Search

Fluorescence lifetime imaging (FLIM) has established itself as a pivotal tool for investigating biological processes within living cells. However, the extensive imaging duration necessary to accumulate sufficient photons for accurate fluorescence lifetime calculations poses a significant obstacle to achieving high-resolution monitoring of cellular dynamics. In this study, we introduce an image reconstruction method based on the edge-preserving interpolation method (EPIM), which transforms rapidly acquired low-resolution FLIM data into high-pixel images, thereby eliminating the need for extended acquisition times. Specifically, we decouple the grayscale image and the fluorescence lifetime matrix and perform an individual interpolation on each. Following the interpolation of the intensity image, we apply wavelet transformation and adjust the wavelet coefficients according to the image gradients. After the inverse transformation, the original image is obtained and subjected to noise reduction to complete the image reconstruction process. Subsequently, each pixel is pseudo-color-coded based on its intensity and lifetime, preserving both structural and temporal information. We evaluated the performance of the bicubic interpolation method and our image reconstruction approach on fluorescence microspheres and fixed-cell samples, demonstrating their effectiveness in enhancing the quality of lifetime images. By applying these techniques to live-cell imaging, we can successfully obtain high-pixel FLIM images at shortened intervals, facilitating the capture of rapid cellular events.

Coordinated control of pollution and carbon reduction is an imperative choice for China’s overall transition towards sustainability. However, China’s environmental policies often treat pollutants and CO2 separately, potentially resulting in imbalanced pollution and carbon reduction. Since several cities are not only critical cities for the Air Pollution Prevention and Control Action Plan (APPCAP) policy but also pilot cities for the Carbon Emissions Trading Scheme (ETS), this study aims to examine the extent to which the policy coordination of APPCAP and ETS can influence air pollutants and CO2 emissions. Using panel data from 2011 to 2019 for China’s 231 prefecture cities, we compare the pollution and carbon reduction effects of separate and coordinated policy implementation of APPCAP and ETS via the difference-in-differences (DID) model and the causal forest model. Research shows that (1) the policy coordination of APPCAP and ETS has significantly reduced both air pollutants and CO2 emissions in dual-policy pilot cities. For non-dual pilot cities, the separate implementation of APPCAP or ETS only exerts significant unilateral effects. (2) Enhancing government supervision, weakening the relationship between government and enterprises, and raising enterprises’ green innovation capabilities are the main mechanisms through which policy coordination can significantly influence pollution and carbon reduction. (3) The combined implementation impacts of APPCAP and ETS are more evident in pollution-intensive cities and cities with weak carbon-peaking trends. Our research inspires the development of a collaborative system of pollution reduction and carbon reduction policies.

Manipulating and real-time monitoring of neuronal activities with cell-type specificity and precise spatiotemporal resolution during animal behavior are fundamental technologies for exploring the functional connectivity, information transmission, and physiological functions of neural circuits in vivo. However, current techniques for optogenetic stimulation and neuronal activity recording mostly operate independently. Here, we report an all-fiber-transmission photometry system for simultaneous optogenetic manipulation and multi-color recording of neuronal activities and the neurotransmitter release in a freely moving animal. We have designed and manufactured a wavelength-independent multi-branch fiber bundle to enable simultaneous optogenetic manipulation and multi-color recording at different wavelengths. Further, we combine a laser of narrow linewidth with the lock-in amplification method to suppress the optogenetic stimulation-induced artifacts and channel crosstalk. We show that the collection efficiency of our system outperforms a traditional epi-fluorescence system. Further, we demonstrate successful recording of dynamic dopamine (DA) responses to unexpected rewards in the nucleus accumbens (NAc) in a freely moving mouse. We also show simultaneous dual-color recording of neuronal Ca2+ signals and DA dynamics in the NAc upon delivering an unexpected reward and the simultaneous optogenetic activating at dopaminergic terminals in the same location. Thus, our multi-function fiber photometry system provides a compatible, efficient, and flexible solution for neuroscientists to study neural circuits and neurological diseases.

Volumetric imaging of a mouse brain in vivo with one-photon and two-photon ultralong anti-diffracting (UAD) beam illumination was performed. The three-dimensional (3D) structure of blood vessels in the mouse brain were mapped to a two-dimensional (2D) image. The speed of volumetric imaging was significantly improved due to the long focal length of the UAD beam. Comparing one-photon and two-photon UAD beam volumetric imaging, we found that the imaging depth of two-photon volumetric imaging (80 μm) is better than that of one-photon volumetric imaging (60 μm), and the signal-to-background ratio (SBR) of two-photon volumetric imaging is two times that of one-photon volumetric imaging. Therefore, we used two-photon UAD volumetric imaging to perform dynamic volumetric imaging of mouse brain blood vessels in vivo, and obtained the blood flow velocity.

Although conventional fluorescence intensity imaging can be used to qualitatively study the drug toxicity of nanodrug carrier systems at the single-cell level, it has limitations for studying nanodrug transport across membranes. Fluorescence correlation spectroscopy (FCS) can provide quantitative information on nanodrug concentration and diffusion in a small area of the cell membrane; thus, it is an ideal tool for studying drug transport across the membrane. In this paper, the FCS method was used to measure the diffusion coefficients and concentrations of carbon dots (CDs), doxorubicin (DOX) and CDs-DOX composites in living cells (COS7 and U2OS) for the first time. The drug concentration and diffusion coefficient in living cells determined by FCS measurements indicated that the CDs-DOX composite distinctively improved the transmembrane efficiency and rate of drug molecules, in accordance with the conclusions drawn from the fluorescence imaging results. Furthermore, the effects of pH values and ATP concentrations on drug transport across the membrane were also studied. Compared with free DOX under acidic conditions, the CDs-DOX complex has higher cellular uptake and better transmembrane efficacy in U2OS cells. Additionally, high concentrations of ATP will cause negative changes in cell membrane permeability, which will hinder the transmembrane transport of CDs and DOX and delay the rapid diffusion of CDs-DOX. The results of this study show that the FCS method can be utilized as a powerful tool for studying the expansion and transport of nanodrugs in living cells, and might provide a new drug exploitation strategy for cancer treatment in vivo.

Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.