Dispersion management and action cross section quantifications for three-photon microscopy of cardiac organoids

Williamson, Lewis (2026) Dispersion management and action cross section quantifications for three-photon microscopy of cardiac organoids. PhD thesis, University of Glasgow.

Abstract

Achieving high-contrast, high-resolution imaging at depth is the principal goal of any multiphoton microscopy system. To advance these capabilities, three-photon microscopy has been developed, exploiting longer excitation wavelengths and three-photon absorption—a higher-order nonlinear excitation mechanism. This approach enables deeper imaging with an improved signal-to-background ratio (SBR) at greater depths. However, the higher-order nonlinearity and longer excitation wavelengths impose constraints on the illumination source, necessitating the use of a laser system capable of producing non-standard wavelengths. Consequently, a pump laser and optical parametric amplifier (OPA) are required to generate infrared wavelengths with short pulse durations, with dispersion compensation provided by a separate pulse compressor.

Within a custom-built two-photon microscope, where a number of highly dispersive elements that significantly affect pulse duration are used. To address this, direct measurement of the pulse duration has been achieved using a novel intensity-only autocorrelator in combination with a custom-built prism compressor. This setup enables precise characterisation of ultrashort pulses at the focal plane of the microscope. Measurements across a range of pre-compensation levels applied via the prism compressor reveal the relationship between dispersion management and temporal pulse compression finding the shortest pulse duration, closely matching the theoretical limit of our Ti:Sa laser source of 139fs.

A custom-built two- and three-photon microscope has been developed and fully characterised in both temporal and spatial domains. The system achieved a spatial resolution of 0.93, 0.89, and 3.44µm along the X, Y, and Z axes respectively at 1300 nm. Temporal characterisation has also been performed, with dispersion management optimised across a range of suitable wavelengths, and the required level of pre-compensation has been determined. For wavelengths of 1300nm a pulse duration of 49fs has been formed and measured at the focal spot of the microscope, from our OPA. A direct measurement of two and three photon absorption has been taken by increasing the illumination power over a sample, taking the logarithmic of measured intensity and applied power.

Organoids represent an emerging area within biomedical research, offering valuable models for drug testing and holding potential clinical applications in tissue growth and repair. Cardiac and heart organoids were supplied, with the heart organoids comprising a more diverse range of cell types. Both cleared and uncleared samples of each tissue type were examined, with all four imaged to a depth of approximately 500 µm from the cleared samples using our custom multiphoton system. These images provided collaborators with essential insights into the structural composition of the cardiac and heart organoids, informing subsequent culturing methods. Quantitatively, the effective attenuation length (EAL) was calculated for the uncleared cardiac and heart organoids, yielding values of 272 µm and 303.5 µm, respectively

Using the in-depth characterisation of our system, the efficacies of three fluorescent dyes; Cy5 (two photon absorption), DAPI, and fluorescein (three photon absorption) in producing multiphoton interactions were measured. The action cross section was determined using the technique outlined by LaViollette et al. [1]. Employing custom optical parametric amplifier (OPA) interactions to narrow the spectral bandwidth of our laser source, measurements were conducted within a photon-counting regime. Despite certain errors associated with this regime, quantitative analysis was achieved for all three dyes across the wavelength range of 1150–1350 nm. When compared with the limited published data, our results show comparable two-photon interaction values for Cy5 and fluorescein, along with a novel finding for DAPI within the near-infrared (NIR) wavelength range.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from UK Research and Innovation (UKRI) under grant number 2813291.
• Subjects: Q Science > QC Physics
• Colleges/Schools: College of Science and Engineering > School of Physics and Astronomy
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Muellenbroich, Dr. Caroline
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-86092
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 Jul 2026 13:36
• Last Modified: 12 Jul 2026 11:42
• Thesis DOI: 10.5525/gla.thesis.86092
• URI: https://theses.gla.ac.uk/id/eprint/86092