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Basal cell carcinoma (BCC) is a common type of skin cancer. Conventional approaches to BCC diagnosis often involve invasive histological examinations that can distort or even destroy information derived from the biomolecules in the sample. Therefore, a non-invasive, label-free examination method for the clinical diagnosis of BCC represents a critical advance. This study combined spectral- and time-resolved two-photon microscopy with a spectral phasor to extract rich biochemical information describing macroscopic tumor morphology and microscopic tumor metabolism. The proposed optical imaging technique achieved the rapid and efficient separation of tumor structures in systematic research conducted on normal and BCC human skin tissues. The results demonstrate that a combination of multidimensional data (e.g., fluorescence intensity, spectrum, and lifetime) with a spectral phasor can accurately identify tumor boundaries and achieve rapid separation. This label-free, real-time, multidimensional imaging technique serves as a complement to the conventional tumor diagnostic toolbox and demonstrates significant potential for the early diagnosis of BCC and wider applications in intraoperative auxiliary imaging.

Cadmium-free quantum-dot (QD) fluorophores can bridge the gap between the macroscopic and microscopic domains in fluorescence super-resolution bioimaging. InP/ZnSe/ZnS QD photoblinking fluorescent probes can improve the performance of reactive super-resolution imaging techniques and spontaneously switch fluorophores between at least two states (open and close) without depending on intense laser light and specialized buffers for bioimaging. Multifocal structured illumination microscopy (MSIM) provides a two-fold resolution enhancement in sub-diffraction imaging, but higher resolutions are limited by the pattern frequency and signal-to-noise ratio. We exploit the synergy between MSIM and spontaneously switching InP/ZnSe/ZnS QD fluorophores to further increase the imaging resolution. We demonstrate the experimental combination of optical-fluctuation-enhanced super-resolution MSIM using ultrasonic-oscillation-assisted organic solvothermal synthesis of narrowband photoblinking InP/ZnSe/ZnS QDs. The InP/ZnSe/ZnS QDs show a monodisperse grain size of approximately 9 nm, fluorescence quantum yields close to 100%, and full width at half maximum below 30 nm. The structural, electronic, and optical properties are characterized through experiments and first-principles calculations. The enhanced MSIM imaging achieves an approximate fourfold improvement in resolution for fixed cells compared with widefield imaging. The proposed InP/ZnSe/ZnS QD fluorescent probes seem promising for super-resolution imaging using MSIM.

In this Letter, we propose a novel triplex-parameter detection method to realize simultaneous radiometric, photoacoustic, and ultrasonic imaging based on single-pulse excitation. The optical attenuation, optical absorption, and acoustic impedance properties can be obtained simultaneously by analyzing the photoacoustic signals and the ultrasonic echo signals. To test the feasibility and accuracy of this method, agar phantoms with different absorption coefficients and elastic coefficients were measured. Then, this method was experimentally verified by imaging a leaf skeleton piece embedded in an agar cylinder. Furthermore, pilot experiments were performed by triplex imaging of pig ear tissue ex vivo to characterize the cartilage and surrounding tissue. Experimental results demonstrated that this technique has future potentials for visualizing and providing the functional and structural information of biological tissues.

Photodynamic therapy (PDT) for hypoxic tumors has attracted wide attention owing to its noninvasiveness, easy maneuverability, and instantaneity. However, hypoxia in tumors and penetration depth of conventional ultraviolet light has greatly weakened its performance. To solve these problems, under NIR-II light irradiation, squaric acid nanoparticles (SQ NPs) with superior reactive oxygen, especially, hydroxyl radicals (•OH) production performance were first utilized for hypoxic tumor therapy. SQ NPs with intense light capture capability, intense NIR emission, and excellent photobleaching resistance show continuous •OH generation capabilities under NIR-II laser excitation. Through the superior PDT performance, the growth of hypoxic tumors was effectively inhibited, and the survival rate of mice was improved. This work highlights the application of NIR-II photoexcitation in deep tissue type I photodynamic therapy of hypoxic tumors, which will facilitate the development of hypoxic tumor PDT in deep depth.

Research on dynamic events in living cells, such as intracellular transportation, is important for understanding cell functions. As movements occur within cells, the microenvironment of the moving vesicles or biomacromolecules may affect the behavior of them. Herein, we propose a method of simultaneously monitoring changes in spatial positions and the local environment related to the fluorescence lifetime, i.e., four-dimensional (4D) multi-particle parallel-tracking in living cells. Based on double-helix point spread function (DH-PSF) microscopy and streak camera, the method combines three-dimensional (3D) localization methods and fluorescence lifetime imaging. By modifying the PSF of the system, the 3D positions and fluorescence lifetime information for several molecules within a depth of a few microns can be acquired simultaneously from a single snapshot. The feasibility of this method is verified by simulating the real-time tracking of a single particle with a given trajectory. In addition, a proof-of-concept 4D tracking system based on the DH-PSF and streak camera was built. The experimental results show that the 3D localization and lifetime precision are σ(x, y, z) = (26 nm, 35 nm, 53 nm) and σ(τ) = 103 ps, respectively, and the effective depth of field is approximately 4 μm. Finally, intracellular endocytosis in a living cell was observed using the system, which demonstrated the successful 4D tracking of two microspheres moving within an axial depth of 4 μm. This work opens a new perspective for research of dynamic processes, by providing information about the chemical (microenvironments) and physical (positions) changes of moving targets in living cells.