tear-Based Vibrational Spectroscopy Applied to Amyotrophic Lateral Sclerosis.

Biofluid analysis by optical spectroscopy techniques is attracting considerable interest due to its potential to revolutionize diagnostics and precision medicine, particularly for neurodegenerative diseases. However, the lack of effective biomarkers combined with the unaccomplished identification of convenient biofluids has drastically hampered optical advancements in clinical diagnosis and monitoring of neurodegenerative disorders. Here, we show that vibrational spectroscopy applied to human tears opens a new route, offering a non-invasive, label-free identification of a devastating disease such as amyotrophic lateral sclerosis (ALS). Our proposed approach has been validated using two widespread techniques, namely, Fourier transform infrared (FTIR) and Raman microspectroscopies. In conjunction with multivariate analysis, this vibrational approach made it possible to discriminate between tears from ALS patients and healthy controls (HCs) with high specificity (∼97% and ∼100% for FTIR and Raman spectroscopy, respectively) and sensitivity (∼88% and ∼100% for FTIR and Raman spectroscopy, respectively). Additionally, the investigation of tears allowed us to disclose ALS spectroscopic markers related to protein and lipid alterations, as well as to a reduction of the phenylalanine level, in comparison with HCs. Our findings show that vibrational spectroscopy is a new potential ALS diagnostic approach and indicate that tears are a reliable and non-invasive source of ALS biomarkers.

Single-Tear Proteomics: A Feasible Approach to Precision Medicine.

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.

Raman spectroscopy to investigate early biochemical alterations in human tears caused by contact lenses.

CLINICAL RELEVANCE: The interaction between contact lenses and tear film plays a critical role in lens biocompatibility and can influence discomfort during wear. Early detection of biochemical changes is important for improving ocular surface health, preventing complications, and developing advanced lens materials to enhance long-term comfort. BACKGROUND: The pursuit of biocompatible lens materials requires a thorough understanding of their interaction with tear environment. The aim of this study is to evaluate different approaches to investigate early alterations in tear biomolecular profiles induced by lens wear. METHODS: Two participants wore different CLs and underwent ocular analysis before CL insertion (T0), after 2-hour wear (T2), and two hours after removing the CL (T4), evaluating non-invasive tear break-up time (NIBUT), tear meniscus height (TMH), and osmolarity. Forty CLs were used. At the same time points, tears were collected and analysed by Raman spectroscopy. RESULTS: Tear film stability, as measured by NIBUT, decreased post-lens wear (T0 vs T2, p = 0.034 and 0.025 for S1 and S2, respectively) but showed recovery within 2 hours (T0 vs T4, p > 0.05 for both S1 and S2), while TMH and osmolarity remained unchanged (T0 vs T2 vs T4, p > 0.05 for both S1 and S2). Raman spectroscopy highlighted notable changes in the biomolecular profile of tears after lens wear, particularly in the 2700-3200 cm-1 spectral window related to C-H stretching, which is sensitive to CH, CH2 and CH3 groups, their functional chemistry group, and the surrounding microenvironment. CONCLUSIONS: Contact lens wear induces subtle biochemical, subclinical alterations in tears that can be detected early by Raman spectroscopy. The method is promising for analysing the subject-specific responses to lenses, also with possible individual variability in the timing of these changes. In addition, this approach can provide insights for designing biocompatible materials and for the early detection of subclinical biomarkers.

Red and green defocus curves and duochrome test in different age groups.

PURPOSE: To compare the eye defocus curves (DCs) obtained with stimuli on red, green, and white backgrounds and to investigate the applicability of the duochrome test (DT) in different age groups. METHODS: 12 elderly (ELD: 59.3 ± 3.9 years) and 8 young (YG: 22.1 ± 1.1 years) subjects were recruited. An optometric assessment with the DT was carried out to obtain the subjective refraction at distance. DCs at distance on green, white, and red backgrounds were measured and the following parameters were deduced: dioptric difference between red-green, green-white, red-white focal positions (minima of the DCs), best corrected visual acuity (BCVA), and widths of the DCs for red, green, and white. RESULTS: The DC difference between the green-white focal positions (mean ± standard deviation) was -0.12±0.17 diopters (D) (ELD, p= 0.012) and -0.11±0.12 D (YG, p= 0.039), while the red-white difference was not statistically significant. The DC red-green difference was 0.20±0.16 D (ELD, p= 0.002) and 0.18±0.18 D (YG, p = 0.008). The ELD BCVA with green background was significantly worse than BCVA with red (p = 0.007) and white (p = 0.007). The mean value of the DC's width in ELD for green (1.01±0.36 D) was higher than for red (0.77±0.21 D) and for white (0.84±0.35 D), but with no statistical significance. CONCLUSION: Both age groups showed a slight focusing preference for red when using white light. Moreover, ELD showed a worse BCVA with a green compared to a red background. Despite these results deduced by DC analyses, these aspects do not compromise the possibility of using the DT in clinical practice both in the young and in the elderly. Furthermore, the difference of about 0.20 D between red-green DC in both groups confirms the clinical appropriateness of the widespread use of 0.25 D step as the standard minimum difference in power between correcting lenses.

Red and green defocus curves and duochrome test in different age groups / Curvas de desenfoque rojo y verde y prueba de duocromo en diferentes grupos de edad

Purpose: To compare the eye defocus curves (DCs) obtained with stimuli on red, green, and white backgrounds and to investigate the applicability of the duochrome test (DT) in different age groups. Methods: 12 elderly (ELD: 59.3 ± 3.9 years) and 8 young (YG: 22.1 ± 1.1 years) subjects were recruited. An optometric assessment with the DT was carried out to obtain the subjective refraction at distance. DCs at distance on green, white, and red backgrounds were measured and the following parameters were deduced: dioptric difference between red-green, green-white, red-white focal positions (minima of the DCs), best corrected visual acuity (BCVA), and widths of the DCs for red, green, and white. Results: The DC difference between the green-white focal positions (mean ± standard deviation) was -0.12±0.17 diopters (D) (ELD, p = 0.012) and -0.11±0.12 D (YG, p = 0.039), while the red-white difference was not statistically significant. The DC red-green difference was 0.20±0.16 D (ELD, p = 0.002) and 0.18±0.18 D (YG, p = 0.008). The ELD BCVA with green background was significantly worse than BCVA with red (p = 0.007) and white (p = 0.007). The mean value of the DC's width in ELD for green (1.01±0.36 D) was higher than for red (0.77±0.21 D) and for white (0.84±0.35 D), but with no statistical significance. Conclusion: Both age groups showed a slight focusing preference for red when using white light. Moreover, ELD showed a worse BCVA with a green compared to a red background. Despite these results deduced by DC analyses, these aspects do not compromise the possibility of using the DT in clinical practice both in the young and in the elderly. Furthermore, the difference of about 0.20 D between red-green DC in both groups confirms the clinical appropriateness of the widespread use of 0.25 D step as the standard minimum difference in power between correcting lenses.(AU)