Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet: (A) Emission profiles for four spectrally distinct fluorescent proteins used to tag specific cell types in C. elegans in combination with Calcein Violet-AM for cell viability and DRAQ5 to identify nucleic acids. (B) Extrachromosomal arrays with cell-type specific promoters driving expression of a fluorescent protein were microinjected into nonfluorescent C. elegans strain N2 worms. Progeny (F1 generation) were segregated to NGM plates and the F2 generation was evaluated for specific and complete fluorescent protein expression in the targeted tissue/cell-type. Lines were established in triplicate using progeny populations that exhibited strong and stable transmission (>75% progeny with positive fluorescence). (C) Extrachromosomal array integration to establish a 100% transmission rate of the transgene was facilitated by TMP-UV integration. 50-70 L4 stage transgenic worms were incubated in M9 medium containing trimethylpsoralen and then exposed to UV irradiation prior to incubation overnight at 15°C with Escherichia coli OP50 (Day 1). Next day, surviving worms were segregated to individual 6cm NGM plates for brood laying. Any surviving adult worms were transferred to a fresh 6cm NGM plate every day for 3 days to segregate progeny laid on days one, two, and three (Day 2-5). Fluorescent F1 progeny were picked into 96-well plates (∼150-200 L4s) containing growth media and incubated at 20°C with shaking until the F2 brood was laid (Day 6-9). All wells of each 96-well plate were screened for fluorescent progeny using the high-content imaging system. Wells where >75% of F2 progeny were fluorescent positive were identified as potential integration events (Day 10-12). Six F2 progeny from each independent integration event were segregated into individual wells of a 96-well plate containing growth media to screen for homozygous fluorescent F3 populations (Day13-15). (D) C. elegans strains expressing tissue-specific fluorescent proteins in the intestine (YFP), body muscle (mCherry), pharyngeal muscle (GFP), and neurons (mKO2; pan-neuronal). Scalebar=100µm.

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Expressing, Transmission Assay, Fluorescence, Transgenic Assay, Incubation, Irradiation, Imaging

Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet: F2 larvae with YFP + intestinal cells giving rise to no fluorescent progeny (left), partial YFP + F3 population (middle), and complete YFP + expression in the intestines of all F3 progeny (right).

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet: (A) Schematic of mating scheme to assemble the CEL eidoscope worm strain. Combining the fluorophores through C. elegans mating required every promoter-driven fluorophore to have randomly integrated on a different chromosome. The integrated array producing ZAM36( vha-6 p::YFP) is located on the X chromosome. Therefore all of the fluorescent markers were consolidated into a single C. elegans strain by subsequent mating of fluorescent males to YFP expressing hermaphrodites. Five YFP-hermaphrodites were mated with 12-15 fluorescent males (GPF/mCherry/mKO2) and co-incubated for 16 hrs and then hermaphrodites were segregated to individual 6 cm NGM plates to identify hermaphrodites that had successfully mated. Successful mates produced F1 progeny with the combined fluorescent profiles (YFP/GFP), which were propagated to new NGM plates (5 L4s/plate for 7 total plates). The F2 population was segregated to individual wells of a 96-well plate (200-384 total L4s) containing growth media and the F3 progeny were screened for wells where all progeny were expressing the desired fluorophores. Homozygous lines were maintained and mated with fluorescent males containing the next fluorophore to be added. (B) Confocal microscopy of young adult CEL eidoscope worm expressing all four tissue-specific fluorescent proteins.

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Expressing, Incubation, Produced, Confocal Microscopy

Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet: (A) Schematic showing the worm strains and cell suspensions used to define the spectral matrix prior to analyzing the CEL eidoscope cell suspension. Non-fluorescent N2 cell suspensions were used as autofluorescence controls as well as single-color technical controls for viability (Calcein Violet-AM) and nucleic acid-containing objects (DRAQ5). The individual fluorescent strains were used to define the YFP, mCherry, GFP, and mKO2 spectral profiles. Additional cell suspensions containing viability and nucleic acid dyes were sorted to compare gene expression profiles with sorted cell suspensions obtained from CEL eidoscope suspensions. (B) Gating strategy based on single-color populations used to identify and sort CEL eidoscope cell populations. (C) Comparison of expected and recovered cell populations based on known cell abundances in L4 stage C. elegans . (D) Imaging of the cell populations show small round neurons, large and spindle-shaped body muscle cells, large round intestine cells, and small oblong pharynx muscle cells.

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Suspension, Gene Expression, Comparison, Imaging

Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet: (A) Expression of tissue-specific genes (TPM; left) and read alignment to fluorescent protein sequences (CPM; right) across CEL eidoscope samples. Tissue markers validate population enrichment, and fluorescent protein alignment confirms construct expression. (B) Hierarchical clustering based on z-score-normalized expression of most expressed C. elegans genes. Clusters 4, 5, 1, and 6 show preferential upregulation in YFP + , GFP + , mCherry + , and mKO2 + populations, respectively. (C) Projection of population-enriched gene clusters onto the CeNGEN single-cell C. elegans reference atlas UMAP using module score analysis (left). Corresponding annotated cell types are shown (right), demonstrating enrichment in neuronal, muscle/mesodermal, intestinal, and pharyngeal muscle populations.

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Expressing, Construct, Single Cell

Journal: bioRxiv

Article Title: CEL eidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

doi: 10.64898/2026.03.25.714250

Figure Lengend Snippet:

Article Snippet: Ligation of the backbone, promoter, and fluorescent protein sequences was performed with T4 DNA Ligase (NEB) per the manufacturer’s instructions.

Techniques: Construct