GEOMScope: Large Field-of-view 3D Lensless Microscopy with Low Computational Complexity

Recent development of lensless imagers has enabled three-dimensional (3D) imaging through a thin piece of optics in close proximity to a camera sensor. A general challenge of wide-field lensless imaging is the high computational complexity and slow speed to reconstruct 3D objects through iterative optimization process. Here, we demonstrated GEOMScope, a lensless 3D microscope that forms image through a single layer of microlens array and reconstructs objects through a geometrical-optics-based pixel back projection algorithm and background suppressions. Compared to others, our method allows local reconstruction, which significantly reduces the required computation resource and increases the reconstruction speed by orders of magnitude. This enables near real-time object reconstructions across a large volume of 23x23x5 mm^3, with a lateral resolution of 40 um and axial resolution of 300 um. Our system opens new avenues for broad biomedical applications such as endoscopy, which requires both miniaturized device footprint and real-time high resolution visualization

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique “Protease ID” for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed “Trypsin ID” and “Chymotrypsin ID” occurred at <i>m</i>/<i>z</i> 761.6 and 711.6. The logarithm value of the intensity ratio of “Protease ID” to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases

Comparison of recombinant CSP from bacteria and yeast.

A, Heparin binding of GFP, GFP-PfCSP, GFP-PfCSPΔN, and GFP-αTSR purified from P. pastoris. Samples for GFP-PfCSP are analyzed by immunoblotting (IB) using antibodies against GFP. All other samples are analyzed by Coomassie staining. A degraded product of GFP-PfCSP is indicated by arrowhead. B, Gel filtration analysis of GFP-PfCSPΔN purified from either E. coli or P. pastoris. Fractions are analyzed by SDS-PAGE and coomassie staining. C, as in B, but with GFP-αTSR.</p

Attachment of CSP to hepatocytes.

<p><b>A</b>, Purified GFP-<i>Pf</i>CSP or mutant proteins were incubated with HepG2 cells. Binding of the proteins to the cells was quantified by using flow cytometry to detect GFP fluorescence. Analyzed data are shown on the right. The data are representative of at least four repetitions. <b>B</b>, Purified GFP-<i>Pf</i>CSP or mutant proteins were incubated with HepG2 cells. GFP on the cell surface was detected by live cell imaging. Nuclei were stained with Hoechst 33258 for identification of individual cells. Approximately 300 cells were counted for each sample. The data represent at least three repetitions.</p

GFP-PfCSP interacts with heparin.

<p><b>A</b>, Domain structures of full-length and recombinant <i>Pf</i>CSP. The termini are indicated. SP, signal peptide; NTD, N-terminal domain; CTD, C-terminal domain; RI, region I; RII+, region II plus; RIII, region III; GPI, GPI anchor sequence. <b>B</b>, Sequence alignment of residues preceding the region I in the NTD of CSP. Residues in <i>Pf</i>CSP are numbered, and residues mutated are highlighted in bold. Mutations that affect heparin binding are colored in red and mutation that does not is colored in cyan. Basic residues in other CSPs that are near region I are colored in orange. The region I is highlighted by a yellow box. Peptides that have been tested for heparin binding are underlined. <b>C</b>,<b>D</b>, Heparin binding of GFP-<i>Pf</i>CSP (<b>C</b>) and GFP alone (<b>D</b>). ~150 μg of purified protein was applied to the heparin column, and samples were analyzed by SDS-PAGE, followed by Coomassie staining. Domain structure of the protein used is shown on the left. I, input; FT, flow-through. <b>E-N</b>, as in <b>C</b>, but with GFP-tagged CSP mutants. Arrowhead indicates a contaminant. All data were confirmed by at least three independent experiments using three independently purified batches of proteins. Data shown are from a representative experiment.</p

Dual Quinone Tagging for MALDI-TOF Mass Spectrometric Quantitation of Cysteine-Containing Peptide

A dual quinone tagging strategy is designed for quantitation of cysteine-containing peptide (CCP) with MALDI-TOF mass spectrometry. The quinone compounds can rapidly and specifically bind to the thiol group of cysteine residues by a Michael addition reaction, which is used to identify both CCP and the number of cysteine residues in CCP through the direct observation of untagged and tagged products. After reduced with DL-dithiothreitol, the intramolecular disulfide bond can also be identified. Using benzoquinone (BQ) and methyl-<i>p</i>-benzoquinone (MBQ) as dual tags and a peptide with an amino acid sequence of SSDQFRPDDCT (C-pep1) as a model target, respectively, the quantitation strategy is performed through the intensity ratio of MBQ-tagged C-pep1 to BQ-tagged C-pep1 as the internal standard. The logarithm value of the intensity ratio is proportional to C-pep1 concentration in a range from 5.0 to 5000 nM. The limit of detection is as low as 2.0 nM. The proposed methodology provides a novel tool for rapid characterization, identification, and quantitation of biomolecules containing thiol reactive sites and has a promising application in the large-scale detection and analysis of cysteine-containing biomolecules

Homotypic interactions between CSP termini.

<p><b>A</b>, <b>B</b>, Purified GFP-<i>Pf</i>CSPΔC or GFP alone was incubated with HA-αTSR. Immunoprecipitation (IP) was performed using anti-GFP (<b>A</b>) or anti-HA (<b>B</b>) antibodies. The samples were analyzed by SDS-PAGE and immunoblotting (IB) with anti-HA or anti-GFP antibodies. <b>C</b>, as in <b>A</b>, but with GFP-<i>Pf</i>CSP instead of GFP. <b>D</b>, as in <b>B</b>, but with GFP-<i>Pf</i>CSP instead of GFP.</p

QUADAS-2 risk of bias assessment.

QUADAS-2 risk of bias assessment.</p

Pooled analysis of the diagnostic performance for choline PET-CT on a per-patient basis and on a per-lesion basis.

Pooled analysis of the diagnostic performance for choline PET-CT on a per-patient basis and on a per-lesion basis.</p

Signal-On Mass Spectrometric Biosensing of Multiplex Matrix Metalloproteinases with a Phospholipid-Structured Mass-Encoded Microplate

The detection of matrix metalloproteinases (MMPs) is of great importance for diagnosis and staging of cancer. This work proposed a signal-on mass spectrometric biosensing strategy with a phospholipid-structured mass-encoded microplate for assessment of multiplex MMP activities. The designed substrate and internal standard peptides were subsequently labeled with the reagents of isobaric tags for relative and absolute quantification (iTRAQ), and DSPE-PEG(2000)maleimide was embedded on the surface of a 96-well glass bottom plate to fabricate the phospholipid-structured mass-encoded microplate, which offered a simulated environment of the extracellular space for enzyme reactions between MMPs and the substrates. The strategy achieved multiplex MMP activity assays by dropping the sample in the well for enzyme cleavages, followed by adding trypsin to release the coding regions for ultrahigh performance liquid chromatography–tandem mass spectrometric (UHPLC–MS/MS) analysis. The peak area ratios of released coding regions and their respective internal standard (IS) peptides exhibited satisfied linear ranges of 0.05–50, 0.1–250, and 0.1–100 ng mL–1 with the detection limits of 0.017, 0.046, and 0.032 ng mL–1 for MMP-2, MMP-7, and MMP-3, respectively. The proposed strategy demonstrated good practicability in inhibition analysis and detections of multiplex MMP activities in serum samples. It is of great potential for clinical applications and can be expanded for multiplex enzyme assays