Library · Biology & lab methods
What Microscopy Sample Cryo-Preparation got wrong, from 28 theses
Summary written by a model from the records below: The reviewed records detail experimental challenges encountered during cryogenic sample preparation and imaging across electron, optical, and microtomy workflows. Failures frequently arose from ice crystallization, substrate adhesion loss during cryosectioning, macromolecular denaturation at the air-water interface, particle orientation bias, beam-induced radiolysis, and additive-mediated imaging artifacts. 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.
Ice crystal formation and freezing rate defects damage specimen morphology during cryofixation
Rapid cryo-quenching, flash freezing, and direct plunge freezing often fail to achieve adequate cooling rates, generating ice crystal artifacts that disrupt structural morphology and tissue integrity. Direct plunge freezing in liquid nitrogen also induces vapour insulation and tissue cracking, prompting researchers to seek alternative coolants or temperatures to prevent morphological destruction.
Tried and failed
cryo-SEM imaging after flash freezing applied to interfacial biofilm morphology characterization. Reason: Slow freezing rate induced artifactual ice crystal formation obscuring true morphology
The effects of air/water interface properties on the formation of biofilm by P. aeruginosa · Texas Tech · PhD
Tried and failed
rapid cryo-quenching for ex-situ structural preservation applied to liquid-vacuum interfaces under electric fields. Outcome: unstable. Reason: supercooling led to slow freezing, pulsating meniscus shutoff, and crystallisation artifacts
Lost to a baseline
Several liver specimens could not be evaluated histologically due to tissue depletion or cryosection freezing artifacts.
Considered and rejected
Considered and rejected: Rejected analyzing frozen-hydrated samples under cryogenic conditions on 3D OrbiSIMS due to ice crystal formation and handling complexity, using freeze-dried/dehydrated samples instead.
Considered and rejected
Considered and rejected: Rejected snap freezing in liquid isopentane (-160 °C) in favour of slow freezing at -20 °C due to inferior tissue morphological integrity observed on H&E staining.
Targeted bio-distribution of drugs to the lymphatic system · Nottingham · PhD
Considered and rejected
Considered and rejected: Rejected snap freezing directly in liquid nitrogen because gaseous vapour insulation creates artefactual tissue cracking, adopting liquid-nitrogen-cooled 2-methylbutane instead.
Analysis of cellular heterogeneity in breast cancer by single cell sequencing · Oxford · PhD
Considered and rejected
Considered and rejected: Direct plunging cryofixation rejected due to water crystallization, splashing, and inhomogeneous freezing with small volumes.
Probing The Dynamics of Biomolecules Using EPR Spectroscopy and Electron Spin Labels · University of Washington · PhD
Cryosectioning causes structural disruption, poor optical resolution, and tissue detachment
Frozen tissue sections frequently suffer from freeze-thaw damage, microtome tearing in distilled water ice, and structural blurring compared to thin paraffin sections. Additionally, cryosections often detach from slides during staining, fail to allow laser penetration when cut too thick, or experience slide coating degradation from solvent fixatives.
Tried and failed
fixed-frozen tissue processing with sucrose cryoprotection applied to brain tissue histology and immunostaining. Outcome: unstable. Reason: tissue sections repeatedly detached from microscope slides during immunostaining procedures
Exploration of post-blast neuropathology and the use of a promising fluid biomarker in a rat model of blast traumatic brain injury · Imperial · PhD
Lost to a baseline
Cryosections (12 µm) yielded blurry, overlapping IF images compared to 3 µm paraffin sections, which provided sharp cytoarchitecture.
Wachstumsverhalten und Charakterisierung von in vitro-3D-Zellkulturen des Harnblasenkarzinoms · Tübingen · PhD
Considered and rejected
Considered and rejected: Abandoned frozen cryosectioning and DAB staining of Matrigel plugs in favor of recommending whole-mount immunofluorescence due to severe freeze-thaw and fixative structural disruption.
Targeting the neuropilin-2 receptor signaling axis · Boston University · Master's
Considered and rejected
Considered and rejected: Rejected generating HE frozen sections from liquid nitrogen cryopreserved tissue due to freeze-thaw cycles (-20°C to -30°C) inducing artifacts and RNA degradation.
Optimierung der Präanalytik für die NGS-basierte (Next Generati-on Sequenzierung) Nukleinsäurediagnostik an Tumorgewebe Optimization of preanalytics for NGS-based (Next Generati-on Sequencing) nucleic acid diagnostics on tumor tissue · University of Marburg · PhD
Considered and rejected
Considered and rejected: Fixation of cryosections with acetone was discarded because it dissolved the silane coating of SuperFrost Plus slides, replacing it with ice-cold isopropanol.
Considered and rejected
Considered and rejected: Rejected cutting thicker cryosections (30 µm) for LCM because the ultraviolet laser could not penetrate the tissue.
Towards understanding the causal relationship between local biomechanics and atherogenesis in a model of human-like advanced coronary plaques · Imperial · PhD
Considered and rejected
Considered and rejected: Distilled water ice embedding for cryosectioning was rejected because tissue folded and tore; replaced with 7.5% HPMC + 2.5% PVP hydrogel.
Mass spectrometry imaging using desorption electrospray ionisation with histological imaging to characterise the chemical composition of burned skin · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected fibre preparation using a cryogenic microtome because it could damage the biofilm matrix and produce unrealistically short, heterogeneous fibres.
Cryo-EM grids exhibit support film aggregation, high background noise, and preferred particle orientation
Proteins often adhere preferentially to carbon support foils or aggregate onto grid bars rather than partitioning into vitreous ice holes. Furthermore, persistent orientation bias and continuous carbon film background noise restrict 3D reconstructions despite stage tilting and grid additives.
Tried and failed
glow discharge and plasma cleaning of grids applied to cryo-EM sample preparation of protein complexes. Reason: particles adhered preferentially to the carbon support foil instead of partitioning into the open ice holes
Structural and functional insights into Functional Amyloid biogenesis in Pseudomonas · Imperial · PhD
Tried and failed
open-hole cryo-EM grid preparation applied to recombinant multiprotein complex imaging. Reason: protein complex aggregated entirely onto the carbon support rather than entering vitreous ice holes
In vitro biochemical reconstitution and electron microscopy of early pre-DNA replication complex intermediates using human proteins · Imperial · PhD
Tried and failed
cryo-EM with stage tilting and nanowire grids applied to heterotrimeric coiled-coil protein complex. Outcome: no signal. Reason: persistent preferred particle orientation and high noise preventing high-resolution 3D reconstruction
Structural and biochemical investigations of collagen-I trimerization · MIT · PhD
Tried and failed
cryo-EM ab initio 3D reconstruction applied to membrane transporter structure determination. Outcome: data insufficient. Reason: high background noise and complete lack of top-down particle orientations prevented reliable 3D map generation
Investigation of the oligomeric state of the mycobacterial lipid transporter, MmpL3: biophysical and computational insights · Imperial · PhD
Tried and failed
combining tilted and additive-treated cryo-EM datasets applied to asymmetric macromolecular complexes with preferred orientation. Outcome: data insufficient. Reason: severe preferential orientation persisted despite grid additives and stage tilting, limiting 3D reconstruction resolution
Molecular Insights into Noncanonical Histone Chaperone Complexes · Harvard · PhD
Macromolecular complexes degrade and dissociate at the cryo-EM air-water interface
Blotting and plunge freezing without chemical stabilization expose fragile protein complexes to the air-water interface, resulting in severe denaturation and subunit disassembly. These interface interactions also exclude particles from the centers of open grid holes or induce severe particle aggregation that impedes classification.
Tried and failed
standard blot-and-plunge cryo-EM vitrification applied to fragile multimeric protein complexes. Outcome: unstable. Reason: blotting caused sample denaturation and complex disassembly at the air-water interface
Tried and failed
cryo-EM vitrification on unsupported holey grids applied to membrane protein-nanodisc complexes. Reason: severe particle aggregation and air-water interface exclusion from hole centers
Disentangling the structural and cellular driving forces of CD59 inhibition of membrane attack complex pore formation · Imperial · PhD
Tried and failed
Cryo-EM grid preparation and plunging optimization applied to heteromeric protein complexes. Outcome: unstable. Reason: Complex particles dissociated during grid freezing despite multiple grid types, additives, crosslinkers, and plunging methods
Biophysical Investigations of the Cytosolic Iron-Sulfur Cluster Assembly Pathway Late Acting Proteins · MIT · PhD
Tried and failed
cryo-EM vitrification without chemical cross-linking applied to membrane protein complexes on nanodiscs. Outcome: unstable. Reason: Sample denaturation at the air-water interface prevented particle alignment during 2D classification.
Disentangling the structural and cellular driving forces of CD59 inhibition of membrane attack complex pore formation · Imperial · PhD
Electron beam irradiation causes radiolysis and severe damage to cryogenic samples
Cryogenic scanning transmission electron microscopy on hydrated samples generates radiolysis species that accelerate polymer degradation relative to dry imaging. High beam doses and high voltage during energy-dispersive X-ray mapping also induce void formation, blistering, and structural radiation damage in cryo-preserved interphases.
Tried and failed
cryogenic scanning transmission electron microscopy applied to hydrated polymer membranes. Outcome: worse than baseline. Reason: ice radiolysis generated reactive species that accelerated polymer degradation compared to dry imaging
Quantitative multiscale methods for scanning transmission electron microscopy of low-dimensional materials in two and three dimensions · UT Austin · PhD
Tried and failed
serial energy-dispersive X-ray spectroscopy mapping applied to cryogenic sample 3D reconstruction. Reason: cumulative electron beam dose caused radiation damage to the cryogenic sample
Understanding the intrinsic structure and composition of sodium battery interfaces using cryo-focused ion beam milling and cryo-electron microscopy · UT Austin · PhD
Tried and failed
high-voltage energy-dispersive X-ray spectroscopy mapping applied to cryogenic beam-sensitive interphases and frozen electrolyte. Outcome: unstable. Reason: electron beam damage caused severe void formation and blistering in sensitive layers
Understanding the intrinsic structure and composition of sodium battery interfaces using cryo-focused ion beam milling and cryo-electron microscopy · UT Austin · PhD
Cryoprotectants and preparation additives introduce background noise and image degradation
Residual density gradient media and plunge-freezing cryoprotectants like glycerol increase ice thickness and worsen resolution during cryo-EM reconstruction. Omitting cryoprotectants in freeze-drying protocols also causes severe membrane disruption and uneven nucleic acid dispersion from thermal shock.
Tried and failed
density gradient centrifugation for sample synchronization applied to bacterial cells for cryo-electron microscopy. Outcome: no signal. Reason: residual gradient medium adhered to membranes, increasing ice thickness and background noise
Characterisation of novel motility proteins in flagellar motors using cryo-electron microscopy · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected omitting cryoprotectant in standard freeze-drying protocols, as Raman imaging demonstrated severe membrane disruption and uneven nucleic acid dispersion from thermal shock.
Study of antimicrobial and antioxidant properties of new materials for development of active food packaging · Politecnico di Torino · PhD
Tried and failed
adding glycerol as cryoprotectant during plunge freezing applied to cryo-EM protein sample preparation. Outcome: worse than baseline. Reason: failed to mimic fast-plunge improvements and worsened reconstruction resolution
Structural and biochemical investigations into the Neisseria gonorrhoeae ribonucleotide reductase · MIT · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Use cryo-fixation with freeze-substitution to validate Rickettsia parkeri-ER tether structures in situ without chemical fixation artifacts. Blocker: Requires wet-lab biological samples, cryo-fixation equipment, freeze-substitution apparatus, and electron microscopy facilities.
Left open
Refine cryoprotectant mixtures using 10%-50% PVA/sucrose/glucose and mount frozen samples directly during cryosectioning to prevent distortion. Blocker: Requires a wet lab, biological specimens (C. elegans), cryostat apparatus, and chemical reagents
Mapping the Spatial Transcriptome Across Whole Organisms · MIT · PhD
Left open
Conduct anchoring and photopolymerization gelation protocols below -20°C to capture dynamic biological processes in cells. Blocker: Requires wet lab equipment, biological specimens, specialized cryo-chemical reagents, and UV photopolymerization apparatus
Toward Ultra-Resolution Biomolecular Mapping in Cells with Expansion Microscopy · MIT · PhD
Left open
Detach etched anisotropic microparticles using ice peeling with frozen water blocks instead of microcontact printing or aqueous retrieval. Blocker: Requires a physical materials fabrication and wet laboratory with lithography and cryo-handling equipment.
Macrophage-hitchhiking Anisotropic Microparticles for Therapeutic and Diagnostic Applications · MIT · PhD
Left open
Adapt standard FFPE protocols or develop cryogenic sample preparation methods for s-SNOM imaging to avoid resin embedding artefacts. Blocker: Requires a wet lab, biological sample preparation facilities, cryogenic equipment, and an s-SNOM instrument
Intracellular mapping of functional chemistry and nanoparticle theranostics with infrared nano-imaging · Imperial · PhD
Left open
Flash-freeze 95 wt% mineral-loaded regolith simulants and low-mineral cryovolcanic solutions and measure their infrared spectra during heating. Blocker: Requires a high-vacuum chamber, liquid nitrogen cryocooling apparatus, syringe injection system, and RAIRS FTIR spectroscopy setup.
Infrared Spectroscopy at Three and Six Microns of Water Ices Mixtures of Astrochemical Interest · Georgia Tech · PhD
Left open
Apply a micromorphology approach to analyze the impact of cryogenic processes on diverse archaeological artifacts embedded in sediments. Blocker: Requires archaeological sediment samples, geological thin-section preparation, and petrographic microscopy lab equipment.
Left open
Test alternative cryogenic glues matched to the thermal contraction of invar from room temperature to 4K to prevent epoxy detachment. Blocker: Requires cryogenic physical lab equipment and materials testing apparatus.
Near-field-scattering-based optical control and Brillouin optomechanics in optical microresonators · Imperial · PhD
Left open
Develop alternative hydrate induction protocols to eliminate optical streak artifacts from residual thawed ice microdroplets without promoting freezing. Blocker: Requires a physical experimental wet lab setup for clathrate hydrate synthesis and optical diagnostics (PLIF/PIV)
Investigations of clathrate hydrate formation using advanced optical diagnostic techniques · Imperial · Doctorate
Left open
Measure ice trapping and release behavior under variable irradiation fluxes and repeated temperature cycles using UHV ice experiment setups. Blocker: Requires a cryogenic ultra-high vacuum (UHV) laboratory apparatus with FTIR, QMS, and VUV irradiation equipment
Decoding Solar System Volatile Compositions: Laboratory Investigations of Ice Entrapment and Deuterium Fractionation · Harvard · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Emma Couves · Imperial · PhD · 2022
Disentangling the structural and cellular driving forces of CD59 inhibition of membrane attack complex pore formation - Alexia Marinna Francoise Simon · Harvard · PhD · 2026
Decoding Solar System Volatile Compositions: Laboratory Investigations of Ice Entrapment and Deuterium Fractionation - Christina Evelina Buffo · Georgia Tech · PhD · 2025
Infrared Spectroscopy at Three and Six Microns of Water Ices Mixtures of Astrochemical Interest - Sorin Asha Srinivasa · MIT · PhD · 2025
Structural and biochemical investigations of collagen-I trimerization - Ruihan Zhang · MIT · PhD · 2025
Mapping the Spatial Transcriptome Across Whole Organisms - Muhammad Afiq Bin Kamel · Imperial · Doctorate · 2025
Investigations of clathrate hydrate formation using advanced optical diagnostic techniques - Samantha Sedor · Harvard · PhD · 2025
Molecular Insights into Noncanonical Histone Chaperone Complexes - Johannes Maximilian Schneider · Tübingen · PhD · 2025
Wachstumsverhalten und Charakterisierung von in vitro-3D-Zellkulturen des Harnblasenkarzinoms - Isobel Evans · Nottingham · 2025
Assessing the influence of fibre type and biofilms on the ingestion and retention of microfibres by freshwater invertebrates - Nanki Singh · Imperial · PhD · 2024
Characterisation of novel motility proteins in flagellar motors using cryo-electron microscopy - Sara Cioccolo · Imperial · PhD · 2024
Investigation of the oligomeric state of the mycobacterial lipid transporter, MmpL3: biophysical and computational insights - Alison Biester · MIT · PhD · 2024
Structural insights into microbial one-carbon metabolic enzymes: Ni–Fe–S-dependent carbon monoxide dehydrogenases and acetyl-CoA synthases
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