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This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 μm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level., (© 2024. The Author(s).)

Significance: Skin color affects light penetration leading to differences in its absorption and scattering properties. COVID-19 highlighted the importance of understanding of the interaction of light with different skin types, e.g., pulse oximetry (PO) unreliably determined oxygen saturation levels in people from Black and ethnic minority backgrounds. Furthermore, with increased use of other medical wearables using light to provide disease information and photodynamic therapies to treat skin cancers, a thorough understanding of the effect skin color has on light is important for reducing healthcare disparities., Aim: The aim of this work is to perform a thorough review on the effect of skin color on optical properties and the implication of variation on optical medical technologies., Approach: Published in vivo optical coefficients associated with different skin colors were collated and their effects on optical penetration depth and transport mean free path (TMFP) assessed., Results: Variation among reported values is significant. We show that absorption coefficients for dark skin are ∼ 6 % to 74% greater than for light skin in the 400 to 1000 nm spectrum. Beyond 600 nm, the TMFP for light skin is greater than for dark skin. Maximum transmission for all skin types was beyond 940 nm in this spectrum. There are significant losses of light with increasing skin depth; in this spectrum, depending upon Fitzpatrick skin type (FST), on average 14% to 18% of light is lost by a depth of 0.1 mm compared with 90% to 97% of the remaining light being lost by a depth of 1.93 mm., Conclusions: Current published data suggest that at wavelengths beyond 940 nm light transmission is greatest for all FSTs. Data beyond 1000 nm are minimal and further study is required. It is possible that the amount of light transmitted through skin for all skin colors will converge with increasing wavelength enabling optical medical technologies to become independent of skin color., (© 2024 The Authors.)

Significance: Rapid advances in medical imaging technology, particularly the development of optical systems with non-linear imaging modalities, are boosting deep tissue imaging. The development of reliable standards and phantoms is critical for validation and optimization of these cutting-edge imaging techniques., Aim: We aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth., Approach: Standards were made using a robust double-network hydrogel matrix consisting of agarose and polyacrylamide. The materials generated ranged from single layers to more complex constructs consisting of up to seven layers, with modality-specific markers embedded between the layers., Results: These standards proved useful in the determination of the axial scaling factor for light microscopy and allowed for depth evaluation for different imaging modalities (conventional one-photon excitation fluorescence imaging, two-photon excitation fluorescence imaging, second harmonic generation imaging, and coherent anti-Stokes Raman scattering) achieving actual depths of 1550, 1550, 1240, and 1240    μ m , respectively. Once fabricated, the phantoms were found to be stable for many months., Conclusions: The ability to image at depth, the phantom's robustness and flexible layered structure, and the ready incorporation of "optical markers" make these ideal depth standards for the validation of a variety of imaging modalities., (© 2023 The Authors.)

Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside analysis to quantify changes in the cells' low-frequency elastic modulus, G 0 ' , and the cell's dynamics over the time window ∼10 -2  s to 10 s. An analogy to optical trapping allows verification of the invariant viscosity of HeLa S3 cells under control conditions and after cytoskeletal disruption. Stiffening of the cell is observed during cytoskeletal rearrangement in the control case, and cell softening when the actin cytoskeleton is disrupted by Latrunculin B. These data correlate with conventional understanding that integrin binding and recruitment triggers cytoskeletal rearrangement. This is, to our knowledge, the first time that cell stiffening has been measured during focal adhesion maturation, and the longest time over which such stiffening has been quantified by any means. STATEMENT OF SIGNIFICANCE: Here, we present an approach for studying mechanical properties of live cells without applying external forces or inserting tracers. Regulation of cellular biomechanics is crucial to healthy cell function. For the first time in literature, we can non-invasively and passively quantify cell mechanics during interactions with functionalised surface. Our method can monitor the maturation of adhesion sites on the surface of individual live cells without disrupting the cell mechanics by applying forces to the cell. We observe a stiffening response in cells over tens of minutes after a bead chemically binds. This stiffening reduces the deformation rate of the cytoskeleton, although the internal force generation increases. Our method has potential for applications to study mechanics during cell-surface and cell-vesicle interactions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Imaging non-invasively into the human body is currently limited by cost (MRI and CT scan), image resolution (ultrasound), exposure to ionising radiation (CT scan and X-ray), and the requirement for exogenous contrast agents (CT scan and PET scan). Optical imaging has the potential to overcome all these issues but is currently limited by imaging depth due to the scattering and absorption properties of human tissue. Skin is the first barrier encountered by light when imaging non-invasively, and therefore a clear understanding of the way that light interacts with skin is required for progress on optical medical imaging to be made. Here we present a thorough review of the optical properties of human skin measured in-vivo and compare these to the previously collated ex-vivo measurements. Both in-vivo and ex-vivo published data show high inter- and intra-publication variability making definitive answers regarding optical properties at given wavelengths challenging. Overall, variability is highest for ex-vivo absorption measurements with differences of up to 77-fold compared with 9.6-fold for the in-vivo absorption case. The impact of this variation on optical penetration depth and transport mean free path is presented and potential causes of these inconsistencies are discussed. We propose a set of experimental controls and reporting requirements for future measurements. We conclude that a robust in-vivo dataset, measured across a broad spectrum of wavelengths, is required for the development of future technologies that significantly increase the depth of optical imaging., Competing Interests: The authors declare no conflicts of interest., (Published by Optica Publishing Group under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.)

In this paper, we show a proof-of-concept method to parallelise phonon microscopy measurements for cell elasticity imaging by demonstrating a 3-fold increase in acquisition speed which is limited by current acquisition hardware. Phonon microscopy is based on time-resolved Brillouin scattering, which uses a pump-probe method with asynchronous optical sampling (ASOPS) to generate and detect coherent phonons. This enables access to the cell elasticity via the Brillouin frequency with sub-optical axial resolution. Although systems based on ASOPS are typically faster compared to the ones built with a mechanical delay line, they are still very slow to study real time changes at the cellular level. Additionally, the biocompatibility is reduced due to long light exposure and scanning time. Using a multi-core fibre bundle rather than a single channel for detection, we acquire 6 channels simultaneously allowing us to speed-up measurements, and open a way to scale-up this method., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Tropomyosin 1 (TPM1) is an essential sarcomeric component, stabilising the thin filament and facilitating actin's interaction with myosin. A number of sarcomeric proteins, such as alpha myosin heavy chain, play crucial roles in cardiac development. Mutations in these genes have been linked to congenital heart defects (CHDs), occurring in approximately 1 in 145 live births. To date, TPM1 has not been associated with isolated CHDs. Analysis of 380 CHD cases revealed three novel mutations in the TPM1 gene; IVS1+2T>C, I130V, S229F and a polyadenylation signal site variant GATAAA/AATAAA. Analysis of IVS1+2T>C revealed aberrant pre-mRNA splicing. In addition, abnormal structural properties were found in hearts transfected with TPM1 carrying I130V and S229F mutations. Phenotypic analysis of TPM1 morpholino-treated embryos revealed roles for TPM1 in cardiac looping, atrial septation and ventricular trabeculae formation and increased apoptosis was seen within the heart. In addition, sarcomere assembly was affected and altered action potentials were exhibited. This study demonstrated that sarcomeric TPM1 plays vital roles in cardiogenesis and is a suitable candidate gene for screening individuals with isolated CHDs., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Congenital heart defects (CHDs) have a neonatal incidence of 0.8-1% (refs. 1,2). Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (∼2.7%), suggesting a considerable role for de novo mutations (DNMs) and/or incomplete penetrance. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of 'syndromic' patients with extra-cardiac manifestations. We exome sequenced 1,891 probands, including both syndromic CHD (S-CHD, n = 610) and nonsyndromic CHD (NS-CHD, n = 1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs but not inherited PTVs in known CHD-associated genes, consistent with recent findings. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study finds evidence for distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Association between the C677T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene and congenital heart disease (CHD) is contentious., Methods and Results: We compared genotypes between CHD cases and controls and between mothers of CHD cases and controls. We placed our results in context by conducting meta-analyses of previously published studies. Among 5814 cases with primary genotype data and 10 056 controls, there was no evidence of association between MTHFR C677T genotype and CHD risk (odds ratio [OR], 0.96 [95% confidence interval, 0.87-1.07]). A random-effects meta-analysis of all studies (involving 7697 cases and 13 125 controls) suggested the presence of association (OR, 1.25 [95% confidence interval, 1.03-1.51]; P=0.022) but with substantial heterogeneity among contributing studies (I(2)=64.4%) and evidence of publication bias. Meta-analysis of large studies only (defined by a variance of the log OR <0.05), which together contributed 83% of all cases, yielded no evidence of association (OR, 0.97 [95% confidence interval, 0.91-1.03]) without significant heterogeneity (I(2)=0). Moreover, meta-analysis of 1781 mothers of CHD cases (829 of whom were genotyped in this study) and 19 861 controls revealed no evidence of association between maternal C677T genotype and risk of CHD in offspring (OR, 1.13 [95% confidence interval, 0.87-1.47]). There was no significant association between MTHFR genotype and CHD risk in large studies from regions with different levels of dietary folate., Conclusions: The MTHFR C677T polymorphism, which directly influences plasma folate levels, is not associated with CHD risk. Publication biases appear to substantially contaminate the literature with regard to this genetic association.

We carried out a genome-wide association study (GWAS) of congenital heart disease (CHD). Our discovery cohort comprised 1,995 CHD cases and 5,159 controls and included affected individuals from each of the 3 major clinical CHD categories (with septal, obstructive and cyanotic defects). When all CHD phenotypes were considered together, no region achieved genome-wide significant association. However, a region on chromosome 4p16, adjacent to the MSX1 and STX18 genes, was associated (P = 9.5 × 10⁻⁷) with the risk of ostium secundum atrial septal defect (ASD) in the discovery cohort (N = 340 cases), and this association was replicated in a further 417 ASD cases and 2,520 controls (replication P = 5.0 × 10⁻⁵; odds ratio (OR) in replication cohort = 1.40, 95% confidence interval (CI) = 1.19-1.65; combined P = 2.6 × 10⁻¹⁰). Genotype accounted for ~9% of the population-attributable risk of ASD.

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10(-5)). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ~5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ~4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Recurrent rearrangements of chromosome 1q21.1 that occur via non-allelic homologous recombination have been associated with variable phenotypes exhibiting incomplete penetrance, including congenital heart disease (CHD). However, the gene or genes within the ~1 Mb critical region responsible for each of the associated phenotypes remains unknown. We examined the 1q21.1 locus in 948 patients with tetralogy of Fallot (TOF), 1488 patients with other forms of CHD and 6760 ethnically matched controls using single nucleotide polymorphism genotyping arrays (Illumina 660W and Affymetrix 6.0) and multiplex ligation-dependent probe amplification. We found that duplication of 1q21.1 was more common in cases of TOF than in controls [odds ratio (OR) 30.9, 95% confidence interval (CI) 8.9-107.6); P = 2.2 × 10(-7)], but deletion was not. In contrast, deletion of 1q21.1 was more common in cases of non-TOF CHD than in controls [OR 5.5 (95% CI 1.4-22.0); P = 0.04] while duplication was not. We also detected rare (n = 3) 100-200 kb duplications within the critical region of 1q21.1 in cases of TOF. These small duplications encompassed a single gene in common, GJA5, and were enriched in cases of TOF in comparison to controls [OR = 10.7 (95% CI 1.8-64.3), P = 0.01]. These findings show that duplication and deletion at chromosome 1q21.1 exhibit a degree of phenotypic specificity in CHD, and implicate GJA5 as the gene responsible for the CHD phenotypes observed with copy number imbalances at this locus.

Background.  Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt-Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design.  In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results.  Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions.  In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts., (© 2011 Wiley Periodicals, Inc.)