Spatiotemporally mapping temperature dynamics of lysosomes and mitochondria using cascade organelle-targeting upconversion nanoparticles.

The intracellular metabolism of organelles, like lysosomes and mitochondria, is highly coordinated spatiotemporally and functionally. The activities of lysosomal enzymes significantly rely on the cytoplasmic temperature, and heat is constantly released by mitochondria as the byproduct of adenosine triphosphate (ATP) generation during active metabolism. Here, we developed temperature-sensitive LysoDots and MitoDots to monitor the in situ thermal dynamics of lysosomes and mitochondria. The design is based on upconversion nanoparticles (UCNPs) with high-density surface modifications to achieve the exceptionally high sensitivity of 2.7% K-1 and low uncertainty of 0.8 K for nanothermometry to be used in living cells. We show the measurement is independent of the ion concentrations and pH values. With Ca2+ ion shock, the temperatures of both lysosomes and mitochondria increased by ∼2 to 4 °C. Intriguingly, with chloroquine (CQ) treatment, the lysosomal temperature was observed to decrease by up to ∼3 °C, while mitochondria remained relatively stable. Lastly, with oxidative phosphorylation inhibitor treatment, we observed an ∼3 to 7 °C temperature increase and a thermal transition from mitochondria to lysosomes. These observations indicate different metabolic pathways and thermal transitions between lysosomes and mitochondria inside HeLa cells. The nanothermometry probes provide a powerful tool for multimodality functional imaging of subcellular organelles and interactions with high spatial, temporal, and thermal dynamics resolutions.

Probing the Nanonewton Mitotic Cell Deformation Force by Ion-Resonance-Enhanced Photonics Force Microscopy.

Mechanical forces are essential for regulating dynamic changes in cellular activities. A comprehensive understanding of these forces is imperative for unraveling fundamental mechanisms. Here, we develop a microprobe capable of facilitating the measurement of biological forces up to nanonewton levels in living cells. This probe is designed by coating the core of anatase titania particles with amorphous titania and silica shells and an upconversion nanoparticles (UCNPs) layer. Leveraging both antireflection and ion resonance effects from the shells, the optically trapped probe attains a maximum lateral optical trap stiffness of 14.24 pN µm-1 mW-1, surpassing the best reported value by a factor of 3. Employing this advanced probe in a photonic force microscope, we determine the elasticity modulus of mitotic HeLa cells as 1.27 ± 0.3 kPa. Nanonewton probes offer the potential to explore 3D cellular mechanics with unparalleled precision and spatial resolution, fostering a deeper understanding of the underlying biomechanical mechanisms.

Exploiting Dynamic Nonlinearity in Upconversion Nanoparticles for Super-Resolution Imaging.

Single-beam super-resolution microscopy, also known as superlinear microscopy, exploits the nonlinear response of fluorescent probes in confocal microscopy. The technique requires no complex purpose-built system, light field modulation, or beam shaping. Here, we present a strategy to enhance this technique's spatial resolution by modulating excitation intensity during image acquisition. This modulation induces dynamic optical nonlinearity in upconversion nanoparticles (UCNPs), resulting in variations of nonlinear fluorescence response in the obtained images. The higher orders of fluorescence response can be extracted with a proposed weighted finite difference imaging algorithm from raw fluorescence images to generate an image with higher resolution than superlinear microscopy images. We apply this approach to resolve single nanoparticles in a large area, improving the resolution to 132 nm. This work suggests a new scope for the development of dynamic nonlinear fluorescent probes in super-resolution nanoscopy.

Quantitatively Monitoring In Situ Mitochondrial Thermal Dynamics by Upconversion Nanoparticles.

Temperature dynamics reflect the physiological conditions of cells and organisms. Mitochondria regulate the temperature dynamics in living cells as they oxidize the respiratory substrates and synthesize ATP, with heat being released as a byproduct of active metabolism. Here, we report an upconversion nanoparticle-based thermometer that allows the in situ thermal dynamics monitoring of mitochondria in living cells. We demonstrate that the upconversion nanothermometers can efficiently target mitochondria, and the temperature-responsive feature is independent of probe concentration and medium conditions. The relative sensing sensitivity of 3.2% K-1 in HeLa cells allows us to measure the mitochondrial temperature difference through the stimulations of high glucose, lipid, Ca2+ shock, and the inhibitor of oxidative phosphorylation. Moreover, cells display distinct response time and thermodynamic profiles under different stimulations, which highlight the potential applications of this thermometer to study in situ vital processes related to mitochondrial metabolism pathways and interactions between organelles.

Upconversion Nonlinear Structured Illumination Microscopy.

Video-rate super-resolution imaging through biological tissue can visualize and track biomolecule interplays and transportations inside cellular organisms. Structured illumination microscopy allows for wide-field super resolution observation of biological samples but is limited by the strong extinction of light by biological tissues, which restricts the imaging depth and degrades its imaging resolution. Here we report a photon upconversion scheme using lanthanide-doped nanoparticles for wide-field super-resolution imaging through the biological transparent window, featured by near-infrared and low-irradiance nonlinear structured illumination. We demonstrate that the 976 nm excitation and 800 nm upconverted emission can mitigate the aberration. We found that the nonlinear response of upconversion emissions from single nanoparticles can effectively generate the required high spatial frequency components in the Fourier domain. These strategies lead to a new modality in microscopy with a resolution below 131 nm, 1/7th of the excitation wavelength, and an imaging rate of 1 Hz.

Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.

Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiological and pathological processes. Mutations in mtDNA cause metabolic diseases and aging. The mtDNA within the human cells are packaged into hundreds of nucleoids within the mitochondrial matrix. Knowledge of how the nucleoids are dynamically distributed and organized within mitochondria is key to understanding mtDNA structure and functions. Therefore, visualizing the distribution and dynamics of mtDNA within mitochondria is a powerful approach to gain insights into the regulation of mtDNA replication and transcription. In this chapter, we describe the methods of observing mtDNA and its replication with fluorescence microscopy in both fixed and live cells using different labeling strategies.

Cortical tension drug screen links mitotic spindle integrity to Rho pathway.

Mechanical force generation plays an essential role in many cellular functions, including mitosis. Actomyosin contractile forces mediate changes in cell shape in mitosis and are implicated in mitotic spindle integrity via cortical tension. An unbiased screen of 150 small molecules that impact actin organization and 32 anti-mitotic drugs identified two molecular targets, Rho kinase (ROCK) and tropomyosin 3.1/2 (Tpm3.1/2), whose inhibition has the greatest impact on mitotic cortical tension. The converse was found for compounds that depolymerize microtubules. Tpm3.1/2 forms a co-polymer with mitotic cortical actin filaments, and its inhibition prevents rescue of multipolar spindles induced by anti-microtubule chemotherapeutics. We examined the role of mitotic cortical tension in this rescue mechanism. Inhibition of ROCK and Tpm3.1/2 and knockdown (KD) of cortical nonmuscle myosin 2A (NM2A), all of which reduce cortical tension, inhibited rescue of multipolar mitotic spindles, further implicating cortical tension in the rescue mechanism. GEF-H1 released from microtubules by depolymerization increased cortical tension through the RhoA pathway, and its KD also inhibited rescue of multipolar mitotic spindles. We conclude that microtubule depolymerization by anti-cancer drugs induces cortical-tension-based rescue to ensure integrity of the mitotic bipolar spindle mediated via the RhoA pathway. Central to this mechanism is the dependence of NM2A on Tpm3.1/2 to produce the functional engagement of actin filaments responsible for cortical tension.

Lanthanide Ion Resonance-Driven Rayleigh Scattering of Nanoparticles for Dual-Modality Interferometric Scattering Microscopy.

Light scattering from nanoparticles is significant in nanoscale imaging, photon confinement. and biosensing. However, engineering the scattering spectrum, traditionally by modifying the geometric feature of particles, requires synthesis and fabrication with nanometre accuracy. Here it is reported that doping lanthanide ions can engineer the scattering properties of low-refractive-index nanoparticles. When the excitation wavelength matches the ion resonance frequency of lanthanide ions, the polarizability and the resulted scattering cross-section of nanoparticles are dramatically enhanced. It is demonstrated that these purposely engineered nanoparticles can be used for interferometric scattering (iSCAT) microscopy. Conceptually, a dual-modality iSCAT microscopy is further developed to identify different nanoparticle types in living HeLa cells. The work provides insight into engineering the scattering features by doping elements in nanomaterials, further inspiring exploration of the geometry-independent scattering modulation strategy.

Studying Mitochondrial Network Formation by In Vivo and In Vitro Reconstitution Assay.

Mitochondria change their morphologies from small isolated vesicles to large continuous networks across the cell cycles. The mitochondrial network formation (MNF) plays an important role in maintaining mitochondrial DNA integrity and interchanging mitochondrial materials. The disruption of the mitochondrial network affects mitochondrial functions, such as ATP production, integration of metabolism, calcium homeostasis, and regulation of apoptosis, leading to the abnormal development and several human diseases including neurodegenerative disease. In this unit, we describe the method of studying MNF, which is driven by microtubule-dependent motor protein, by in vivo imaging and single-molecule in vitro reconstitution assays.

Stratified Disk Microrobots with Dynamic Maneuverability and Proton-Activatable Luminescence for in Vivo Imaging.

Microrobots can expand our abilities to access remote, confined, and enclosed spaces. Their potential applications inside our body are obvious, e.g., to diagnose diseases, deliver medicine, and monitor treatment efficacy. However, critical requirements exist in relation to their operations in gastrointestinal environments, including resistance to strong gastric acid, responsivity to a narrow proton variation window, and locomotion in confined cavities with hierarchical terrains. Here, we report a proton-activatable microrobot to enable real-time, repeated, and site-selective pH sensing and monitoring in physiological relevant environments. This is achieved by stratifying a hydrogel disk to combine a range of functional nanomaterials, including proton-responsive molecular switches, upconversion nanoparticles, and near-infrared (NIR) emitters. By leveraging the 3D magnetic gradient fields and the anisotropic composition, the microrobot can be steered to locomote as a gyrating "Euler's disk", i.e., aslant relative to the surface and along its low-friction outer circumference, exhibiting a high motility of up to 60 body lengths/s. The enhanced magnetomotility can boost the pH-sensing kinetics by 2-fold. The fluorescence of the molecular switch can respond to pH variations with over 600-fold enhancement when the pH decreases from 8 to 1, and the integration of upconversion nanoparticles further allows both the efficient sensitization of NIR light through deep tissue and energy transfer to activate the pH probes. Moreover, the embedded down-shifting NIR emitters provide sufficient contrast for imaging of a single microrobot inside a live mouse. This work suggests great potential in developing multifunctional microrobots to perform generic site-selective tasks in vivo.

Optical tweezers beyond refractive index mismatch using highly doped upconversion nanoparticles.

Optical tweezers are widely used in materials assembly1, characterization2, biomechanical force sensing3,4 and the in vivo manipulation of cells5 and organs6. The trapping force has primarily been generated through the refractive index mismatch between a trapped object and its surrounding medium. This poses a fundamental challenge for the optical trapping of low-refractive-index nanoscale objects, including nanoparticles and intracellular organelles. Here, we report a technology that employs a resonance effect to enhance the permittivity and polarizability of nanocrystals, leading to enhanced optical trapping forces by orders of magnitude. This effectively bypasses the requirement of refractive index mismatch at the nanoscale. We show that under resonance conditions, highly doping lanthanide ions in NaYF4 nanocrystals makes the real part of the Clausius-Mossotti factor approach its asymptotic limit, thereby achieving a maximum optical trap stiffness of 0.086 pN µm-1 mW-1 for 23.3-nm-radius low-refractive-index (1.46) nanoparticles, that is, more than 30 times stronger than the reported value for gold nanoparticles of the same size. Our results suggest a new potential of lanthanide doping for the optical control of the refractive index of nanomaterials, developing the optical force tag for the intracellular manipulation of organelles and integrating optical tweezers with temperature sensing and laser cooling7 capabilities.

Increased Formation of Follicular Antrum in Aquaporin-8-Deficient Mice Is Due to Defective Proliferation and Migration, and Not Steroidogenesis of Granulosa Cells.

Aquaporin-8 (AQP8) is a water channel protein expressed exclusively in granulosa cells (GCs) in mouse ovary. Our previous studies of AQP8-deficient (AQP8-/-) mice demonstrated that AQP8 participates in folliculogenesis, including in the formation of follicles, ovulation, and atresia. However, its physiological function in formation of the antral follicle is still largely unknown. In the present study, we observed significantly increased numbers of antral follicles in AQP8-/- ovaries as well as significantly increased follicular antrum formation in in vitro 3D culture of AQP8-/- follicles. Functional detection of AQP8-/- GCs indicated that cell proliferation is impaired with FSH treatment, and wound healing and Transwell migration are also impaired with or without FSH treatment, compared with that in WT. However, the biosynthesis of estradiol and progesterone as well as the mRNA levels of key steroidogenic enzyme genes (CYP19A1 and StAR) in AQP8-/- GCs did not change, even with addition of FSH and/or testosterone. In order to estimate the influence of the impaired proliferation and migration on the density of GC mass, preantral follicles were injected with FITC-dextran, which distributes only in the intercellular space, and analyzed by confocal microscopy. The micrographs showed significantly higher transmission of fluorescence in AQP8-/- follicles, suggesting increased intercellular space of GCs. Based on this evidence, we concluded that AQP8 deficiency leads to increased formation of follicular antra in vivo and in vitro, and the mechanism may be associated with increased intercellular space of GCs, which may be caused by defective proliferation and migration of GCs. This study may offer new insight into the molecular mechanisms of the formation of follicular antrum.

Analysis of serum ß-amyloid peptides, α2-macroglobulin, complement factor H, and clusterin levels in APP/PS1 transgenic mice during progression of Alzheimer's disease.

As a progressive age-related neurodegenerative disorder, Alzheimer's disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of ß-amyloid proteins, α2-macroglobulin (α-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum Aß40, Aß42, α-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum Aß42 levels and Aß42/Aß40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The α-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum Aß42 level, Aß42/Aß40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD.