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A 2023 Science study showed that individual glycan-decorated molecules can be imaged with low-temperature scanning tunnelling microscopy (STM). The team first electrosprayed intact ions and gently soft-landed them on a surface, then used cryogenic STM images and density-functional-theory calculations to interpret glycan and biomolecule features.
The result is molecularly specific information that ensemble measurements usually average together: which glycans are present, where they sit on an individual protein or lipid, and how occupancy varies from molecule to molecule. It is a laboratory demonstration, not a clinical test or an imaging method performed inside living cells.
What the 2023 study demonstrated
Glycans are chains or branches of sugars attached to proteins and lipids. Those attachments can alter folding, stability, trafficking, molecular recognition and immune interactions. A sample described as one glycoprotein species can still contain many subtly different glycoforms: molecules with different sugar compositions, linkages or occupied attachment sites.
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1Scan for outdated or missing drivers - takes under a minute2Repair Windows errors before they cause bigger problems3Fix the driver behind crashes, sound loss and screen glitchesThe study by Anggara and colleagues reported direct, single-molecule imaging of glycoconjugates—molecules in which a glycan is bonded to a protein, peptide or lipid. The paper, “Direct observation of glycans bonded to proteins and lipids at single molecule level,” used low-temperature STM after soft-landing intact electrosprayed ions on a surface. Images were interpreted with structural calculations, including density functional theory (DFT).
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The authors’ motivation was the limitation of ensemble-averaged measurements. Bulk assays can provide valuable composition or abundance information, but they generally combine signals from many molecules. The STM workflow instead asks what one deposited molecule looks like and where its glycan features are located on that molecule. That distinction matters when a heterogeneous sample contains several glycoforms at once.
Why molecule-by-molecule glycan information matters
Glycosylation is not a simple label added uniformly to every copy of a protein. Occupancy can differ between sites, and the same nominal glycan composition can be arranged in different structures. Averaging those molecules can hide minority forms or blur the relationship between a sugar feature and its exact attachment site.
Single-molecule imaging does not automatically replace established glycomic, proteomic or lipid-analysis methods. Its potential value is complementary: it can show spatial relationships on an individual intact molecule rather than only a population-level signal. For a glycoprotein, that means examining whether a glycan appears at the expected residue and whether neighboring structural features vary among copies.
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Outbyte Driver Updater FREEScan for outdated or missing drivers - takes under a minuteDriver Scan →Outbyte PC Repair FREEClear out junk files and repair common Windows errorsFree Scan →The approach also extends beyond proteins. The reported examples span glycopeptides, glycolipids, an N-glycoprotein and an O-glycosylated reporter fragment. Together, they demonstrate that the same general strategy can be applied to chemically different glycoconjugates, provided the molecules can be prepared and imaged under the required conditions.
How the imaging workflow works
- Prepare intact glycoconjugates. The molecules remain associated with their attached glycans rather than being reduced to a mixture of free sugars and peptide fragments.
- Generate ions by electrospray. Electrospray ionization transfers the sample into the gas phase as charged molecular ions. The study used an ion-beam deposition arrangement suitable for transporting those intact species.
- Soft-land the ions. The ions are deposited gently onto a conductive surface so that the glycoconjugate can remain sufficiently intact for imaging. This is different from a high-energy impact that could fragment or rearrange a fragile glycan.
- Select a surface appropriate to the chemistry. Copper was used for some samples. For sulfated molecules, the authors used a more inert silver surface to help preserve labile sulfate groups during deposition and imaging.
- Cool the sample to cryogenic temperature. Low-temperature operation reduces molecular motion and helps stabilize deposited molecules while the microscope scans them.
- Scan with STM. A sharply prepared conductive tip moves across the surface while the tunnelling current is measured. Submolecular contrast can reveal parts of the deposited biomolecule and attached glycan.
- Interpret the contrast chemically. The team compared observed features with structural models and DFT calculations. Differences such as stereoconfiguration and side groups can contribute to distinguishable image patterns, but an image is interpreted within the complete experimental and computational context rather than read as a simple photograph.
This sequence explains both the power and the boundaries of the method. The microscope examines molecules that have been transferred, deposited and immobilized under vacuum-compatible cryogenic conditions. It does not watch glycosylation occurring in a cell, and it does not image a living tissue in situ.
What the images revealed
| Sample class | Reported observation | Why it matters |
|---|---|---|
| Glycopeptides | Individual peptide-bound glycan features were imaged and chemically interpreted. | Shows that the method can examine a smaller, defined protein fragment while retaining the attached sugar. |
| Glycolipids | The gangliosides GM3 and GD3 were among the examples. | Extends the demonstration beyond protein-linked glycans to lipid-linked glycoconjugates. |
| RNase B | Individual N-glycoproteins were imaged, with the attached N-glycan localized near residue 34. | Provides a molecule-level check against the known Asn34 attachment site. |
| MUC1 reporter fragment | Images showed multiple O-glycan occupancy states on an engineered fragment containing 34 potential sites. | Illustrates site-to-site and molecule-to-molecule heterogeneity in a densely O-glycosylated construct. |
RNase B: locating an N-glycan on individual proteins
RNase B is an N-glycosylated protein with a well-established glycosylation site at Asn34. In the analysis, 33 individual N-glycoproteins were examined. The reported attachment position was residue 34 ± 2, consistent with the known site.
The value of this result is not that it discovers an unknown RNase B sequence. Instead, it shows that the imaging and interpretation workflow can recover a site-specific feature on separate molecules and obtain a distribution centered on the expected location. That is a more demanding demonstration than observing only an average signal from a large population.
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The ±2 value describes the reported positional spread in this analysis; it is not a universal spatial-accuracy specification for every glycoprotein or every surface. Molecular size, orientation, deposition behavior and image interpretation can all affect what can be resolved in another sample.
MUC1: heterogeneous O-glycosylation in an engineered reporter
The MUC1 experiment used a designed reporter fragment with 34 potential O-glycosylation sites. The reported images included glycoforms carrying 27, 21 and 20 O-glycans. Across the construct, the paper reported an average of 3.4 O-glycans per tandem repeat and variable occupancy at individual sites.
Those numbers describe the engineered reporter used in the experiment. They should not be read as a measurement of every native human MUC1 molecule, a patient population or a general rule for mucin glycosylation. Native mucins are large, heterogeneous biological materials, whereas a reporter fragment is selected and designed to make a controlled structural question tractable.
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MUC1 is often discussed in connection with cancer biomarkers, and an accessible October 17, 2023 report in Chemistry World described that context. The imaging result itself, however, does not validate a cancer diagnostic. No clinical sensitivity, specificity, patient cohort or regulatory performance was established by this laboratory demonstration.
Glycopeptides and gangliosides
Examples involving glycopeptides and the gangliosides GM3 and GD3 show that the signal is not limited to one protein scaffold. Gangliosides are glycolipids, so their inclusion demonstrates that the workflow can preserve and image glycans attached to a lipid as well as sugars attached to peptide or protein frameworks.
The authors also reported distinguishing component monosaccharides using image features associated with stereoconfiguration and side groups. Such assignments rely on comparison with chemical structures and calculations; they are not claims that every sugar can always be identified from an STM image without supporting information.
How this differs from ensemble glycan analysis
The study did not establish a head-to-head performance ranking against other analytical platforms. The practical differences are best understood by asking what each approach measures and what preparation it requires.
| Question | Single-molecule STM workflow | Ensemble-oriented measurements |
|---|---|---|
| What is observed? | Deposited individual glycoconjugate molecules with submolecular image features. | Signals averaged over many molecules, often reporting composition, abundance or mass-related information. |
| Can site relationships be retained? | Potentially, because the intact molecule is imaged and a glycan can be related to a position on that molecule. | Depends on the technique and preparation; population averaging can obscure which features coexist on one molecule. |
| Sample preparation | Electrospray, intact-ion transport, soft landing on a conductive surface and cryogenic STM-compatible handling. | Varies widely; some methods use solution-phase separations, enzymatic releases, labeling or other preparation. |
| Throughput and routine use | Not established by this study; the workflow requires specialized instrumentation and molecule-by-molecule interpretation. | Often selected for the specific need to process populations, quantify signals or survey many samples, but capabilities differ by method. |
| Clinical status | No clinical diagnostic use was demonstrated. | Some other glycan assays may have validated applications, but those claims cannot be transferred to this STM method. |
The table is a framework, not a numerical benchmark. The reported sources do not establish comparative sensitivity, cost, sample consumption, throughput or clinical usefulness.
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Practical constraints and failure points
Keeping the molecule intact
Soft landing is central because a fragmented ion would no longer answer the same structural question. Fragile glycans, especially sulfated species, can be damaged during ion transport or surface contact. The use of silver for sulfated molecules reflects this chemical constraint rather than a universal rule that silver is the best surface for every sample.
Controlling surface behavior
Once deposited, a molecule must remain sufficiently stable and positioned for scanning. Strong movement, unfavorable orientation or crowding could make submolecular features harder to assign. Cryogenic temperatures help reduce motion, but they do not remove the need for careful deposition and interpretation.
Separating image contrast from chemical certainty
STM records electronic tunnelling behavior, not a direct elemental label for each pixel. Structural models and DFT calculations therefore support the assignment of glycan features. A credible interpretation requires agreement between the image, known chemistry and calculated structures; contrast alone should not be treated as an unrestricted molecular identification system.
Managing sample heterogeneity
Single-molecule measurements expose heterogeneity instead of averaging it away. That is scientifically useful, but it also means that many molecules may need to be examined before the distribution of glycoforms becomes clear. The RNase B result involved 33 analyzed molecules; that count supports the reported observation but is not a universal sample-size requirement for future studies.
What the method could enable next
The team indicated that further development could help identify previously unknown glycoproteins and glycolipids. The conditional wording matters: the 2023 paper demonstrates feasibility on selected examples, not a completed unknown-compound identification service.
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Future work would need to establish how broadly the method works across larger proteins, more complex branched glycans, mixtures and chemically fragile modifications. It would also need reproducible preparation, validated assignment criteria and a practical way to compare many molecules across samples. None of those requirements is answered by the reported RNase B or MUC1 demonstrations alone.
For basic biology, the approach could be especially informative when the question is about coexisting glycoforms: whether a particular site is occupied, whether occupancy varies among copies, or whether a lipid-linked glycan adopts a distinct molecular arrangement. Those are questions for controlled laboratory analysis, not yet for routine patient testing.
Publication details
The primary paper appeared in Science, volume 382, issue 6667, pages 219–223, dated October 12, 2023. Its DOI is 10.1126/science.adh3856. Authors listed for the paper include Kelvin Anggara, Laura Sršan, Thapakorn Jaroentomeechai, Xu Wu, Stephan Rauschenbach, Yoshiki Narimatsu, Henrik Clausen, Thomas Ziegler, Rebecca L. Miller and Klaus Kern. Chemistry World’s October 17, 2023 coverage identified Kelvin Anggara as a co-lead researcher and summarized the prospective applications.
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Random freezes, missing sound and display glitches usually trace back to one bad driver. Find and replace yours safely.Free scan · under a minuteThe paper’s abstract describes the work as “single molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy.” That wording accurately captures the achievement: direct imaging of prepared, deposited molecules under cryogenic laboratory conditions.
The bottom line
Low-temperature STM combined with electrospray soft landing gave researchers a way to inspect individual protein- and lipid-bound glycans instead of viewing only a population average. RNase B images placed the N-glycan near the known Asn34 site across 33 molecules, while an engineered MUC1 reporter exposed several O-glycan occupancy states across 34 potential sites. The work is a powerful structural demonstration and a possible foundation for future glycoanalysis, but it is not yet a clinical diagnostic, a living-cell imaging technique or a validated replacement for established glycan measurements.
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