Library · Biology & lab methods

What Fluorescence In Situ Hybridization (FISH) got wrong, from 14 theses

Summary written by a model from the records below: The records document technical limitations and experimental failures encountered when implementing fluorescence in situ hybridization protocols across diverse biological specimens. Reported difficulties include optical resolution limits, autofluorescence interference, non-specific background amplification, probe penetration barriers, and physical sample loss. These records come from graduate theses, 2021 to 2024, 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.

theses

institutions

failure records

failure modes

Plus 8 problems the authors left open, listed at the end.

Failure modes, by theses reporting them

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Optical resolution limits and signal crowding impede target identification

5 theses · 3 institutions · 5 records

Host autofluorescence and weak signals obscure target detection

4 theses · 4 institutions · 4 records

Non-specific probe binding and amplification generate high background

3 theses · 2 institutions · 3 records

Probe size and matrix density restrict tissue penetration

3 theses · 2 institutions · 3 records

Extended processing causes sample detachment and substrate drying

1 theses · 1 institutions · 1 records

Optical resolution limits and signal crowding impede target identification

5 theses · 3 institutions

In dense transcript clusters or high-abundance RNA targets, optical crowding and diffraction limits prevent resolving individual signals without physical sample expansion. Inadequate optical resolution also prevents detecting spatial colocalization between sub-chromosomal loci and protein marks, falling short compared to single-cell sequencing.

Considered and rejected

Considered and rejected: Rejected standard in situ hybridization (ISH) and immunostaining assays for comparing accelerated vs control replacement conditions due to low resolution and lack of specificity compared to single-cell sequencing

From Cells to Molecules: An Overview on the Dynamics of Accelerated Whole Tooth Regeneration · Georgia Tech · PhD

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 · PhD

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 · PhD

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 · PhD

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.

Towards genome wide RNA imaging in single cells · Harvard · PhD

Host autofluorescence and weak signals obscure target detection

4 theses · 4 institutions

Host tissue autofluorescence, optical losses, and confocal photon noise frequently obscure genuine hybridization signals across low-copy targets. Standard single-probe or unamplified approaches yield weak staining and insufficient signal-to-noise ratios, leading to failure even after chemical quenching treatments.

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 · PhD

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 · PhD

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 · Johns Hopkins · Master's

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

Development of Optical Tools and Techniques Toward a Functional Connectomic Understanding of C. elegans · MIT · PhD

Non-specific probe binding and amplification generate high background

3 theses · 2 institutions

Non-split amplification methods and branched DNA can generate high background signals in negative controls through non-specific reactions. Secondary antibody labeling and repetitive genomic sequences similarly introduce severe non-specific background outside of target chromosomal regions.

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 · Master's

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 · PhD

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

Genome-scale imaging: from the subcellular structure of chromatin to the 3D organization of the peripheral olfactory system · Harvard · PhD

Probe size and matrix density restrict tissue penetration

3 theses · 2 institutions

Probes fail to penetrate effectively into the deeper layers of thick intact specimens, causing substantial loss of signal to noise. Extending probe concatemer lengths for signal amplification similarly impedes migration through hydrogel matrices and leads to sharp drops in signal.

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 · PhD

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 · PhD

Tried and failed

increasing concatemer length for signal amplification applied to in situ fluorescence hybridization. Outcome: worse than baseline. Reason: signal dropped off significantly beyond an optimal concatemer length threshold

Development of bipolar interneuron subtypes within the retinal space-time continuum · Harvard · PhD

Extended processing causes sample detachment and substrate drying

1 theses · 1 institutions

Standard wash steps cause severe detachment and loss when handling non-adherent suspension cells. In addition, extending hybridization durations past sixteen hours leads to slide drying and excessively high background.

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 · PhD

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 · Master's

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 · PhD

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 · PhD

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 · Master's

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 · PhD

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 · PhD

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 · PhD

Left open

Validate bacterial tissue presence and localisation using fluorescence in situ hybridization (FISH) and qPCR on trophoblast tissue samples. Blocker: Requires wet lab equipment, biological tissue samples, and specialized assay reagents (FISH/qPCR).

The reproductive tract microbiota and miscarriage · Imperial · Doctorate