Amanda J. Wright

Despite questionable accuracy, subjective methods to categorize skin color are heavily relied upon in research and medicine. Objective skin color determination is expensive requiring specialized instrumentation and interpretation. We compare three subjective approaches, i) Fitzpatrick Skin Type Scale (FST), ii) Pantone SkinTone Guide (PST) and, iii) Monk Skin Tone Scale (MST), with objectively measured skin color from a spectrophotometer in 87 volunteers to understand the limitations of each method. In agreement with others, we show that the popular FST questionnaire correlates poorly with the objective approach. However, PST color swatches provide good correlation with spectrophotometer measurements. PST consists of 110+ swatches that are inexpensive and easy to use, however, similar to other reports, the volunteers found the number of swatches overwhelming and/or excessive. We found that the recently introduced MST is not representative of reality with only 3 of the 10 color groups representing our volunteers and published populations of volunteers. In future, we propose using 9 color swatches to split the spectrum of human skin color into 10 groupings (Nottingham Skin Categories - NSC) that are representative of the global population. This new approach would be easy to implement and inexpensive in research, healthcare and cosmetics settings, and maps directly to objective, quantitative, measures taken with a spectrophotometer. For the testing and development of new optical devices, NSC would provide increased comparability between studies and ensure studies are representative of local/global populations. In the clinic NSC would be useful for dermatology, photodynamic therapy and dosage assessment for topical medicine, for example.

In dividing cells, chromosomes are coated in a sheath of proteins and RNA called the mitotic chromosome periphery. This sheath is thought to confer biophysical properties to chromosomes, critical for successful cell division. However, the details of chromosome mechanics, and specifically, if and how the chromosome periphery contributes to them, remain poorly understood. In this study, we present a comprehensive characterisation of single-chromosome mechanics using optical tweezers and an improved broadband microrheology analysis. We extend this analysis to direct measurements of the chromosome periphery by manipulating levels of Ki-67, its chief organiser, and apply a rheological model to isolate its contribution to chromosome mechanics. We report that the chromosome periphery governs dynamic self-reorganisation of chromosomes and acts as a structural constraint, providing force-damping properties. This work provides significant insight into chromosome mechanics and will inform our understanding of the mitotic chromosome periphery’s role in cell division.

Adaptive optics is a technique for correcting aberrations and improving image quality. When adaptive optics was first used in microscopy, it was common to rely on iterative approaches to determine the aberrations present. It is advantageous to avoid iteration, and therefore there has been a shift to deep learning for aberration prediction. However, issues remain regarding the practicalities of machine learning for adaptive optics, an important one being the requirement for a large training dataset. Here, we explore transfer learning to overcome this need for data by pre-training a network on a large simulated dataset and fine-tuning it with reduced experimental data for application in an experimental setting. We demonstrate that the pre-trained network can make noticeable improvements with fine-tuning on just 24 experimental samples. To further enhance practicality, we significantly extend the range of aberrations present, predicting up to 25 Zernike modes with each coefficient ranging from −1 to 1, and perform a thorough analysis of the type and magnitude of phase-diversity required in the input data for a successful network. Our approach demonstrates substantial aberration reduction on experimental data for 10 Zernike modes, with an average 73% decrease in RMS wavefront error from 1.81 to 0.48 rad when correction is applied. This method achieves complete experimental image capture and aberration inference at rates comparable to the image acquisition time of a typical laser scanning microscope. Additionally, we consider the benefits of further improvements via an iterative step. As such, this work addresses some of the key practical hurdles that remain in the use of deep learning for aberration prediction and correction.

Optical tweezers have emerged as a powerful, versatile approach for a range of studies in cellular and molecular biology. A particular highlight has been its use for microrheology measurements in miniscule sample volumes. In this study, we demonstrate the application of optical tweezers to investigate the viscosity of the extracellular matrix surrounding mammalian oocytes and determine whether this is associated with oocyte developmental potential. By analysing the motion of a trapped particle, we can quantify the mechanical properties of this matrix, thus overcoming limitations of traditional passive microrheology techniques that rely on free diffusion. We utilise two maturation methods—in vivo and in vitro—to generate oocytes with differing developmental potential. Our findings indicate that oocytes matured in vivo exhibit higher viability post-fertilisation compared to their in vitro counterparts and we establish a positive correlation between extracellular matrix viscosity and oocyte developmental potential. This work demonstrates that optical tweezers are a novel, non-invasive tool for assessing oocyte quality, contributing to valuable insights to the field of reproductive biology.

Mitotic chromosomes are specialised packets of condensed genetic material with dynamic mechanical properties. Each chromosome is coated by a sheath of proteins and RNA, called the mitotic chromosome periphery (MCP). The MCP is widely considered as an essential chromosome compartment where its multiple functions bestow material properties important for successful cell division. However, the details of the micromechanical properties of mitotic chromosomes, and specifically if and how the MCP contributes to these features, remain poorly understood. In this study, we present the most comprehensive characterisation of single-chromosome mechanics to date spanning a broadband frequency range, using optical tweezers and a novel microrheology technique. We extend this analysis to the first direct measurements of MCP micromechanics by manipulating levels of Ki-67, the chief organiser of this compartment, and apply a rheological model to isolate its contribution to chromosome dynamics. We report that the MCP governs high-frequency self-reorganisation dynamics and acts as a structural constraint, providing force-damping properties that mitigate mitotic stress. This work significantly advances our understanding of chromosome micromechanics and how the MCP contributes to the fundamental properties of chromosomes.

Microrheology with optical tweezers (MOT) is an all-optical technique that allows the user to investigate a materials’ viscoelastic properties at microscopic scales, and is particularly useful for those materials that feature complex microstructures, such as biological samples. MOT is increasingly being employed alongside 3D imaging systems and particle tracking methods to generate maps showing not only how properties may vary between different points in a sample but also how at a single point the viscoelastic properties may vary with direction. However, due to the diffraction limited shape of focussed beams, optical traps are inherently anisotropic in 3D. This can result in a significant overestimation of the fluids’ viscosity in certain directions. As such, the rheological properties can only be accurately probed along directions parallel or perpendicular to the axis of trap beam propagation. In this work, a new analytical method is demonstrated to overcome this potential artefact. This is achieved by performing principal component analysis on 3D MOT data to characterise the trap, and then identify the frequency range over which trap anisotropy influences the data. This approach is initially applied to simulated data for a Newtonian fluid where the trap anisotropy induced maximum error in viscosity is reduced from ~ 150% to less than 6%. The effectiveness of the method is corroborated by experimental MOT measurements performed with water and gelatine solutions, thus confirming that the microrheology of a fluid can be extracted reliably across a wide frequency range and in any arbitrary direction. This work opens the door to fully spatially and angularly resolved 3D mapping of the rheological properties of soft materials over a broad frequency range.

Optical technologies have transformed medical diagnostics, surgery, and therapeutics in recent decades and the medical device industry provides vast opportunity for optical technology. Optical fields in medicine account for $73 billion of the global market and include ophthalmic optics, robotics, lasers, optical surgery, microscopy, and endoscopy, making it the largest technology sector in medicine.¹ Given the size of this sector, it is likely that there will be advances in optical fibers, making them thinner and smaller; use of imaging techniques such as optical coherence tomography (OCT) will become more widespread with greater resolution and providing noninvasive imaging; and laser-based therapeutics will become more accurate and widespread.² Optical devices are used by the majority of medical disciplines and do not consist only of microscopes; they range from otoscopes and ophthalmoscopes to endoscopes and colonoscopes, including surgical microscopes and imaging during robotically assisted surgery.

SignificanceRapid 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.AimWe aim to design and fabricate flexible, multi-layered hydrogel-based optical standards and evaluate advanced optical imaging techniques at depth.ApproachStandards 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.ResultsThese 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 μm, respectively. Once fabricated, the phantoms were found to be stable for many months.ConclusionsThe 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.

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.