Chemical & Biomedical Imaging最新文献

The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses (CoV), particularly the highly conserved nonstructural CoV proteins (nsp). Among these, the nsp13 helicase of severe pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV is one of the most preserved CoV nsp. Utilizing single-molecule FRET, we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed. If a different nucleotide is introduced, these steps diminish to 3–4 bp. Dwell-time analysis revealed 3–4 concealed steps within each unwinding process, which suggests the hydrolysis of 3–4 dTTP. Combining our observations with previous studies, we propose an unwinding model of CoV nsp13 helicase. This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA, even as the helicase core domain has advanced over 3–4 bp, thereby inducing accumulated strain on the nsp13-DNA complex. Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.

The COVID-19 pandemic has underscored the importance of in-depth research into the proteins encoded by coronaviruses (CoV), particularly the highly conserved nonstructural CoV proteins (nsp). Among these, the nsp13 helicase of severe pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV is one of the most preserved CoV nsp. Utilizing single-molecule FRET, we discovered that MERS-CoV nsp13 unwinds DNA in distinct steps of about 9 bp when ATP is employed. If a different nucleotide is introduced, these steps diminish to 3-4 bp. Dwell-time analysis revealed 3-4 concealed steps within each unwinding process, which suggests the hydrolysis of 3-4 dTTP. Combining our observations with previous studies, we propose an unwinding model of CoV nsp13 helicase. This model suggests that the elongated and adaptable 1B-stalk of nsp13 may enable the 1B remnants to engage with the unwound single-stranded DNA, even as the helicase core domain has advanced over 3-4 bp, thereby inducing accumulated strain on the nsp13-DNA complex. Our findings provide a foundational framework for determining the unwinding mechanism of this unique helicase family.

Bacterial resistance, primarily stemming from misdiagnosis, misuse, and overuse of antibacterial medications in humans and animals, is a pressing issue. To address this, we focused on developing a fluorescent probe for the detection of bacteria, with a unique feature-an exceptionally long fluorescence lifetime, to overcome autofluorescence limitations in biological samples. The polymyxin-based probe (ADOTA-PMX) selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium (with a reported fluorescence lifetime of 19.5 ns). Evaluation of ADOTA-PMX's bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria, and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications. The probe exhibited a lifetime of 4.5 ns when bound to bacteria, possibly indicating interactions with the bacterial outer membrane. Furthermore, the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes. This study represents the first targeted probe for fluorescence lifetime imaging, offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.

Bacterial resistance, primarily stemming from misdiagnosis, misuse, and overuse of antibacterial medications in humans and animals, is a pressing issue. To address this, we focused on developing a fluorescent probe for the detection of bacteria, with a unique feature─an exceptionally long fluorescence lifetime, to overcome autofluorescence limitations in biological samples. The polymyxin-based probe (ADOTA-PMX) selectively targets Gram-negative bacteria and used the red-emitting fluorophore azadioxatriangulenium (with a reported fluorescence lifetime of 19.5 ns). Evaluation of ADOTA-PMX’s bacterial labeling efficacy revealed strong specificity for Gram-negative bacteria, and full spectral fluorescence lifetime imaging microscopy demonstrated the potential of ADOTA-PMX for bacterial imaging applications. The probe exhibited a lifetime of 4.5 ns when bound to bacteria, possibly indicating interactions with the bacterial outer membrane. Furthermore, the fluorescence lifetime measurements of ADOTA-PMX labeled bacteria could be performed using a benchtop fluorimeter without the need of sophisticated microscopes. This study represents the first targeted probe for fluorescence lifetime imaging, offering sensitivity for detecting Gram-negative bacteria and enabling multiplexing via fluorescence lifetime imaging.

Enzyme catalytic activities are critical biomarkers of tissue states under physiological and pathophysiological conditions. However, the direct measurement and imaging of enzyme activity in vivo remains extremely challenging. We report the synthesis and characterization of the first stable triarylmethyl (TAM) radical substrate of alkaline phosphatase (TAM-ALPs). The enzymatic dephosphorylation of TAM-ALPs results in a drastic change in its electron paramagnetic resonance (EPR) spectrum that can be used to image enzyme activity using EPR-based technologies. TAM-ALPs and their enzyme products were fully characterized using EPR and HPLC-MS techniques. A proof of concept of imaging enzyme activity using Overhauser-enhanced magnetic resonance imaging was demonstrated in vitro. This study clearly demonstrates the potential of EPR-based imaging technologies associated with TAM spin probes to map enzyme activity in vivo in future studies.

Enzyme catalytic activities are critical biomarkers of tissue states under physiological and pathophysiological conditions. However, the direct measurement and imaging of enzyme activity in vivo remains extremely challenging. We report the synthesis and characterization of the first stable triarylmethyl (TAM) radical substrate of alkaline phosphatase (TAM-ALPs). The enzymatic dephosphorylation of TAM-ALPs results in a drastic change in its electron paramagnetic resonance (EPR) spectrum that can be used to image enzyme activity using EPR-based technologies. TAM-ALPs and their enzyme products were fully characterized using EPR and HPLC-MS techniques. A proof of concept of imaging enzyme activity using Overhauser-enhanced magnetic resonance imaging was demonstrated in vitro. This study clearly demonstrates the potential of EPR-based imaging technologies associated with TAM spin probes to map enzyme activity in vivo in future studies.

Electrochemiluminescence (ECL) is nowadays a powerful technique widely used in biosensing and imaging, offering high sensitivity and specificity for detecting and mapping biomolecules. Screen-printed electrodes (SPEs) offer a versatile and cost-effective platform for ECL applications due to their ease of fabrication, disposability, and suitability for large-scale production. This research introduces a novel method for improving the ECL characteristics of screen-printed carbon electrodes (SPCEs) through the application of CO₂ laser treatment following fabrication. Using advanced ECL microscopy, we analyze three distinct carbon paste-based electrodes and show that low-energy laser exposure (ranging from 7 to 12 mJ·cm^-2) enhances the electrochemical performance of the electrodes. This enhancement results from the selective removal of surface binders and contaminants achieved by the laser treatment. We employed ECL microscopy to characterize the ECL emission using a bead-based system incorporating magnetic microbeads, like those used in commercial platforms. This approach enabled high-resolution spatial mapping of the electrode surface, offering valuable insights into its electrochemical performance. Through quantitative assessment using a photomultiplier tube (PMT), it was observed that GST electrodes could detect biomarkers with high sensitivity, achieving an approximate detection limit (LOD) of 11 antibodies per μm². These findings emphasize the potential of laser-modified GST electrodes in enabling highly sensitive electrochemiluminescent immunoassays and various biosensing applications.

Electrochemiluminescence (ECL) is nowadays a powerful technique widely used in biosensing and imaging, offering high sensitivity and specificity for detecting and mapping biomolecules. Screen-printed electrodes (SPEs) offer a versatile and cost-effective platform for ECL applications due to their ease of fabrication, disposability, and suitability for large-scale production. This research introduces a novel method for improving the ECL characteristics of screen-printed carbon electrodes (SPCEs) through the application of CO₂ laser treatment following fabrication. Using advanced ECL microscopy, we analyze three distinct carbon paste-based electrodes and show that low-energy laser exposure (ranging from 7 to 12 mJ·cm^–2) enhances the electrochemical performance of the electrodes. This enhancement results from the selective removal of surface binders and contaminants achieved by the laser treatment. We employed ECL microscopy to characterize the ECL emission using a bead-based system incorporating magnetic microbeads, like those used in commercial platforms. This approach enabled high-resolution spatial mapping of the electrode surface, offering valuable insights into its electrochemical performance. Through quantitative assessment using a photomultiplier tube (PMT), it was observed that GST electrodes could detect biomarkers with high sensitivity, achieving an approximate detection limit (LOD) of 11 antibodies per μm². These findings emphasize the potential of laser-modified GST electrodes in enabling highly sensitive electrochemiluminescent immunoassays and various biosensing applications.

Nanoscale surface topography is an effective approach in modulating cell-material interactions, significantly impacting cellular and nuclear morphologies, as well as their functionality. However, the adaptive changes in cellular metabolism induced by the mechanical and geometrical microenvironment of the nanotopography remain poorly understood. In this study, we investigated the metabolic activities in cells cultured on engineered nanopillar substrates by using a label-free multimodal optical imaging platform. This multimodal imaging platform, integrating two photon fluorescence (TPF) and stimulated Raman scattering (SRS) microscopy, allowed us to directly visualize and quantify metabolic activities of cells in 3D at the subcellular scale. We discovered that the nanopillar structure significantly reduced the cell spreading area and circularity compared to flat surfaces. Nanopillar-induced mechanical cues significantly modulate cellular metabolic activities with variations in nanopillar geometry further influencing these metabolic processes. Cells cultured on nanopillars exhibited reduced oxidative stress, decreased protein and lipid synthesis, and lower lipid unsaturation in comparison to those on flat substrates. Hierarchical clustering also revealed that pitch differences in the nanopillar had a more significant impact on cell metabolic activity than diameter variations. These insights improve our understanding of how engineered nanotopographies can be used to control cellular metabolism, offering possibilities for designing advanced cell culture platforms which can modulate cell behaviors and mimic natural cellular environment and optimize cell-based applications. By leveraging the unique metabolic effects of nanopillar arrays, one can develop more effective strategies for directing the fate of cells, enhancing the performance of cell-based therapies, and creating regenerative medicine applications.

Nanoscale surface topography is an effective approach in modulating cell-material interactions, significantly impacting cellular and nuclear morphologies, as well as their functionality. However, the adaptive changes in cellular metabolism induced by the mechanical and geometrical microenvironment of the nanotopography remain poorly understood. In this study, we investigated the metabolic activities in cells cultured on engineered nanopillar substrates by using a label-free multimodal optical imaging platform. This multimodal imaging platform, integrating two photon fluorescence (TPF) and stimulated Raman scattering (SRS) microscopy, allowed us to directly visualize and quantify metabolic activities of cells in 3D at the subcellular scale. We discovered that the nanopillar structure significantly reduced the cell spreading area and circularity compared to flat surfaces. Nanopillar-induced mechanical cues significantly modulate cellular metabolic activities with variations in nanopillar geometry further influencing these metabolic processes. Cells cultured on nanopillars exhibited reduced oxidative stress, decreased protein and lipid synthesis, and lower lipid unsaturation in comparison to those on flat substrates. Hierarchical clustering also revealed that pitch differences in the nanopillar had a more significant impact on cell metabolic activity than diameter variations. These insights improve our understanding of how engineered nanotopographies can be used to control cellular metabolism, offering possibilities for designing advanced cell culture platforms which can modulate cell behaviors and mimic natural cellular environment and optimize cell-based applications. By leveraging the unique metabolic effects of nanopillar arrays, one can develop more effective strategies for directing the fate of cells, enhancing the performance of cell-based therapies, and creating regenerative medicine applications.