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.