Targeted Enzyme Activity Imaging with Quantitative Phase Microscopy.

Quantitative phase imaging (QPI) is a powerful optical imaging modality for label-free, rapid, and three-dimensional (3D) monitoring of cells and tissues. However, molecular imaging of important intracellular biomolecules such as enzymes remains a largely unexplored area for QPI. Herein, we introduce a fundamentally new approach by designing QPI contrast agents that allow sensitive detection of intracellular biomolecules. We report a new class of bio-orthogonal QPI-nanoprobes for in situ high-contrast refractive index (RI) imaging of enzyme activity. The nanoprobes feature silica nanoparticles (SiO2 NPs) having higher RI than endogenous cellular components and surface-anchored cyanobenzothiazole-cysteine (CBT-Cys) conjugated enzyme-responsive peptide sequences. The nanoprobes specifically aggregated in cells with target enzyme activity, increasing intracellular RI and enabling precise visualization of intracellular enzyme activity. We envision that this general design of QPI-nanoprobes could open doors for spatial-temporal mapping of enzyme activity with direct implications for disease diagnosis and evaluating the therapeutic efficacy.

Ancient administrative handwritten documents: X-ray analysis and imaging.

Handwritten characters in administrative antique documents from three centuries have been detected using different synchrotron X-ray imaging techniques. Heavy elements in ancient inks, present even for everyday administrative manuscripts as shown by X-ray fluorescence spectra, produce attenuation contrast. In most cases the image quality is good enough for tomography reconstruction in view of future applications to virtual page-by-page `reading'. When attenuation is too low, differential phase contrast imaging can reveal the characters from refractive index effects. The results are potentially important for new information harvesting strategies, for example from the huge Archivio di Stato collection, objective of the Venice Time Machine project.

Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.

The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.

Accelerated Fourier ptychographic diffraction tomography with sparse annular LED illuminations.

Fourier ptychographic diffraction tomography (FPDT) is a recently developed label-free computational microscopy technique that retrieves high-resolution and large-field three-dimensional (3D) tomograms by synthesizing a set of low-resolution intensity images obtained with a low numerical aperture (NA) objective. However, in order to ensure sufficient overlap of Ewald spheres in 3D Fourier space, conventional FPDT requires thousands of intensity measurements and consumes a significant amount of time for stable convergence of the iterative algorithm. Herein, we present accelerated Fourier ptychographic diffraction tomography (aFPDT), which combines sparse annular light-emitting diode (LED) illuminations and multiplexing illumination to significantly decrease data amount and achieve computational acceleration of 3D refractive index (RI) tomography. Compared with existing FPDT technique, the equivalent high-resolution 3D RI results are obtained using aFPDT with reducing data requirement by more than 40 times. The validity of the proposed method is experimentally demonstrated on control samples and various biological cells, including polystyrene beads, unicellular algae and clustered HeLa cells in a large field of view. With the capability of high-resolution and high-throughput 3D imaging using small amounts of data, aFPDT has the potential to further advance its widespread applications in biomedicine.

Graphene-Based Confocal Refractive Index Microscopy for Label-Free Differentiation of Living Epithelial and Mesenchymal Cells.

Label-free imaging and investigation of living cells are significant for many biomedical studies. It has been challenging to detect the epithelial-mesenchymal transition of cells in situ without affecting cellular activity. Here, we present a common-path differential confocal microscope based on the polarization-sensitive absorption of graphene to realize high-performance refractive index imaging and differentiation of living colorectal cancer cells (HCT116) with large detecting depth (1.29 µm), excellent refractive index resolution (2.86 × 10-5 RIU), and high spatial resolution (727 nm) simultaneously. Compared with epithelial (parental HCT116) cells, mesenchymal (paclitaxel-resistant HCT116) cells manifest generally lower refractive index values through the refractive index statistics, which is due to the stronger migration ability and weaker surface adherence of mesenchymal cells. The graphene-based microscopy provides an effective label-free approach to high-resolution imaging and study of living cell kinetics, and we expect it to be widely used in the research fields of pathology, tumorigenesis, and chemotherapy.