Imaging Single Biomolecules Research Articles

Quantitative Single‐Molecule Electrochemiluminescence Bioassay

AbstractA single‐molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single‐molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single‐molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single‐molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

Quantitative Single-Molecule Electrochemiluminescence Bioassay.

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.

THz Near‐Field Imaging: Near‐Field Nanoscopic Terahertz Imaging of Single Proteins (Small 3/2021)

In article number 2005814, Jiang Li, Huabin Wang, and co-workers demonstrate the imaging of single protein molecules with a scattering-type terahertz (THz) scanning near-field optical microscope, taking advantage of a graphene substrate with high THz reflectivity and atomic smoothness, and a long platinum probe with high scattering ability. This strategy opens new routes to imaging single biomolecules with THz techniques.

Near-Field Nanoscopic Terahertz Imaging of Single Proteins.

Terahertz (THz) biological imaging has attracted intense attention due to its capability of acquiring physicochemical information in a label-free, noninvasive, and nonionizing manner. However, extending THz imaging to the single-molecule level remains a challenge, partly due to the weak THz reflectivity of biomolecules with low dielectric constants. Here, the development of graphene-mediated THz scattering-type scanning near-field optical microscope for direct imaging of single proteins is reported. Importantly, it is found that a graphene substrate with high THz reflectivity and atomic flatness can provide high THz contrast against the protein molecules. In addition, a platinum probe with an optimized shaft length is found enabling the enhancement of the amplitude of the scattered THz near-field signals. By coupling these effects, the topographical and THz scattering images of individual immunoglobulin G (IgG) and ferritin molecules with the size of a few nanometers are obtained, simultaneously. The demonstrated strategy thus opens new routes to imaging single biomolecules with THz.

Time-Resolved Nanometer Scale AFM Imaging of Antimicrobial Peptide Activity on Live Escherichia coli Cells

Bacterial membranes have a much more complex structure than mammalian cell membranes. As such, knowledge of bacterial membrane composition and organization, as well as characterization of the molecular-level responses to drug interactions, is critical to the development and assessment of effective antibacterial drug formulations. Cellular drug responses involve highly dynamic processes. However, the ability to image live cells with nanometer resolution on timescales relevant to dynamic cellular events has proven challenging. With traditional AFM systems, the typically longer image acquisition times required to obtain a single high-resolution image (~minutes) has limited the ability to investigate dynamic biological processes. While recent years have shown significant progress in the development of high-speed atomic force microscopy (HS-AFM), the nature of the instrumentation that has been developed has several drawbacks in specimen size, requiring small scan sizes and flat sample surfaces. As such, the majority of biologically-related HS-AFM studies have concentrated on imaging single biomolecules with little focus on using HS-AFM to examine cellular processes.

Non-destructive imaging of an individual protein

The mode of action of proteins is to a large extent given by their ability to adopt different conformations. This is why imaging single biomolecules at atomic resolution is one of the ultimate goals of biophysics and structural biology. The existing protein database has emerged from X-ray crystallography, NMR or cryo-TEM investigations. However, these tools all require averaging over a large number of proteins and thus over different conformations. This of course results in the loss of structural information. Likewise it has been shown that even the emergent X-FEL technique will not get away without averaging over a large quantity of molecules. Here we report the first recordings of a protein at sub-nanometer resolution obtained from one individual ferritin by means of low-energy electron holography. One single protein could be imaged for an extended period of time without any sign of radiation damage. Since ferritin exhibits an iron core, the holographic reconstructions could also be cross-validated against TEM images of the very same molecule by imaging the iron cluster inside the molecule while the protein shell is decomposed.

Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins

Single-molecule (SM) microscopy allows outstanding insight into biomolecular mechanisms in cells. However, selective detection of single biomolecules in their native environment remains particularly challenging. Here, we introduce an easy methodology that combines specific targeting and nanometer accuracy imaging of individual biomolecules in living cells. In this method, named complementation-activated light microscopy (CALM), proteins are fused to dark split-fluorescent proteins (split-FPs), which are activated into bright FPs by complementation with synthetic peptides. Using CALM, the diffusion dynamics of a controlled subset of extracellular and intracellular proteins are imaged with nanometer precision, and SM tracking can additionally be performed with fluorophores and quantum dots. In cells, site-specific labeling of these probes is verified by coincidence SM detection with the complemented split-FP fusion proteins or intramolecular single-pair Förster resonance energy transfer. CALM is simple and combines advantages from genetically encoded and synthetic fluorescent probes to allow high-accuracy imaging of single biomolecules in living cells, independently of their expression level and at very high probe concentrations.

Physics: Toward imaging single biomolecules

Science NewsVolume 171, Issue 16 p. 253-254 Of Note Physics: Toward imaging single biomolecules First published: 30 September 2009 https://doi.org/10.1002/scin.2007.5591711616AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume171, Issue1621 April 2007Pages 253-254 RelatedInformation

Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction.

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 A. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.

High resolution 3-D X-ray diffraction microscopy and its potential of imaging single biomolecules

High resolution 3-D X-ray diffraction microscopy and its potential of imaging single biomolecules