What Immunofluorescence & Confocal Microscopy got wrong, from 44 theses

Tried and failed

immunofluorescence with commercial polyclonal antibodies applied to target protein histological detection. Outcome: no signal. Reason: antibodies showed broad non-specific or inconclusive staining patterns

Profiling Host Respiratory Responses to SARS-CoV-2 Infection · MIT · PhD

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

immunofluorescence co-staining with commercial antibodies applied to transcription factor detection in embryonic tissue. Outcome: no signal. Reason: commercial antibodies lacked reliability and signal compatibility under multiplex fixation conditions

Investigating the role of microRNA-7 in pancreatic islet development · Imperial · PhD

Tried and failed

immunofluorescence on hydrogels embedded with fluorescent beads applied to focal adhesion quantification in cultured cells. Outcome: no signal. Reason: non-specific dotty background staining obscured target signal preventing quantification

Mechanotaxis and mechanoresistance in cancer · Imperial · PhD

Lost to a baseline

Monovalent NHS-functionalized M41 (M41647_NHS) performed worse in immunofluorescence detection of GFP-Miro1 compared to indirect detection using unlabelled monovalent M41 plus secondary anti-VHH antibody.

Nano- and chromobodies for the structural and functional analysis of the mitochondrial outer membrane associated components, Miro1 and DRP1 · Tübingen · PhD

Considered and rejected

Considered and rejected: Pivoted away from immunofluorescence imaging to subcellular fractionation and western blotting due to poor antibody staining quality and weak signal.

Sex Differences In Endothelial FoxO1 Response To Nutrient And Oxidative Stress · York University · Master's

Tried and failed

immunofluorescence assay for protein detection applied to isolated primary cells. Outcome: no signal. Reason: non-viable primary antibody from improper storage and microscope hardware malfunction

Role of ATP in activating NLRP3 inflammasome in human placental trophoblasts · Nottingham · Master's

Tried and failed

polyclonal antibody immunofluorescence detection applied to bacterial detection in tissue sections. Outcome: no signal. Reason: nonspecific cross-reactivity in negative-control tissue samples lacking the target organism

Fusobacterium-Stromal Cell Interactions in Colorectal Cancer · Harvard · Master's

Tried and failed

immunofluorescence quantification using target-specific antibodies applied to intracellular viral particle quantification. Outcome: no signal. Reason: nonspecific cross-reactivity and particle size heterogeneity prevented accurate measurement

Spatiotemporal host and viral transcriptomics, host restriction and DNA sensing after HSV-1 infection · Imperial · PhD

Tried and failed

phosphospecific antibody immunofluorescence detection applied to target protein phosphorylation in yeast cells. Outcome: no signal. Reason: antibody exhibited non-specific staining failing to differentiate wild-type from deletion mutant cells

The spatiotemporal control of CDK activity · Imperial · PhD

Tried and failed

multiplexed cyclic immunofluorescence staining applied to candidate antibodies on tissue microarrays. Outcome: no signal. Reason: candidate antibodies failed initial staining validation and sensitivity criteria across iterative cycles

Using Cyclic Immunofluorescence (CyCIF), Deep-learning, and Human Tumor Microarrays (hTMAs) for Antibody Validation and Creation of an Atlas of Cancers Emphasizing Unique Phenotype · Harvard · Master's

Tried and failed

immunofluorescence with commercial antibodies applied to host protein localization during viral infection. Outcome: no signal. Reason: antibodies exhibited high variability, low signal, and cross-reactivity with viral antigens

BAF CHROMATIN REMODELING COMPLEXES DURING HERPES SIMPLEX VIRUS 1 INFECTION · Cornell · PhD

Considered and rejected

Considered and rejected: Staining fibronectin as a proxy for collagen in dECM due to technical difficulties with collagen antibodies in immunofluorescence.

THE PLANTAR MECHANICAL MICROENVIRONMENT PROMOTES PROLIFERATION AND INVASION OF ACRAL MELANOMAS · Johns Hopkins · PhD

Tried and failed

reducing antibody concentration and increasing wash duration applied to immunofluorescence imaging of tissue sections. Reason: failed to reduce endogenous background autofluorescence in tissue sections

Change, Source, and Effect of GDF11 in Mouse Spleen · Harvard · Master's

Considered and rejected

Considered and rejected: Standard immunofluorescence with AP-Fast Red on archived paraffin sections (rejected due to inconsistent staining/autofluorescence in favor of standard IHC).

Peripheral contributions to the development and maintenance of inflammation and pain in arthritis Peripheral contributions to the development and maintenance of inflammation and pain in arthritis - A VEGF-A story · Nottingham · PhD

Considered and rejected

Considered and rejected: Rejected automated confocal microscopy imaging and plate-reader fluorescence assays in favor of PBS recovery followed by flow cytometry due to scaffold autofluorescence and optical interference

Impact of the Tumor ECM Niche On AML Drug Sensitivity: A Comparative Screening Study · York University · Master's

Tried and failed

direct multi-channel segmentation without common reference channel applied to fluorescence microscopy cell counting. Outcome: no signal. Reason: wide fluorophore intensity disparities, cytoplasmic fluorophore leakage, and autofluorescence prevented reliable object detection

Development of oblique plane microscopy and its application to time-lapse high-content imaging of live oncogene-expressing spheroids · Imperial · PhD

Tried and failed

three-marker immunofluorescence gating applied to spatial transcriptomics region selection. Reason: unquenchable intrinsic tissue autofluorescence caused cell phenotype misclassification

Deciphering Virus-Mediated Remodeling of the Lymphoma Tumor Microenvironment Through Spatial Multi-Omics · Harvard · PhD

Tried and failed

fluorescence microscopy membrane integrity assay applied to fungal hyphae cytoplasmic retraction. Outcome: no signal. Reason: sample autofluorescence at 544 nm obscured the fluorescence signal needed to assess membrane integrity

Spores-on-a-Chip: investigations on arbuscular mycorrhizal fungi using novel microfluidic technology · Imperial

Tried and failed

changing secondary antibody to reduce autofluorescence applied to immunofluorescence tissue staining. Outcome: no signal. Reason: endogenous tissue autofluorescence persisted independently of the secondary antibody used

Change, Source, and Effect of GDF11 in Mouse Spleen · Harvard · Master's

Tried and failed

DRAQ5 with FITC Cy3 immunofluorescence multiplexing applied to FFPE tissue section imaging. Outcome: no signal. Reason: High tissue autofluorescence and overlapping emission spectra corrupted signal separation

Analysing the relationship between immune infiltration and tissue architecture in high grade serous ovarian cancer · Cambridge · PhD

Tried and failed

fluorophore-tagged single-molecule tracking microscopy applied to bacterial cellular protein imaging. Outcome: no signal. Reason: excitation wavelengths generated high cellular autofluorescence background, obscuring fluorophore signals

METAL TRAFFICKING DURING LIMITATION AND EXCESS IN BACILLUS SUBTILIS · Cornell · PhD

Considered and rejected

Considered and rejected: Rejected widefield epifluorescence microscopy due to cuticle autofluorescence obscuring epithelial structures.

Optimisation of a protocol for the study of subcellular cAMP compartmentalisation in epithelial cells of Drosophila Melanogaster · Nottingham · Master's

Considered and rejected

Considered and rejected: Performing immunofluorescence staining directly on cells lodged inside microfluidic chips, rejected due to reagent diffusion gradients, cell-debris colocalisation, and insufficient cell numbers for statistics.

Biomechanical activation of pro-metastatic programs in circulating tumour cells by vasculature constriction forces · Imperial · PhD

Tried and failed

immunofluorescence staining with primary antibodies applied to 3D cell culture spheroids. Outcome: no signal. Reason: antibodies failed across a wide dilution range from 1:1000 to 1:50

Investigation into the interplay between cancer cells and their associated fibroblasts in malignant pleural mesothelioma: therapeutic implications · Imperial · PhD

Tried and failed

3D immunofluorescence staining of encapsulated cells applied to intracellular proteins in synthetic hydrogels. Outcome: no signal. Reason: Staining remained diffuse with poor penetration and background, obscuring distinct focal adhesions and stress fibers.

DEVELOPMENT OF A 3D TRACTION FORCE MICROSCOPY PLATFORM USING PEG-4MAL HYDROGELS FOR THE STUDY OF 3D VINCULIN MECHANOTRANSDUCTION · Georgia Tech · PhD

Tried and failed

immunofluorescent staining quantification applied to dense 3D hydrogel cell cultures. Outcome: no signal. Reason: unreliable antibody penetration and staining in dense 3D culture prevented quantification

Generation of microvascular endothelium in induced pluripotent stem cell-derived myocardium · Imperial · PhD

Tried and failed

immunofluorescence staining in hydrogel sandwich culture applied to cells encapsulated between hydrogel layers. Outcome: no signal. Reason: antibodies could not penetrate the dense hydrogel network to reach the interface

Dissecting Notch ligand-induced muscle stem cell fates via engineered ex vivo niches. · Cornell · PhD

Tried and failed

tissue clearing combined with antibody fluorescence microscopy applied to deep tissue fungal visualization. Outcome: no signal. Reason: ineffective staining penetration, slow, expensive, and unable to detect fungal conidia

In pursuit of the antifungal basis of recombinant interferon gamma immunotherapy: a quantitative approach · Imperial · PhD

Tried and failed

fluorescent probe staining for cell viability applied to cells in dense 3D hydrogel matrices. Outcome: no signal. Reason: inconsistent diffusion of fluorescent probes through dense 3D matrix prevented reliable quantification

Generation of microvascular endothelium in induced pluripotent stem cell-derived myocardium · Imperial · PhD

Tried and failed

conventional immunofluorescence microscopy applied to dense cell colony surface glycans. Outcome: no signal. Reason: optical crowding and limited resolution masked target epitopes in dense tissue and colony centers

Roles for cell surface glycans in guiding human pluripotent stem cell fate · MIT · PhD

Considered and rejected

Considered and rejected: Discarded TIRF and confocal microscopy (including PLCδ1-PH-GFP translocations) in Xenopus oocytes due to high cytosolic GFP background and lack of inter-oocyte reproducibility in favor of the chemiluminescent HA-tag surface assay.

Charakterisierung der Voltage Sensitive Phosphatase von Säugetieren in Xenopus laevis Oozyten Characterization of mammalian voltage-sensitive phosphatase in Xenopus laevis oocytes · University of Marburg · PhD

Considered and rejected

Considered and rejected: Epifluorescence and confocal microscopy were rejected for focal adhesion visualization due to excessive background and cytosolic fluorescence obscuring the cell-substrate interface

Mechanotaxis and mechanoresistance in cancer · Imperial · PhD

Considered and rejected

Considered and rejected: Rejected HEK293T cells in favor of DF-1 chicken fibroblast cells for indirect immunofluorescence assays because DF-1 cells offer a spread-out morphology allowing clearer optical separation between nucleus and cytoplasm.

Functional characterization of SARS-CoV-2 nucleocapsid protein: nuclear trafficking and its impact on host transcriptional regulation · Boston University · PhD

Tried and failed

single fluorescent protein endogenous tagging applied to low-abundance protein quantification in live tissue. Outcome: no signal. Reason: Fluorescence intensity from a single fluorophore tag was too weak to distinguish from background on confocal microscopy

Cell-specific spindle alignment in epithelial tissue via RASSF proteins · Imperial · PhD

Considered and rejected

Considered and rejected: Rejected standard confocal microscopy for visualizing and quantifying in vivo syncytiotrophoblast fusion due to insufficient optical resolution to clearly resolve syncytiotrophoblast boundaries.

Interferon Inhibits Trophoblast Fusion and Contributes to Male-female Dimorphism at Mouse Placenta During Maternal Immune Activation · Harvard · Master's

Tried and failed

computational adaptive optics with focus scanning applied to deep tissue optical coherence microscopy. Outcome: no signal. Reason: multiple scattering scrambled optical phase at depths exceeding seven scattering mean free paths

Computational and Hardware Adaptive Optics for Biomedical Imaging with Optical Coherence Tomography · Cornell · PhD

Tried and failed

near-infrared continuous wave optical microscopy applied to deep scattering tissue imaging. Outcome: no signal. Reason: Severe optical scattering at shorter wavelengths prevented resolving deep structural features despite high power.

Deep tissue imaging with short-wave infrared light and adaptive optics · Cornell · PhD

Considered and rejected

Considered and rejected: Rejected evaluating individual myelin sheath pathologies via spectral confocal reflectance (SCoRe) microscopy in thick monkey white matter due to excessive light reflectance from dense fiber packs

Neuroinflammation and T cells in the aging monkey brain: relationships with white matter damage and cognitive decline · Boston University · PhD

Considered and rejected

Considered and rejected: Discontinued 2-Photon microscopy due to insufficient signal intensity and slow point-by-point scanning speed for capturing dynamic z-stacks

Mesenchymal-to-Epithelial Transition of the Drosophila melanogaster embryonic midgut Mesenchymaler-zu-Epithel-Übergang des embryonalen Mitteldarms von Drosophila melanogaster · University of Marburg · PhD

Tried and failed

calcofluor white fluorescent staining for timelapse imaging applied to live fungal cell imaging in microfluidics. Outcome: no signal. Reason: dye aggregation and severe background fluorescence fluctuations in media over time

Microbes-on-a-chip: deciphering the responsiveness of microbes using microfluidic chemostats · Imperial · PhD

Considered and rejected

Considered and rejected: Rejected confocal microscopy for live intracellular pH quantification due to rapid SNARF-AM dye fluorescence decay and high cell mortality during setup.

Using pH to understand species-specific coccolithophore dynamics: a multi-disciplinary approach · Oxford · PhD

Considered and rejected

Considered and rejected: Rejected using fluorescence/confocal microscopy for long-term topography timelapses due to photobleaching, refractive index mismatch at air interface, and inability to label non-model isolates.

Quantitifaction and Modeling of Biofilm Development through Interferometry · Georgia Tech · PhD

Tried and failed

Dual-colour total internal reflection fluorescence microscopy applied to single-molecule membrane translocation tracking. Outcome: no signal. Reason: Severe fluorophore photobleaching in the evanescent field during prolonged single-molecule observation events

Improving droplet interface platforms for nanopore sensing · Imperial · PhD

Tried and failed

confocal microscopy with custom flow cell applied to in situ biofilm growth monitoring. Reason: standard confocal instruments were physically incompatible with the custom flow cell geometry

The effects of air/water interface properties on the formation of biofilm by P. aeruginosa · Texas Tech · PhD

Tried and failed

direct embedding of liquid suspension in hydrogel applied to liquid biopsy cellular imaging. Reason: sample dispersed across the Z-axis, severely hindering optical microscopy tracking

Developing Tools to Physically Magnify Biological Substrates for Clinical Applications · MIT · PhD

Considered and rejected

Considered and rejected: Quantifying macrophage transwell migration on cut-out membranes by fluorescence microscopy was abandoned because excised membranes did not stay flat in a single optical plane.

Expression and function of human macrophage heparan sulfate proteoglycans · Oxford · PhD

Left open

Perform immunofluorescence staining for FSP-1, podoplanin, and periostin CAF markers in colorectal cancer tumor microenvironment models. Blocker: Requires a wet biology lab, mouse CRC tissue sections, specific antibodies, and immunofluorescence imaging equipment.

Fusobacterium-Stromal Cell Interactions in Colorectal Cancer · Harvard · Master's

Left open

Evaluate alternative cellular stains and fluorescently tagged antibodies to improve signal uniformity and brightness over SYTO-9 staining. Blocker: Requires wet lab access, biological samples, staining reagents, and fluorescence microscopy equipment.

Density-shift Immunomagnetic Separation for Pathogen Retrieval from Complex Media · MIT · PhD

Left open

Perform immunofluorescence analysis on spleen and CNS tissues to measure spatial proximity between TIGIT+ Tregs and Th1 cells. Blocker: Requires wet-lab animal tissue harvesting, sectioning, and immunofluorescence microscopy

PD-1 function on T cells in spontaneous and anti-PD-1-mediated autoimmunity · Harvard · PhD

Left open

Probe additional EGFR phosphorylation sites such as Y992 using western blot or immunofluorescence across lung tissue regions. Blocker: Requires wet lab facilities, biological samples, and wet-lab experimental protocols (western blotting, immunofluorescence).

Epidermal growth factor receptor signalling in the pulmonary immune response - diverging roles in early life and adulthood? · Imperial · PhD

Left open

Use distinct fluorescent emission wavelengths (red in gel, green in suspension) to eliminate background noise in confocal channels during traction rheoscopy. Blocker: Requires a wet lab, flow cell apparatus, fluorescent particles/gel preparation, and confocal microscope

Spatio-temporal Measurements of Dynamic Suspensions · Harvard · PhD

Left open

Determine if HDAC7 isoforms are differentially localized using immunofluorescence or fluorescent protein fusions in unstimulated versus stimulated conditions. Blocker: Requires a wet lab and cell culture to perform immunofluorescence or fluorescent protein fusion experiments.

Role Of Pre-Mrna Alternative Splicing In Protein Diversity And Epigenetics · Penn · PhD

Left open

Perform immunofluorescence microscopy with organelle-specific markers to assess intracellular trafficking and localization of VWF-HRP fusion constructs in ER, Golgi, and WPBs. Blocker: Requires wet-lab facilities, cell culture, recombinant VWF-HRP constructs, and fluorescence microscopy equipment.

The impact of multiple myeloma and carfilzomib on the endothelium · Imperial · PhD

Left open

Determine if STING colocalizes and interacts with TLR8 and IKKε in the Myddosome using immunofluorescence microscopy and co-immunoprecipitation. Blocker: Requires a wet laboratory, physical cell cultures, and biological assays (immunofluorescence microscopy and co-immunoprecipitation).

ELUCIDATING THE INTRICATE CROSSTALK BETWEEN PATTERN RECOGNITION RECEPTORS AND HOST-MICROBIOME INTERACTIONS FOR DISEASE THERAPEUTICS · Cornell · PhD

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