Chapter Four · failure evidence
What Fluorescence In Situ Hybridization (FISH) got wrong, from 25 dissertations
The records describe technical challenges and procedural rejections encountered when applying fluorescence in situ hybridization across diverse biological samples. Investigators frequently faced issues including autofluorescence interference, optical resolution limits, non-specific background binding, probe penetration barriers, and sample loss during processing. These records come from PhD theses at 15 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.
Autofluorescence and weak staining obscure hybridization signals
Researchers encountered overwhelming host tissue and fixed specimen autofluorescence that obscured target signals despite chemical quenching treatments. Unamplified detection and optical losses further prevented target signals from being distinguished from background noise.
Tried and failed
un均为amplified fluorescence in situ hybridization applied to low-copy plasmid DNA detection. Outcome: no signal. Reason: Confocal photon noise and optical losses prevented separation between target and background signals.
MAPPING BACTERIA AND MOBILE GENETIC ELEMENTS IN HUMAN MICROBIOMES · Cornell
Tried and failed
chemical quenching and helper-assisted FISH applied to intracellular symbiont tissue localization. Outcome: no signal. Reason: overwhelming host tissue autofluorescence obscured hybridization signal despite multiple chemical quenching treatments
Evolutionary and metabolic consequences of symbiont addition and replacement in Brazilian treehoppers · Texas Tech
Considered and rejected
Considered and rejected: Single-probe ISH or FISH was rejected in favor of LISH-LNR rolling circle amplification because FISH spots are 10-fold smaller and lack adequate signal-to-noise ratio in autofluorescent FFPE tissue.
SCALING OF HIGH-PLEX, SPATIAL TRANSCRIPTOMICS IN ENDOMETRIAL CARCINOMAS USING NOVEL AUTOMATED DETECTION · JScholarship
Tried and failed
multiplexed fluorescent in situ hybridization applied to specific target gene detection in neurons. Outcome: no signal. Reason: Target gene yielded extremely low signal-to-noise ratio and weak staining intensity under imaging
Optical diffraction and crowding prevent resolution of dense targets
High transcript density and dense transcriptional clusters caused signal overlap and optical crowding that standard imaging could not resolve without physical sample expansion. Insufficient optical resolution also hindered the detection of spatial colocalization between genetic loci and protein markers.
Tried and failed
sequential multiplexed fluorescence in situ hybridization applied to high abundance RNA transcripts. Outcome: worse than baseline. Reason: optical crowding and signal overlap severely reduced detection efficiency without physical sample expansion
Visualizing organization of RNAs and proteins in cells using advanced optical imaging methods · Harvard
Tried and failed
combined immunofluorescence and fluorescence in situ hybridization applied to sub-chromosomal locus colocalization. Outcome: no signal. Reason: optical resolution was insufficient to detect spatial overlap between target loci and protein marks
Small chromosomal structural variants are detected by the meiotic machinery in C. elegans · Harvard
Tried and failed
reducing probe concentration and hybridization target length applied to single-molecule RNA fluorescence in situ hybridization. Reason: optical resolution limit prevented resolving individual transcripts within dense transcriptional clusters
Spatiotemporal analysis of Herpesvirus genome presentation and transcriptional progression · Imperial
Considered and rejected
Considered and rejected: Rejected relying solely on increasing sequential hybridization rounds without sample expansion to dilute spot density, because sample expansion additionally reduces total fluidic time and background fluorescence.
Non-specific probe binding and amplification generate high background noise
Hybridization assays produced high background signals due to secondary fluorophore-conjugated antibodies or non-specific amplification in negative controls. Probe libraries targeting repetitive genomic sequences also resulted in widespread non-specific binding.
Tried and failed
immuno-FISH combining protein immunolabeling and fluorescent probes applied to plant meiotic pollen cells. Reason: secondary fluorophore-conjugated antibody caused high non-specific background fluorescence outside chromosomes
Apply Immuno-FISH in Zea Maize · Cornell
Tried and failed
branched DNA and non-split hybridization chain reaction applied to single-molecule DNA fluorescence in situ hybridization. Reason: non-specific amplification produced high background signal in negative controls
MAPPING BACTERIA AND MOBILE GENETIC ELEMENTS IN HUMAN MICROBIOMES · Cornell
Tried and failed
multiplexed fluorescence in situ hybridization library design applied to genome-scale chromatin and RNA imaging. Reason: non-specific binding and high background from repetitive genomic target sequences
Physical diffusion barriers impede probe penetration into thick matrices
Hybridization probes suffered from poor penetration into the deeper layers of thick intact specimens despite extended incubation times. In hydrogels, increasing probe lengths restricted matrix penetration and led to variable signal intensity.
Tried and failed
whole-mount fluorescence in situ hybridization applied to thick intact tissue specimens. Outcome: no signal. Reason: poor probe penetration into deeper layers causing severe signal-to-noise loss despite increased hybridization time
Multi-omics characterisation of mouse gastrulation and organogenesis at single-cell resolution · Cambridge
Tried and failed
primer exchange reaction signal amplification applied to fluorescence in situ hybridization in hydrogels. Reason: extended probe lengths impeded hydrogel matrix penetration and created variable fluorescence signal per probe
Towards projectionFISH, a Method for Efficient Spatial Transcriptomics · Harvard
Protocol washing and extended hybridization cause sample loss and slide drying
Repeated washing steps during hybridization protocols caused extensive detachment and loss of non-adherent suspension cells. Hybridization steps extending beyond sixteen hours resulted in slide drying and excessively high background levels.
Tried and failed
standard wash steps for in situ hybridization applied to suspension cells. Reason: repeated washing caused extensive loss of non-adherent cells
Towards projectionFISH, a Method for Efficient Spatial Transcriptomics · Harvard
Tried and failed
extended duration in situ hybridization applied to tissue sections on slides. Reason: hybridization extending past 16 hours caused slide drying and excessively high background
Spatial and temporal characterization of genes expressed early in root and embryo development of plants · Iowa State
Investigators exclude in situ hybridization in favor of immunohistochemistry
In situ hybridization approaches were rejected for the evaluation of HER2 status and gene amplification. Protocols instead relied exclusively on immunohistochemistry scoring systems.
Considered and rejected
Considered and rejected: Rejected in-situ hybridization (FISH/CISH) for HER2 status evaluation, relying solely on immunohistochemistry with ultralow categorization
Expression und prognostische Aussagekraft von TROP2, FOLR1 und HER2 in Ovarialkarzinomen Expression and prognostic significance of TROP2, FOLR1 and HER2 in ovarian cancer · open_UMR Marburg DSpace 10.0
Considered and rejected
Considered and rejected: Excluded in situ hybridization (FISH/CISH) for HER2 gene amplification, relying exclusively on IHC scoring (0-3+).
Ductales Carcinoma in situ. Einfluss der molekularen Subtypen auf Ausbreitungsmuster und Resektionsstrategie · Publikationssystem UB Tuebingen
Left open by the authors
Problems the authors named and did not get to.
Left open
Employ fluorescence in situ hybridization to validate the presence and enhance specificity of Fusobacterium detection on FFPE mouse colon tissues. Blocker: Requires wet lab access, FFPE mouse tissue samples, and specialized histological/FISH reagents.
Fusobacterium-Stromal Cell Interactions in Colorectal Cancer · Harvard
Left open
Perform fluorescence in situ hybridization and quantify GRBV titers in dissected Spissistilus festinus salivary glands over time to determine tissue tropism. Blocker: Requires a wet biology lab, live insect colonies, infected plant tissues, dissection equipment, and FISH reagents.
TRANSMISSION BIOLOGY OF GRAPEVINE RED BLOTCH VIRUS BY SPISSISTILUS FESTINUS · Cornell
Left open
Measure H3 histone levels across cell sizes using fluorescence microscopy with fluorescent tags instead of western blotting. Blocker: Requires a wet biology laboratory, yeast strains with fluorescent tags, and fluorescence microscopy equipment
Coordination between cell size and chromatin structure · Imperial
Left open
Use fluorescence in situ hybridization (FISH) to visualize and determine niche-specific contributions of indole and serotonin combinations. Blocker: Requires a wet biology lab and fluorescence in situ hybridization (FISH) imaging equipment and reagents.
Interplay Between Tryptophan Metabolites and the Virulence Factors of Enteric Pathogens · DSpace at UTSWMED
Left open
Determine whether itb6 and ita7 are coexpressed in the same mesenchymal cells using single-cell RNA-seq or double fluorescent in situ hybridization. Blocker: Requires wet lab double FISH experiments or validation across specific Hofstenia miamia single-cell datasets.
Stem Cell Migration in the Early Stages of Regeneration in Hofstenia miamia. · Harvard
Left open
Combine single-cell sequencing genomics with multiplex fluorescence in situ hybridization (FISH) to reconstruct spatial anatomical locations of cells. Blocker: Requires wet lab experimentation, tissue samples, imaging hardware, and single-cell/FISH experimental assays.
Left open
Quantify Fgf signaling disruption in lrp13b zebrafish mutants using qPCR and single-molecule RNA in situ hybridization. Blocker: Requires wet lab facilities, zebrafish mutant lines, and reagents for qPCR and smFISH
A novel low-density lipoprotein receptor-related protein protects against environmental teratogens · UT Austin
Left open
Profile RNA transcripts of engineered bidirectional promoters using RNA-seq and single-molecule fluorescence in situ hybridization (smFISH). Blocker: Requires wet lab experiments, specialized biological assays (RNA-seq, smFISH), and engineered cell lines.
Advancing Genetic Circuit Design with Prediction, Reversible Logic, and Thermal Induction · Georgia Tech
Left open
Validate RNA-seq transcriptomic findings using secondary assays such as qPCR or in situ hybridization. Blocker: Requires wet lab facilities, tissue samples, and experimental assays (qPCR or in situ hybridization)
Mechanisms Driving the Skeletal Response to Mechanical Loading and Parathyroid Hormone · Cornell
Left open
Develop mixed culture assays paired with flow cytometry and fluorescent DNA tags to assess community-level viability after treatment. Blocker: Requires a wet lab, biological cultures, flow cytometers, and fluorescent DNA tags
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