Two-photon synthetic aperture microscopy for minimally invasive fast 3D imaging of native subcellular behaviors in deep tissue.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.

Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale.

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.

Harnessing artificial intelligence to reduce phototoxicity in live imaging.

Fluorescence microscopy is essential for studying living cells, tissues and organisms. However, the fluorescent light that switches on fluorescent molecules also harms the samples, jeopardizing the validity of results - particularly in techniques such as super-resolution microscopy, which demands extended illumination. Artificial intelligence (AI)-enabled software capable of denoising, image restoration, temporal interpolation or cross-modal style transfer has great potential to rescue live imaging data and limit photodamage. Yet we believe the focus should be on maintaining light-induced damage at levels that preserve natural cell behaviour. In this Opinion piece, we argue that a shift in role for AIs is needed - AI should be used to extract rich insights from gentle imaging rather than recover compromised data from harsh illumination. Although AI can enhance imaging, our ultimate goal should be to uncover biological truths, not just retrieve data. It is essential to prioritize minimizing photodamage over merely pushing technical limits. Our approach is aimed towards gentle acquisition and observation of undisturbed living systems, aligning with the essence of live-cell fluorescence microscopy.

New approaches for low phototoxicity imaging of living cells and tissues.

Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Phototoxicity of cyclometallated Ir(III) complexes bearing a thio-bis-benzimidazole ligand, and its monodentate analogue, as potential PDT photosensitisers in cancer cell killing.

Two novel cyclometallated iridium(III) complexes have been prepared with one bidentate or two monodentate imidazole-based ligands, 1 and 2, respectively. The complexes showed intense emission with long lifetimes of the excited state. Femtosecond transient absorption experiments established the nature of the lowest excited state as 3IL state. Singlet oxygen generation with good yields (40% for 1 and 82% for 2) was established by detecting 1O2 directly, through its emission at 1270 nm. Photostability studies were also performed to assess the viability of the complexes as photosensitizers (PS) for photodynamic therapy (PDT). Complex 1 was selected as a good candidate to investigate light-activated killing of cells, whilst complex 2 was found to be toxic in the dark and unstable under light. Complex 1 demonstrated high phototoxicity indexes (PI) in the visible region, PI > 250 after irradiation at 405 nm and PI > 150 at 455 nm, in EJ bladder cancer cells.

Comparison of two biological systems used for phototoxicity testing: Cellular and tissue.

The OECD has approved two similar methods for testing the phototoxic potency of chemicals. The first method, OECD 432, is based on the cytotoxicity properties of materials to the mouse 3T3 (clone A31) cell line (fibroblasts) after exposure to light. The second method, OECD 498, is based on the same properties but using reconstructed human epidermis - EpiDerm (stratified keratinocytes). The aim of this study was to compare these two methods using statistical tests (specificity, sensitivity, negative predictive value, positive predictive value and accuracy) and non-statistical characteristics (e.g. price and experimental duration, amount of material, level of complications, cell type, irradiation dose). Both tests were performed according to the relevant guidelines using the same 11 control substances. Higher performance values were observed for OECD 432 in both phototoxic and non-phototoxic classifications. The accuracy of OECD 432 was 90.9%, while that of OECD 498 was 72.7%. OECD 432 was also shorter and less expensive. On the other hand, OECD 498 was less complicated, and used human cells with stratum corneum, which better reflects real skin. This method can also be used with oily substances that are poorly soluble in water. However, both methods are important for testing the phototoxic properties of materials, and can be used alone or in a tiered strategy.

Platinum(II)-Salphen Complexes as DNA Binders and Photosensitisers.

Current anticancer therapies suffer from issues such as off-target side effects and the emergence of drug resistance; therefore, the discovery of alternative therapeutic approaches is vital. These can include the development of drugs with different modes of action, and the exploration of new biomolecular targets. For the former, there has been increasing interest in drugs that are activated by an external stimulus to generate cytotoxic species. For the latter, significant efforts are being directed to explore non-canonical DNA and RNA structures (e.g. guanine-quadruplexes), as alternative biomolecular targets. Herein we report the synthesis of a library of 21 new platinum(II)-Salphen complexes, investigation of their photophysical and photochemical properties, their interactions with duplex and quadruplex DNA, and their cytotoxicity against HeLa cancer cells in the dark and upon light irradiation. Thanks to the intrinsic phosphorescence of the platinum(II) complexes, confocal microscopy was used for six of the complexes to determine their cellular permeability and localisation in two cancer cell lines. These studies have allowed us to identify two lead platinum(II) complexes with high guanine-quadruplex DNA affinity and selectivity, good cell permeability and nuclear localisation, and high cytotoxicity against HeLa cancer cells upon irradiation with no detected cytotoxicity in the dark.

Fabrication of Carbon Dots with Singlet Oxygen Generation and Their Potential Photodynamic Therapy Applications.

Due to the unique chemical and biomedical properties of carbon dots (CDs), they have increasingly obtained the attention in many research fields, for example, bioimaging, fluorescence sensing, and drug delivery, etc. Recently, it was found that, under light excitation, CDs can also be exploited as a novel photosensitizer to prepare reactive oxygen species (ROS), which expand their applications in the field of photodynamic therapy for cancer treatment. Nevertheless, the high cost and complex fabrication approach of CDs significantly limit their applications. To address this issue, bottom-up routes usually utilize sustainable and inexpensive carbon precursor as starting materials, employed N,N-dimethylformamide (DMF) or ethanol as an environmental-friendly solvent. Bottom-up approach was energy efficient, and the purification process was relatively simple by dialysis. Therefore, carbon dots (CDs) were facilely fabricated in a one-pot solvothermal process using 1-aminoanthraquinone as a precursor, and their application as photosensitizers for in vitro antitumor cells, especially photodynamic therapy (PDT) was established. Then the photophysical and nanoscale dimensions properties of the fabricated CDs were characterized via TEM, UV-visible, fluorescence, and FT-IR spectroscopy. The synthesized N-doped CDs can easily dissolve in water, possess very low biotoxicity, yellow-light emission (maximum peak at 587 nm). More importantly, PDT studies demonstrated that the obtained CDs possess a high singlet oxygen yield of 35%, and exhibit significant phototoxicity to cancer cells upon 635 nm laser irradiation. These studies highlight that N-doped CDs can be facilely synthesized from only one precursor, and are a potentially novel theranostic agent for in vivo PDT.

A Review of Phototoxic Plants, Their Phototoxic Metabolites, and Possible Developments as Photosensitizers.

This study provides a comprehensive overview of the current knowledge regarding phototoxic terrestrial plants and their phototoxic and photosensitizing metabolites. Within the 435,000 land plant species, only around 250 vascular plants have been documented as phototoxic or implicated in phototoxic occurrences in humans and animals. This work compiles a comprehensive catalog of these phototoxic plant species, organized alphabetically based on their taxonomic family. The dataset encompasses meticulous details including taxonomy, geographical distribution, vernacular names, and information on the nature and structure of their phototoxic and photosensitizing molecule(s). Subsequently, this study undertook an in-depth investigation into phototoxic molecules, resulting in the compilation of a comprehensive and up-to-date list of phytochemicals exhibiting phototoxic or photosensitizing activity synthesized by terrestrial plants. For each identified molecule, an extensive review was conducted, encompassing discussions on its phototoxic activity, chemical family, occurrence in plant families or species, distribution within different plant tissues and organs, as well as the biogeographical locations of the producer species worldwide. The analysis also includes a thorough discussion on the potential use of these molecules for the development of new photosensitizers that could be used in topical or injectable formulations for antimicrobial and anticancer phototherapy as well as manufacturing of photoactive devices.

Toxic potential assessment of hair dye developer 2,4,5,6-tetraaminopyrimidine sulfate exposed under ambient UVB radiation.

Synthetic cosmetics, particularly hair dyes, are becoming increasingly popular among people of all ages and genders. 2,4,5,6-tetraaminopyrimidine sulfate (TAPS) is a key component of oxidative hair dyes and is used as a developer in several hair dyes. TAPS has previously been shown to absorb UVB strongly and degrade in a time-dependent manner, causing phototoxicity in human skin cells. However, the toxic effects of UVB-degraded TAPS are not explored in comparison to parent TAPS. Therefore, this research work aims to assess the toxicity of UVB-degraded TAPS than TAPS on two different test systems, that is, HaCaT (mammalian cell) and Staphylococcus aureus (a bacterial cell). Our result on HaCaT has illustrated that UVB-degraded TAPS is less toxic than parent TAPS. Additionally, UVB-exposed TAPS and parent TAPS were given to S. aureus, and the bacterial growth and their metabolic activity were assessed via CFU and phenotype microarray. The findings demonstrated that parent TAPS reduced bacterial growth via decreased metabolic activity; however, bacteria easily utilized the degraded TAPS. Thus, this study suggests that the products generated after UVB irradiation of TAPS is considered to be safer than their parent TAPS.

In Silico Phototoxicity Prediction of Drugs and Chemicals by using Derek Nexus and QSAR Toolbox.

Phototoxicity testing is crucial for evaluating the potential harmful effects of pharmaceuticals and chemicals on human skin when exposed to sunlight. Traditional in vivo models involving mice, rats, guinea pigs, as well as in vitro assays such as the 3T3 Neutral Red Uptake phototoxicity assay and methods based on the use of reconstructed human epidermis, have been established for phototoxicity testing. While these approaches are extremely valuable, they are costly in terms of both time and resources. Consequently, in silico approaches based on the use of predictive software tools can offer more rapid and cost-effective phototoxicity screening solutions. With this goal in mind, the current study evaluated two in silico tools - Derek Nexus 6.1.0/Derek Knowledge Base 2020 1.0 (Lhasa Limited, UK) and the QSAR Toolbox (v 4.5) developed by the Organisation for Economic Co-operation and Development (OECD) - for their capacity to predict the phototoxicity of several substances from diverse classes. Derek Nexus and the QSAR Toolbox were both found to be very useful for predicting the phototoxicity of drugs and other chemicals. Derek Nexus predicted phototoxicity of the compounds, with a sensitivity of 63%, specificity of 93%, Positive Predictive Values of 90% and Negative Predictive Value of 69%, overall accuracy of 77% and balanced accuracy of 78%. The QSAR Toolbox achieved sensitivity of 73%, specificity of 85%, Positive Predictive Value of 85% and Negative Predictive Value of 74%, overall accuracy of 79% and balanced accuracy of 79%. The results show that Derek Nexus and the QSAR Toolbox can be usefully incorporated in the workflow of phototoxicity testing for pharmaceuticals and chemicals.

Development of Sensitive In Vitro Protocols for the Biocompatibility Testing of Medical Devices and Pharmaceuticals Intended for Contact with the Eyes: Acute Irritation and Phototoxicity Assessment.

This study introduces a novel in vitro methodology that employs the 3-D reconstructed tissue model, EpiOcular, to assess the irritation and phototoxicity potential of medical devices and drugs in contact with the eye. Our study evaluated diverse test materials, including medical devices, ophthalmological solutions and an experimental drug (cemtirestat), for their potential to cause eye irritation and phototoxicity. The protocols used in this study with the EpiOcular tissue model were akin to those used in the ultra-mildness testing of cosmetic formulations, which is challenging to predict with standard in vivo rabbit tests. To design these protocols, we leveraged experience gained from the validation project on the EpiDerm skin irritation test for medical devices (ISO 10993-23:2021) and the OECD TG 498 method for photo-irritation testing. The predictions were based on the tissue viability and inflammatory response, as determined by IL-1α release. By developing and evaluating these protocols for medical devices, we aimed to expand the applicability domain of the tests referred to in ISO 10993-23. This will contribute to the standardisation and cost-effective safety evaluation of ophthalmic products, while reducing reliance on animal testing in this field. The findings obtained from the EpiOcular model in the photo-irritation test could support its implementation in the testing strategies outlined in OECD TG 498.

Phototoxic Reactions Inducted by Hydrochlorothiazide and Furosemide in Normal Skin Cells-In Vitro Studies on Melanocytes and Fibroblasts.

Hypertension is known to be a multifactorial disease associated with abnormalities in neuroendocrine, metabolic, and hemodynamic systems. Poorly controlled hypertension causes more than one in eight premature deaths worldwide. Hydrochlorothiazide (HCT) and furosemide (FUR), being first-line drugs in the treatment of hypertension, are among others the most frequently prescribed drugs in the world. Currently, many pharmacoepidemiological data associate the use of these diuretics with an increased risk of adverse phototoxic reactions that may induce the development of melanoma and non-melanoma skin cancers. In this study, the cytotoxic and phototoxic potential of HCT and FUR against skin cells varied by melanin pigment content was assessed for the first time. The results showed that both drugs reduced the number of metabolically active normal skin cells in a dose-dependent manner. UVA irradiation significantly increased the cytotoxicity of HCT towards fibroblasts by approximately 40% and melanocytes by almost 20% compared to unirradiated cells. In the case of skin cells exposed to FUR and UVA radiation, an increase in cytotoxicity by approximately 30% for fibroblasts and 10% for melanocytes was observed. Simultaneous exposure of melanocytes and fibroblasts to HCT or FUR and UVAR caused a decrease in cell viability, and number, which was confirmed by microscopic assessment of morphology. The phototoxic effect of HCT and FUR was associated with the disturbance of redox homeostasis confirming the oxidative stress as a mechanism of phototoxic reaction. UVA-irradiated drugs increased the generation of ROS by 10-150%, and oxidized intracellular thiols. A reduction in mitochondrial potential of almost 80% in melanocytes exposed to HCT and UVAR and 60% in fibroblasts was found due to oxidative stress occurrence. In addition, HCT and FUR have been shown to disrupt the cell cycle of normal skin cells. Finally, it can be concluded that HCT is the drug with a stronger phototoxic effect, and fibroblasts turn out to be more sensitive cells to the phototoxic effect of tested drugs.

Current Advances in Photodynamic Therapy (PDT) and the Future Potential of PDT-Combinatorial Cancer Therapies.

Photodynamic therapy (PDT) is a two-stage treatment that implies the use of light energy, oxygen, and light-activated compounds (photosensitizers) to elicit cancerous and precancerous cell death after light activation (phototoxicity). The biophysical, bioengineering aspects and its combinations with other strategies are highlighted in this review, both conceptually and as they are currently applied clinically. We further explore the recent advancements of PDT with the use of nanotechnology, including quantum dots as innovative photosensitizers or energy donors as well as the combination of PDT with radiotherapy and immunotherapy as future promising cancer treatments. Finally, we emphasize the potential significance of organoids as physiologically relevant models for PDT.

Targeting Mitochondrial Guanine Quadruplexes for Photoactivatable Chemotherapy in Normoxic and Hypoxic Environments.

A first example of a mitochondrial G-quadruplex (mitoG4s) targeted Ru(II) photooxidant complex is reported. The complex, Ru-TAP-PDC3 induces photodamage toward guanine quadruplexes (G4s) located in the mitochondrial genome under hypoxic and normoxic conditions. Ru-TAP-PDC3 shows high affinity for mitoG4s and localises within mitochondria of live HeLa cells. Immunolabelling with anti-G4 antibody, BG4, confirms Ru-TAP-PDC3 associates with G4s within the mitochondria of fixed cells. The complex induces depletion of mtDNA in live cells under irradiation at 405 nm, confirmed by loss of PicoGreen signal from mitochondria. Biochemical studies confirm this process induces apoptosis. The complex shows low dark toxicity and an impressive phototoxicity index (PI) of >89 was determined in Hela under very low intensity irradiation, 5 J/cm2. The phototoxicity is thought to operate through both Type II singlet oxygen and Type III pathways depending on normoxic or hypoxic conditions from live cell imaging and plasmid DNA cleavage. Overall, we demonstrate targeting mitoG4s and mtDNA with a photooxidant is a potent route to achieving apoptosis under hypoxic conditions that can be extended to phototherapy.

A Semi-Automated, High-Throughput Approach for the Synthesis and Identification of Highly Photo-Cytotoxic Iridium Complexes.

The discovery of new compounds with pharmacological properties is usually a lengthy, laborious and expensive process. Thus, there is increasing interest in developing workflows that allow for the rapid synthesis and evaluation of libraries of compounds with the aim of identifying leads for further drug development. Herein, we apply combinatorial synthesis to build a library of 90 iridium(III) complexes (81 of which are new) over two synthesise-and-test cycles, with the aim of identifying potential agents for photodynamic therapy. We demonstrate the power of this approach by identifying highly active complexes that are well-tolerated in the dark but display very low nM phototoxicity against cancer cells. To build a detailed structure-activity relationship for this class of compounds we have used density functional theory (DFT) calculations to determine some key electronic parameters and study correlations with the experimental data. Finally, we present an optimised semi-automated synthesise-and-test protocol to obtain multiplex data within 72 hours.

Nutraceutical strategies for alleviation of UVB phototoxicity.

Ultraviolet B exposure to keratinocytes promotes carcinogenesis by inducing pyrimidine dimer lesions in DNA, suppressing the nucleotide excision repair mechanism required to fix them, inhibiting the apoptosis required for the elimination of initiated cells, and driving cellular proliferation. Certain nutraceuticals - most prominently spirulina, soy isoflavones, long-chain omega-3 fatty acids, the green tea catechin epigallocatechin gallate (EGCG) and Polypodium leucotomos extract - have been shown to oppose photocarcinogenesis, as well as sunburn and photoaging, in UVB-exposed hairless mice. It is proposed that spirulina provides protection in this regard via phycocyanobilin-mediated inhibition of Nox1-dependent NADPH oxidase; that soy isoflavones do so by opposing NF-κB transcriptional activity via oestrogen receptor-beta; that the benefit of eicosapentaenoic acid reflects decreased production of prostaglandin E2; and that EGCG counters UVB-mediated phototoxicity via inhibition of the epidermal growth factor receptor. The prospects for practical nutraceutical down-regulation of photocarcinogenesis, sunburn, and photoaging appear favourable.

Photochemical defense as trait of fungi from Cortinarius subgenus Dermocybe.

The photobiological activity of ten colorful species belonging to subgenus Dermocybe of the basidiomycete genus Cortinarius was investigated. Extracts of all species produced singlet oxygen and are thus photoactive. Pigment analysis was performed and showed similarities of the anthraquinone pigments across the species in dependency to their respective pigmentation types. Detailed content analysis of the pigments in the whole agaricoid fruiting body compared to the three different tissue types (pileus, stipe, and lamellae) revealed that the pigments emodin, dermocybin, and dermorubin, as well as their respective glycosides, are enhanced in the gills. In an independent experiment, the gills were shown to be the most photoactive tissues of the fruiting body. Photobiological experiments with invertebrates (i.e., glassworm Chaoborus crystallinus) proved a phototoxic effect of the methanolic extract of the red blood webcap (Cortinarius sanguineus var. aurantiovaginatus). This work adds further evidence to a common photobiological trait in Cortinarius subgenus Dermocybe and underpins the possibility of a photochemical defense mechanism in fungi.

Photosensitizing properties and subcellular localisation of 3,4-dihydro-ß-carbolines harmaline and harmalol.

Harmaline (1) and harmalol (2) represent two 3,4-dihydro-ß-carboline (DHßCs) most frequently reported in a vast number of living systems. Fundamental aspects including the photosensitizing properties, cellular uptake, as well as the cyto- and phototoxicity of 1 and 2 were investigated herein. The molecular basis underlying the investigated processes are elucidated. Data reveal that both alkaloids show a distinctive pattern of extracellular DNA photodamage. Compound 1 induces a DNA photodamage profile dominated by oxidised purines and sites of base loss (AP sites), whereas 2 mostly induces single-strand breaks (SSBs) in addition to a small extent of purine oxidative damage. In both cases, DNA oxidative damage would occur through type I mechanism. In addition, a concerted hydrolytic attack is suggested as an extra mechanism accounting for the SSBs formation photoinduced by 2. Subcellular internalisation, cyto- and phototoxicity of 1 and 2 and the corresponding full-aromatic derivatives harmine (3) and harmol (4) also showed quite distinctive patterns in a structure-dependent manner. These results are discussed in the framework of the potential biological, biomedical and/or pharmacological roles reported for these alkaloids. The subtle structural difference (i.e., the exchange of a methoxy group for a hydroxyl substituent at C(7)) between harmaline and harmalol, gives rise to distinctive photosensitizing and subcellular localisation patterns.