Yike Xie1,9, Abbas Habibalahi2, Ayad G. Anwer2, Kanu Wahi3, Jacqueline Bailey4, Francis Lin4, Catherine Gatt1, Emma M.V. Johansson5, Tatyana Chtanova4, Jeff Holst3, Ewa Goldys2,8 and Fabio Zanini1,6,7,8 1School of Clinical Medicine, UNSW Sydney, Sydney, New South Wales 2052, Australia; 2School of Biomedical Engineering, UNSW Sydney, Sydney, New South Wales 2033, Australia; 3School of Biomedical Sciences, UNSW Sydney, Sydney, New South Wales 2052, Australia; 4School of Biotechnology and Biomolecular Sciences, Faculty of Science, UNSW Sydney, Sydney, New South Wales 2052, Australia; 5Flow Cytometry Unit, Mark Wainwright Analytical Centre, UNSW Sydney, Sydney, New South Wales 2033, Australia; 6UNSW Cellular Genomics Futures Institute, UNSW Sydney, Sydney, New South Wales 2052, Australia; 7Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Sciences, UNSW Sydney, Sydney, New South Wales 2033, Australia

↵8 These authors contributed equally to this work.

↵9 Present address: Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg SE-413 90, Sweden

Corresponding authors: fabio.zaniniunsw.edu.au, e.goldysunsw.edu.au Abstract

Microscopy and omics are complementary approaches to probe cellular molecular states in health and disease, combining granularity with scalability. However, integrating both imaging- and sequencing-based assays on the same cell has proven challenging. This study demonstrates a new approach called SpectralSeq that combines hyperspectral autofluorescence imaging with transcriptomics on the same cell. SpectralSeq is applied to Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and identifies a subpopulation of cells exhibiting bright autofluorescence rings at the plasma membrane in optical channel 13 (λex = 431 nm, λem = 594 nm). Correlating the presence of a ring with the gene expression in the same cell indicates that ringed cells show higher expression of apoptosis-related genes and lower expression of ATP production genes. Furthermore, correlation of cell morphology with gene expression reveals downregulation of multiple spliceosome members in larger MCF-7 cells. Multiple genes exhibit consistent expression across cell sizes but varied exon usage. Finally, correlation between gene expression and fluorescence within the spectral range of nicotinamide adenine dinucleotide hydrogen (NADH) provides insights into the metabolic states of MCF-7 cells. Overall, SpectralSeq links optical spectrum with internal molecular states, offering a single streamlined workflow for single-cell resolution studies integrating spectral, morphological, and transcriptomic analyses.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.280014.124.

Freely available online through the Genome Research Open Access option.

Received September 11, 2024. Accepted June 6, 2025.