Biophotonics-the interdisciplinary fusion of light-based technologies with biology and medicine-is rapidly transforming research, diagnostics, and therapy across various domains. This white paper, developed in conjunction with the International Congress on Biophotonics 2024, offers a comprehensive overview of the current landscape and future potential of biophotonics. It discusses core technologies such as bioimaging, biosensing, and photonic-based therapies, while highlighting novel applications in oncology, infectious diseases, neurology, cardiovascular health, agriculture, food safety, and environmental monitoring. The document also explores key enablers, including artificial intelligence, novel materials, and quantum biophotonics, along with critical challenges related to standardization, regulation, and clinical translation. A SWOT analysis and recommendations are provided to guide future research, commercialization, and interdisciplinary collaboration, underscoring biophotonics as a cornerstone of next-generation precision medicine and the One Health approach.

Visual proteomics (VP) aims to allow researchers to visualise, measure and analyse proteins in the context of cell and tissue structure in health and disease. VP is becoming a reality through technological advances across several domains, including in situ structural biology and correlative light and electron microscopy (CLEM). However, widespread adoption remains limited due to the complexity and cost of the various VP approaches reported to date. Here we present the VP-CLEM-Kit, a disruptive cost-effective pipeline for super resolution volume CLEM (SR-vCLEM) that can be implemented with minimal advanced electron microscopy expertise and equipment, making it accessible to light microscopy facilities and research labs. SR-vCLEM is based on in-resin fluorescence (IRF), where fluorophores are preserved through processing into resin. The easyIRF protocol reported here reduces the requirement for complex costly sample preparation equipment and toxic chemicals compared to standard IRF protocols. easyIRF blocks are cut into ultrathin sections that are imaged using ‘tomoSTORM’, a new modular and cost-effective openFrame -based light microscope controlled by the open-source software package Micro-Manager that provides serial single molecule localisation microscopy in array tomography format. Sections are then post-stained and imaged using a tabletop scanning electron microscope controlled by open-source SBEMimage software to run in array tomography format. We demonstrate the potential of the VP-CLEM-Kit by imaging organelle reporters in human cell lines and stem cell derived neurons, fluorescently labelled protein in neurons, and immunolabelled cells in human kidney biopsy tissue from transplant patients. The VP-CLEM-Kit delivers a ∼5-fold improvement in resolution at a ∼7-fold lower cost, and thus provides new technical capability as well as a blueprint for more equitable access to advanced imaging workflows. ### Competing Interest Statement The authors have declared no competing interest.

Optical microscopy is a ubiquitous tool in the physical and life sciences and in histopathology, where visible light microscopy is used to analyse clinical tissue sections at the micron scale to help diagnose diseases. In recent years, microscope technologies have dramatically evolved, but these have generally come with increased cost and complexity. To widen access to advanced microscopy capabilities, we have developed a cost-effective modular platform for optical microscopy (www.openscopes. com). Many of these instruments can be based around a new low-cost and flexible microscope stand, 'open Frame', for which the core components are open source. openFrame can support implementations of a wide range of microscope modalities for diverse applications, including research, pathology and training. Unlike many commercial microscopes that are often designed for specific applications and cannot be easily upgraded or adapted for different imaging modalities, openFramebased instruments can be relatively easily maintained, upgraded or adapted to another modality without requiring manufacturer support. To this end, openFramebased instruments are envisaged to operate with open source software, enabling researchers to assemble and modify their microscopes with minimal challenges presented by proprietary (closed) hardware or software. Here, we describe the implementation of a low-cost, research-grade modular optical microscope applicable to research and pathology.

Spectrally resolved imaging is typically realised using bandpass filters, which are inefficient when they reject "out-of-band" photons or using angularly dispersive devices with at least one image dimension requiring scanning to acquire a full hyperspectral dataset and therefore sequential data acquisition, unless cascades of dichroic beamsplitters are employed, for which cost and experimental complexity scale with the number of spectral channels. Here we present a new approach, "PolSpec", to realise rapid and flexible widefield hyperspectral imaging with lower cost and complexity using polarisation optics instead of dielectric coatings or dispersive devices. PolSpec utilises Lyot filters that provide continuously varying transmission across the desired spectral range to generate orthogonal "spectral modulation vectors" that can represent specific spectral signatures with significantly lower data volumes than full spectral profiles. We demonstrate single-shot widefield hyperspectral imaging using a polarisation-resolving camera and rapid, electronically reconfigurable, more photon efficient, hyperspectral imaging using a liquid crystal variable retarder. ### Competing Interest Statement The authors are intending to commercialise aspects of PolSpec.

A better understanding of the signalling mechanisms underlying transitions from drug-sensitive to drug-tolerant states is required to overcome therapy failure. We combined single-cell biosensor imaging with functional perturbations to investigate the regulation of oncogenic signalling in EGFR-mutant lung adenocarcinoma. We find that despite the constant presence of the mutant oncogene, ERK signalling exhibits pulsatile dynamics, with pulse characteristics determined by the endocytic machinery. Analysis of drug-tolerant persisters (DTPs) revealed that, after an initial phase of complete pathway shut-down, signalling was rewired leading to renewed ERK pulses that drive cell cycle progression. FAK- and SRC-regulated adhesion complexes replace mutant EGFR as the driver of reactivated ERK pulses in DTPs, yet they remain controlled by the membrane trafficking machinery. We show that DTPs rely on additional survival pathways including YAP signalling, and that the phosphatase PTPRS represents a key node in therapy resistant cells, coordinating regulation of ERK, the cytoskeleton, and YAP.### Competing Interest StatementAcknowledgements A.L.M. is a recipient of post-doctoral funding by AstraZeneca through the Crick/AZ Alliance. D.R.C is supported by the Francis Crick Institute, which receives its core funding form Cancer Research UK (FC001169), the UK Medical Research Council (FC002269), and the Wellcome Trust (FC001169), as well as an NC3Rs training fellowship (NC/S001832/1). K.V. is supported by a CRUK Accelerator doctoral studentship (C7408/A28450). E.S. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2040), the UK Medical Research Council (CC2040), and the Wellcome Trust (CC2040) and the European Research Council (ERC Advanced Grant CAN_ORGANISE, Grant agreement number 101019366). C.S. is a Royal Society Napier Research Professor (RSRP\R\210001). His work is supported by the Francis Crick Institute that receives its core funding from Cancer Research UK (CC2041), the UK Medical Research Council (CC2041), and the Wellcome Trust (CC2041) and the European Research Council under the European Union Horizon 2020 research and innovation program (ERC Advanced Grant PROTEUS Grant agreement no. 835297). C.D. is supported by EPSRC (EP/T003103/1), CRUK Accelerator (C10441/A29368) and Imperial EPSRC Impact Acceleration Account (EP/R511547/1). P.F. is supported by a CRUK MDA award (C7408/A28450). F.F. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2242), the UK Medical Research Council (CC2242), and the Wellcome Trust (CC2242). J.D. is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001070), the UK Medical Research Council (FC001070), and the Wellcome Trust (FC001070), the European Research Council (ERC Advanced RASImmune), and from a Wellcome Trust Senior Investigator Award 103799/Z/14/Z. Competing interests E.S. reports grants from Novartis, Merck Sharp Dohme, AstraZeneca and personal fees from Phenomic outside the submitted work. F.F. receives consulting fees from Deep Origin outside the submitted work. C.S reports grants and personal fees from Bristol Myers Squibb, AstraZeneca, BoehringerIngelheim, Roche-Ventana, personal fees from Pfizer, grants from Ono Pharmaceutical, Personalis, grants, personal fees, and other support from GRAIL, other support from AstraZeneca and GRAIL, personal fees and other support from Achilles Therapeutics, Bicycle Therapeutics, personal fees from Genentech, Medixci, China Innovation Centre of Roche (CiCoR) formerly Roche Innovation Centre, Metabomed, Relay Therapeutics, Saga Diagnostics, Sarah Canon Research Institute, Amgen, GlaxoSmithKline, Illumina, MSD, Novartis, other support from Apogen Biotechnologies and Epic Bioscience outside the submitted work; in addition, C.S. has a patent for PCT/ US2017/028013 licensed to Natera Inc, UCL Business, a patent for PCT/EP2016/059401 licensed to Cancer Research Technology, a patent for PCT/EP2016/071471 issued to Cancer Research Technology, a patent for PCT/GB2018/051912 pending, a patent for PCT/ GB2018/052004 issued to Francis Crick Institute, University College London, Cancer Research Technology Ltd, a patent for PCT/ GB2020/050221 issued to Francis Crick Institute, University College London, a patent for PCT/EP2022/077987 pending to Cancer Research Technology, a patent for PCT/GB2017/053289 licensed, a patent for PCT/EP2022/077987 pending to Francis Crick Institute, a patent for PCT/EP2023/059039 pending to Francis Crick Institute, and a patent for PCT/GB2018/051892 pending to Francis Crick Institute. C.S is Co-chief Investigator of NHS Galleri trial funded by GRAIL. He is Chief Investigator for the AstraZeneca MeRmaiD I and II clinical trials and Chair of the Steering Committee. C.S is cofounder of Achilles Therapeutics and holds stock options. M.M. is an employee and shareholder of AstraZeneca. C.D. has a granted patent on oblique plane microscopy (OPM) (PCT/GB2009/001802) licensed to Leica Microsystems and sublicensed to ASI, and has filed a patent application (PCT/GB2020/052279) on dual-view oblique plane microscopy (dOPM). J.D. reports grants from Bristol Myers Squibb, Revolution Medicines, Novartis, Vividion, AstraZeneca and personal fees from Jubilant, Theras, Roche, Curve Therapeutics.

We recently demonstrated polarisation differential phase contrast microscopy (pDPC) as a robust, low-cost single-shot implementation of (semi)quantitative phase imaging based on differential phase microscopy. pDPC utilises a polarisation-sensitive camera to simultaneously acquire four obliquely transilluminated images from which phase images mapping spatial variation of optical path difference can be calculated. pDPC microscopy can be implemented on existing or bespoke microscopes and can utilise radiation at a wide range of visible to near infrared wavelengths and so is straightforward to integrate with fluorescence microscopy. Here we present a low-cost open-source pDPC module that is designed for use with the modular open-source microscope stand “openFrame”. With improved hardware and software, this new pDPC implementation provides a real-time readout of phase across a field of view that facilitates optimisation of system alignment. We also provide protocols for background subtraction and correction of crosstalk

Optical projection tomography (OPT) is a three-dimensional mesoscopic imaging modality that can use absorption or fluorescence contrast, and is widely applied to fixed and live samples in the mm-cm scale. For fluorescence OPT, we present OPT implemented for accessibility and low cost, an open-source research-grade implementation of modular OPT hardware and software that has been designed to be widely accessible by using low-cost components, including light-emitting diode (LED) excitation and cooled complementary metal-oxide-semiconductor (CMOS) cameras. Both the hardware and software are modular and flexible in their implementation, enabling rapid switching between sample size scales and supporting compressive sensing to reconstruct images from undersampled sparse OPT data, e.g. to facilitate rapid imaging with low photobleaching/phototoxicity. We also explore a simple implementation of focal scanning OPT to achieve higher resolution, which entails the use of a fan-beam geometry reconstruction method to account for variation in magnification. This article is part of the Theo Murphy meeting issue 'Open, reproducible hardware for microscopy'.

Optical super-resolution microscope is a powerful tool for the life sciences, including cell biology and pathology yielding information on, e.g., biological structure and protein distribution in the nano-scale range. In recent years, there has been an exponential rise in the interest to apply super-resolution microscopes in diverse areas, but its wider utility is hindered by the cost of purchasing and maintaining super-resolved microscopes. In this paper we present the implementation of easySTORM, an accessible implementation of stochastic optical reconstruction microscopy (STORM) at Indian Institute of Technology Guwahati (IITG), India, in a flexible, user friendly and cost-effective manner using a state-of-the-art, modular, open-source, optical microscope platform called: "openFrame". Providing comparable imaging performance to commercial optical super-resolution microscopes, the openFrame-based implementation of easySTORM uses in-expensive multimode diode lasers and industry grade CMOS cameras, and the open-source and modular nature of the instrument makes it easy to maintain and to upgrade. To demonstrate its successful implementation at IITG, we image quantum dots and actin-tubulin structure in both normal and cancer cells, resolved features separated by a few tens of nanometers. This work demonstrates that openFrame-enabled easySTORM instrumentation can be widely accessible to provide affordable, research grade super-resolution microscopy capability for academic and medical research.

We present a single-shot adaptation of Optical Projection Tomography (OPT) for high-speed volumetric snapshot imaging of dynamic mesoscopic samples. Conventional OPT has been applied to in vivo imaging of animal models such as D. rerio but the sequential acquisition of projection images required for volumetric reconstruction typically requires samples to be immobilised during the acquisition of an OPT data set. We present a proof-of-principle system capable of single-shot imaging of a 1 mm diameter volume, demonstrating camera-limited rates of up to 62.5 volumes/second, which we have applied to 3D imaging of a freely-swimming zebrafish embryo. This is achieved by recording 8 projection views simultaneously on 4 low-cost CMOS cameras. With no stage required to rotate the sample, this single-shot OPT system can be implemented with a component cost of under £5,000. The system design can be adapted to different sized fields of view and may be applied to a broad range of dynamic samples, including fluid dynamics.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene ( Dmd ), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd , and its paralogue Utrn , in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd -deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd- mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.

'openFrame' is a modular, low-cost, open-hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open-source and proprietary hardware components or software enables low- cost, yet research- grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long-term time-lapse image data acquisition, slide-scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single-shot closed-loop focus lock, which we have demonstrated to accommodate defocus up to +/- 37 mu mwith <200 nm accuracy, and a two-step autofocus mode which we have shown can operate with defocus up to +/- 68 mu m. We have used this to implement automated single molecule localisation microscopy (SMLM) in a relatively low-cost openFrame-based instrument using multimode diode lasers for excitation and cooled CMOS cameras.

Lung cancer is a leading cause of cancer-related deaths. Despite enhanced characterization of genomic complexity and intra-tumor heterogeneity that underpin therapy failure, the mechanistic and predictive factors shaping and steering evolutionary dynamics remain less clear. In this study, we combined experimental, computational, and tumor molecular data analysis approach to investigate factors and rules governing evolutionary dynamics in lung cancer. A lineage tracing study in a BL/6 KRASG12D TP53mutant lung adenocarcinoma cell line model uncovers unexpected variation in the long-term fate of clones with no selective advantage or disadvantage, with clones arising near the edge of tumor colonies being favored to expand. An agent-based computational model of cell proliferation and cell-cell physical interaction, incorporating the experimentally driven assumption of compression-induced non-proliferation, recapitulated the growth patterns of tumor colonies and unequal expansion of clones. By further implementing cell random motility, the model predicts that enhanced motility destabilizes cell-cell cohesion and causes spatial clone intermixing with altered size distributions. Corroborating the observations in silico, experimental manipulations that increase the migratory capabilities of cancer cells relieve density-driven growth arrest and lead to clone intermixing in vitro. Furthermore, lung cancer cell lines with more migratory properties exhibit less propensity for suppression of proliferation. To relate findings in our models to tumor data and to infer the degree of clone intermixing in patients, we implemented a mutational process in the computational model and extracted features of variant allele frequency that enable to distinguish model runs with higher or lower degree of clone intermixing. Inference in lung adenocarcinomas in the TRACERx (TRAcking Cancer Evolution through therapy (Rx)) Lung study using these features, cross-referenced with RNA sequencing data in the same tumors, reveals notable correlation between mechanisms driving cell migration in our experimental models and inter-subclone mixing in clinical samples. Finally, modelling the competition between sensitive and mutagenesis-derived resistant clones under a virtual cytotoxic therapy predicts an accelerated selection of therapy resistant clones under conditions with enhanced cell motility. In summary, our integrated study demonstrates that intra-epithelia cell dynamics dictates the fates of clones both under neutral evolution and under selective pressures imposed by therapy. Citation Format: Xiao Fu, Ajay Bhargava, Sasha Bailey, Dhruva Biswas, Carlos Martinez Ruiz, Sunil Kumar, Paul French, Nicholas McGranahan, Charles Swanton, Paul A. Bates, Erik Sahai. Intra-epithelia cell dynamics shape evolutionary dynamics and selection of therapy resistant clones in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A040.

ABSTRACT We present a robust, “real-time” optical autofocus system for microscopy that provides high accuracy (<230 nm) and long range (∼130 µm) with a 1.4 numerical aperture oil immersion objective lens. This autofocus can operate in a closed loop, single-shot functionality over a range of ±37.5 µm and can also operate as a 2-step process up to ±68 µm. A real-time autofocus capability is useful for experiments with long image data acquisition times, including single molecule localization microscopy, that may be impacted by defocusing resulting from drift of components, e.g., due to changes in temperature or mechanical drift. It is also vital for automated slide scanning or multiwell plate imaging where the sample may not be in the same horizontal plane for every field of view during the image data acquisition. To realise high precision and long range, we implement orthogonal optical readouts using cylindrical lenses. We demonstrate the performance of this new optical autofocus system with automated multiwell plate imaging and single molecule localisation microscopy and illustrate the benefit of using a superluminescent diode as the autofocus light source.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region ( sDMR ). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.

Reduced nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolysis and oxidative metabolism and is thus recognized as a key biomarker for probing metabolic state. While the fluorescence characteristics of NADH have been investigated extensively, there are discrepancies in the published data due to diverse experimental conditions, instrumentation and microenvironmental parameters that can affect NADH fluorescence. Using a cuvette-based time-resolved spectrofluorimeter employing one-photon excitation at 375 nm, we characterized the fluorescence intensity, lifetime, spectral response, anisotropy and time-resolved anisotropy of NADH in aqueous solution under varying microenvironmental conditions, namely temperature, pH, and binding to lactate dehydrogenase (LDH). Our results demonstrate how temperature, pH, and binding partners each impact the fluorescence signature of NADH and highlight the complexity of the fluorescence data when different parameters produce competing effects. We hope that the data presented in this study will provide a reference for potential sources of variation in experiments measuring NADH fluorescence.

The success of research institutions heavily relies upon identifying the right researchers "for the job": researchers may need to identify appropriate collaborators, often from across disciplines; students may need to identify suitable supervisors for projects of their interest; administrators may need to match funding opportunities with relevant researchers, and so on. Usually, finding potential collaborators in institutions is a time-consuming manual search task prone to bias. In this paper, we propose a novel query-based framework for searching, scoring, and exploring research expertise automatically, based upon processing abstracts of academic publications. Given user queries in natural language, our framework finds researchers with relevant expertise, making use of domain-specific knowledge bases and word embeddings. It also generates explanations for its recommendations. We evaluate our framework with an institutional repository of papers from a leading university, using, as baselines, artificial neural networks and transformer-based models for a multilabel classification task to identify authors of publication abstracts. We also assess the cross-domain effectiveness of our framework with a (separate) research funding repository for the same institution. We show that our simple method is effective in identifying matches, while satisfying desirable properties and being efficient.