Towards simplified spectral fingerprinting via optical phasor analysis
Overlapping fluorescence spectra in biological imaging often obscure the identity of individual components, making reliable spectral unmixing essential for accurate cellular and tissue analysis. Current approaches, such as hyperspectral imaging and fluorescence lifetime imaging (FLIM), provide relia...
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| Tipo de recurso: | tesis de maestría |
| Fecha de publicación: | 2025 |
| País: | España |
| Institución: | Universitat Politècnica de Catalunya (UPC) |
| Repositorio: | UPCommons. Portal del coneixement obert de la UPC |
| Idioma: | inglés |
| OAI Identifier: | oai:upcommons.upc.edu:2117/452662 |
| Acceso en línea: | https://hdl.handle.net/2117/452662 |
| Access Level: | acceso abierto |
| Palabra clave: | Fluorescence Microscopy Imaging systems Imaging Phasor Analysis Spectral Unmixing Microscòpia de fluorescència Imatgeria (Tècnica) Àrees temàtiques de la UPC::Enginyeria de la telecomunicació::Telecomunicació òptica::Fotònica |
| Sumario: | Overlapping fluorescence spectra in biological imaging often obscure the identity of individual components, making reliable spectral unmixing essential for accurate cellular and tissue analysis. Current approaches, such as hyperspectral imaging and fluorescence lifetime imaging (FLIM), provide reliable unmixing but are time-consuming, expensive, and technically demanding. These limitations motivate the need for simpler and faster alternatives to conventional spectral and lifetime imaging methods for unmixing overlapping emission spectra. In this work, we investigate a hardware-based method based on phasor analysis by using custom-designed sine and cosine filters. By recording only three images (sine, cosine, and unfiltered), phasor coordinates are computed pixel-wise to generate spectral distributions without the need for full spectral acquisition or lifetime fitting. The method was validated on biological samples, including HeLa cells stained with multiple fluorophores, as well as on Convallaria autofluorescence. Additionally, the method was validated for the separation of the second harmonic generation (SHG) and two photon excitation fluorescence (TPEF) in zebrafish tissue. Results demonstrate that filter-based phasor analysis achieves separability, while offering fast acquisition and simple implementation. |
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