Library · Signals, sensing & imaging
What Super-Resolution Microscopy got wrong, from 16 theses
Summary written by a model from the records below: The records document technical limitations and experimental bottlenecks encountered when applying super-resolution microscopy and related nanoscale expansion methods. Practitioners frequently faced difficulties including sample distortion, probe photobleaching, temporal constraints, and sub-optimal optical configurations that compromised target resolution. These records come from graduate theses, 2021 to 2025, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 10 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Suboptimal labeling strategies and bulky probe assemblies degrade localization precision
Direct unencapsulated labeling and live stains suffer from low photon budgets, poor blinking, and overlapping point spread functions. Furthermore, bulky antibody reagents or branched DNA assemblies either require destructive sectioning or degrade nanoscale spatial fidelity.
Tried and failed
direct fluorophore labelling with live-cell DNA stain applied to chromatin localization super-resolution microscopy. Outcome: no signal. Reason: poor fluorophore blinking and overlapping point spread functions causing high localisation uncertainty
Super Resolution Microscopy to investigate epigenetic mechanisms driving platinum resistance in ovarian High Grade Serous Carcinoma · Imperial · PhD
Tried and failed
direct fluorophore labelling without encapsulation applied to optical super-resolution localization microscopy. Outcome: worse than baseline. Reason: lower photon budget and significantly larger localization uncertainty compared to encapsulated probes
INFORMED DESIGN OF POLYMER-SELECTIVE NANOPROBES FOR OPTICAL SUPER-RESOLUTION MICROSCOPY · Cornell · PhD
Considered and rejected
Considered and rejected: Rejected STORM super-resolution imaging in favor of PALM because STORM requires fluorescent antibody labeling that necessitates physical sectioning of spores.
Investigating proteins that influence membrane-associated germination processes in Bacillus subtilis spores · Virginia Tech · Doctorate
Considered and rejected
Considered and rejected: Rejected branched DNA (bDNA) signal amplification due to physical size of branch assemblies degrading super-resolution spatial fidelity
Methods for visualizing a whole human chromosome in super resolution and distinguishing maternal and paternal homologs genome-wide · Harvard · PhD
Extended acquisition times and environmental constraints hinder live-cell super-resolution imaging
Long acquisition durations cause phototoxicity, motion blur artifacts, and background noise accumulation from non-specific binding. Additionally, microscope setups often lack physiological environmental controls or require chemical fixation that introduces disruptive sample artifacts.
Tried and failed
increasing acquisition duration at low tracer concentration applied to super-resolution localization imaging. Outcome: worse than baseline. Reason: noise accumulation and non-specific binding over time degraded local image quality
Effect of agent concentration in ultrasound super- resolution imaging at clinically low frequency · Imperial · Master's
Considered and rejected
Considered and rejected: Rejected live-cell STORM imaging in favour of deconvolved widefield microscopy due to long acquisition times causing motion artefacts, phototoxicity, and poor image quality with SiR dyes.
Light microscopy applied to study defective macrophage phagocytosis in chronic obstructive pulmonary disease · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected super-resolution microscopy for live co-culture oxygen mapping because the microscope stage lacked physiological environmental control (5% CO2, 37°C), reverting to wide-field microscopy.
Development and application of oxygen nanosensors for the assessment of molecular oxygen in 2D and 3D cell culture · Nottingham · PhD
Considered and rejected
Considered and rejected: Ruled out using chemical fixation techniques (such as electron microscopy or super-resolution microscopy) to directly verify TAT-MORN peptide disruption of dyadic clefts due to potential artifacts incompatible with the peptide's mode of action.
The Complexities of cAMP Signaling in the Heart · University of Nevada, Reno · PhD
Incomplete digestion and anchoring degradation cause structural distortions during expansion microscopy
Residual extracellular matrix networks prevent isotropic gel expansion when enzymatic matrix degradation is omitted. In addition, imaging buffers and re-embedding procedures degrade anchoring moieties and trigger random nanoscale displacements.
Tried and failed
chemical softening without enzymatic matrix degradation applied to expansion microscopy of dense extracellular matrix. Reason: residual collagen network prevented isotropic gel expansion, leading to structural distortion
Developing Tools to Physically Magnify Biological Substrates for Clinical Applications · MIT · PhD
Tried and failed
sequential super-resolution and expansion microscopy applied to in situ DNA FISH imaging. Outcome: no signal. Reason: imaging buffer and exposure degraded gel-anchoring acrydite moieties, preventing target retention after expansion
In situ genome sequencing: genomic measurement at the convergence of structure and molecular identity · MIT · PhD
Tried and failed
hydrogel re-embedding after sample expansion applied to super-resolution imaging of nanoscale molecular structures. Outcome: unstable. Reason: sample denaturation causes random anchor displacements and nanometer-scale structural distortions below 30 nm
Tetrahedral expansion gel enables superior preservation of molecular structure for super-resolution imaging · Harvard · PhD
Fluorophore photobleaching and improper switching kinetics impede stochastic reconstruction
Rapid fluorophore photobleaching during acquisition or sample storage destroys signal fidelity and prevents multi-angle three-dimensional reconstruction. Conversely, probes with excessive photostability fail to undergo the photoswitching required for single-molecule localization microscopy.
Considered and rejected
Considered and rejected: Rejected mounting cells in Poly-D-Lysine/CFM3 or Poly-D-Lysine/water due to severe photobleaching (>50-67% loss after first rotation) compromising 3D super-resolution reconstruction.
Translocating under the radar: an investigation into the secretion pathway of EspC, a ‘classical’ autotransporter from enteropathogenic E. coli · Nottingham · PhD
Considered and rejected
Considered and rejected: Direct stochastic optical reconstruction microscopy (dSTORM) for BCR cluster characterization was abandoned due to sample degradation during storage, rapid photobleaching, and severe spatial artifacts.
Engineered Systems of the Germinal Center and Bone Marrow for Antigen-Specific B Cell Generation and Longevity · Georgia Tech · PhD
Considered and rejected
Considered and rejected: Rejected stochastic optical reconstruction microscopy (STORM) due to low photobleaching and high photostability of ZnPcTS.
Preparation and Characterisation of Photoactive Supramolecular Soft Materials · Nottingham · PhD
Optical depletion and patterned illumination defects degrade resolution enhancement
Three-dimensional STED configurations failed to achieve focal volume reductions beyond standard two-dimensional baselines, while optical bandpass filtering caused spectral overlap and unwanted re-excitation. In computational sub-Rayleigh imaging, parasitic reflections from illumination arrays corrupted phase retrieval convergence.
Tried and failed
3D STED microscopy for focal volume reduction applied to fluorescence correlation spectroscopy observation volumes. Outcome: worse than baseline. Reason: failed to reduce focal width below 100 nm compared to 2D STED
Analysis of liquid-liquid phase separated systems for artificial cell applications · Georgia Tech · PhD
Tried and failed
optical bandpass filtering for laser depletion applied to super-resolution fluorescence microscopy. Reason: filter band edge overlapped with fluorophore absorption spectrum, causing unwanted re-excitation
Extending Spatiotemporal Resolution of Super-Resolution Microscopy · Georgia Tech · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform immunofluorescent co-staining of amyloid and microglia to quantify co-localization and microglial morphology at higher resolution. Blocker: Requires a wet lab, biological tissue samples, and high-resolution fluorescence microscopy equipment.
Mechanistic investigation of Temporal Interference brain stimulation and its translational application · Imperial · PhD
Left open
Perform superresolution imaging studies across physiological contexts to experimentally estimate r* and excluded volume parameters. Blocker: Requires wet lab facilities, superresolution microscopy apparatus, and biological samples
Modeling and controlling resource loading in bacterial genetic circuits · MIT · PhD
Left open
Functionalize dye-doped core-shell nanoparticles with targeting proteins like Biotin-BSA and anti-CD71 for live-cell super-resolution imaging and tracking. Blocker: Requires wet lab chemical synthesis, biological cell cultures, and super-resolution fluorescence microscopy equipment
Synthesis and characterization of dye-doped nanoparticles for use in super-resolution fluorescence microscopy · Texas Tech · PhD
Left open
Combine super-resolution imaging and two-fluorophore FRET to quantify absolute copy numbers of cell-surface glycoRNAs per cell. Blocker: Requires wet lab biological samples and super-resolution microscopy apparatus
Spatial imaging of glycoRNA with aptamer and exploration of thrombin-interactive DNA in neutrophil extracellular traps · UT Austin · PhD
Left open
Validate the silent-synapse rapid acquisition hypothesis in vivo using super-resolution imaging alongside voltage indicators and GluSnFR activity reporters. Blocker: Requires a wet lab, live animal models, and in-vivo super-resolution optical imaging setups.
Functional and ultrastructural investigation of mouse and human dendritic spines · MIT · PhD
Left open
Combine umExM with super-resolution microscopy or 16x expansion methods to achieve higher resolution brain imaging. Blocker: Requires a wet lab, chemical synthesis reagents, biological samples, and optical super-resolution microscopy equipment.
Scalable Chemical Tool for Analyzing Brain: Electron Microscope-Like Images with Fluorescent Microscope · MIT · PhD
Left open
Develop fluorescent chloride and voltage sensors achieving single-synapse spatial resolution and submillisecond temporal resolution. Blocker: Requires molecular biology wet lab, protein engineering, and live-cell optical imaging facilities
Interaction of Quantal Inhibitory and Excitatory Neurotransmission in Regulation of Synaptic Homeostasis via Molecular Signaling · UT Southwestern · PhD
Left open
Resolve Plasmodium basal complex subcompartments by combining ultrastructure expansion microscopy (U-ExM) with STED microscopy or iterative U-ExM. Blocker: Requires wet-lab parasite culturing, U-ExM sample preparation, and specialized STED super-resolution microscopy equipment.
Structure and function of the basal complex: the unique mechanisms of Plasmodium cytokinesis · Harvard · PhD
Left open
Apply super-resolution microscopy (STORM and SIM) to verify if high integrated density RAP1 foci represent longer telomeres or telomeric clusters. Blocker: Requires wet lab access, HeLa cell samples, and super-resolution microscopy equipment (STORM/SIM).
Telomeric localization of the TElomeric Repeat-containing RNA TERRA impairs telomerase activity in human cancer cells · University of Trento · PhD
Left open
Construct single-synapse resolution maps of neurotransmitters and receptors across C. elegans and Drosophila nervous systems. Blocker: Requires wet-lab experimental imaging, genetic tagging, and electron microscopy data collection.
Neural Sequences Underlying Directed Turning in C. elegans · MIT · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Dean Lee · Harvard · PhD · 2025
Methods for visualizing a whole human chromosome in super resolution and distinguishing maternal and paternal homologs genome-wide - Talya Kramer · MIT · PhD · 2025
Neural Sequences Underlying Directed Turning in C. elegans - Alexander A Morano · Harvard · PhD · 2024
Structure and function of the basal complex: the unique mechanisms of Plasmodium cytokinesis - Liana Kramer · Georgia Tech · PhD · 2024
Engineered Systems of the Germinal Center and Bone Marrow for Antigen-Specific B Cell Generation and Longevity - Shaun Smith · Nottingham · PhD · 2024
Preparation and Characterisation of Photoactive Supramolecular Soft Materials - Tasdiq Ahmed · Georgia Tech · PhD · 2023
Analysis of liquid-liquid phase separated systems for artificial cell applications - Mi Qi Lim · Imperial · PhD · 2023
Super Resolution Microscopy to investigate epigenetic mechanisms driving platinum resistance in ovarian High Grade Serous Carcinoma - Patrycja Dzialecka · Imperial · PhD · 2023
Mechanistic investigation of Temporal Interference brain stimulation and its translational application - Jenna L. Aronson · MIT · PhD · 2023
Developing Tools to Physically Magnify Biological Substrates for Clinical Applications - Dimitra Vardalaki · MIT · PhD · 2023
Functional and ultrastructural investigation of mouse and human dendritic spines - Tay Won Shin · MIT · PhD · 2023
Scalable Chemical Tool for Analyzing Brain: Electron Microscope-Like Images with Fluorescent Microscope - Matthew Jose Flores · Virginia Tech · Doctorate · 2023
Investigating proteins that influence membrane-associated germination processes in Bacillus subtilis spores
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