Chapter Four · failure evidence
What Microscopy Sample Cryo-Preparation got wrong, from 38 dissertations
Cryogenic preparation for microscopy frequently encounters structural artifacts ranging from ice crystallization and sectioning damage to particle denaturation at the air-water interface. In addition, specimens often suffer from preferential orientation bias during grid preparation and severe beam-induced radiolysis during cryogenic imaging. These records come from PhD theses at 16 institutions, 2021 to 2026. 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.
Ice crystal formation and freezing artifacts compromise specimen morphology during cryofixation
Rapid freezing and plunge methods can lead to ice crystallization, tissue cracking from vapor insulation, or freeze-thaw damage that disrupts cellular integrity. These freezing artifacts obscure biofilm architecture, destroy biopsy histology, and hinder high-resolution cryogenic surface analysis.
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
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
Two muscle biopsy samples from the trained group (reducing trained sample size to n=8; 3 men, 5 women) and all muscle biopsy samples from the control group (n=8) were lost due to ice crystal artifact damage during freezing.
Histochemical changes in skeletal muscle after 12-weeks of resistance training in elderly men and women · Iowa State
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.
Delivery of cannabinoids to specific anatomical regions of the brain following oral administration to reach the site of action for glioblastoma multiforme and multiple sclerosis · University of Nottingham Repository
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 · University of Nottingham Repository
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 · open_UMR Marburg DSpace 10.0
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
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 · ResearchWorks
Protein complexes denature, aggregate, or adhere to support foils during cryo-EM vitrification
Exposure to the air-water interface during grid blotting and plunge freezing causes multimeric complexes to dissociate, denature, or aggregate outside the ice holes. Grid preparation treatments and support films can also promote particle binding to carbon surfaces or increase background noise, degrading final reconstruction resolution.
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
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
Considered and rejected
Considered and rejected: Rejected continuous amorphous carbon support films for cryo-EM due to excessive background noise and contrast reduction on small membrane proteins.
Cryo-Electron Microscopy of Nicotinic Acetylcholine Receptors · DSpace at UTSWMED
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
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
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.
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
Cryosectioning and mounting produce tissue tearing, detachment, and poor optical resolution
Frozen tissue sections frequently detach from coated glass slides during staining or tear when embedded in unsuitable matrices such as pure water ice. Furthermore, cryosectioning can yield thick or blurry sections that degrade immunofluorescence resolution and mechanical handling can disrupt delicate matrix fibers.
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
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 · Publikationssystem UB Tuebingen
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.
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.
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.
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.
Assessing the influence of fibre type and biofilms on the ingestion and retention of microfibres by freshwater invertebrates · University of Nottingham Repository
Preferred particle orientation hinders three-dimensional reconstruction in cryo-EM
Macromolecular samples often adopt severe orientation bias on cryo-EM grids, lacking views from specific angles such as top-down perspectives. Attempts to resolve this bias using grid additives, nanowires, or stage tilting fail to eliminate the preferred orientation and leave high background noise that limits map resolution.
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
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
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
Electron beam radiation causes radiolysis and structural degradation in cryogenic specimens
Cumulative electron exposure during cryogenic scanning transmission imaging and energy-dispersive X-ray mapping causes rapid radiolysis of surrounding ice. This beam-induced damage generates reactive species that degrade sensitive polymers and creates blistering or void formation in beam-sensitive layers.
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
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
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
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
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
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
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
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
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
Left open
Determine the high-resolution structure and stoichiometry of the native CRY:HK complex using cryo-EM. Blocker: Requires wet-lab protein purification and physical access to a cryo-EM facility
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
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