Laser Scanning Multiphoton Microscopy – Focusing on Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging for Biomedical Applications
Laser scanning multiphoton microscopy (MPM) is considered as a non-invasive technology for three-dimensional imaging of complex biological tissues. The quantitative potential of the MPM is not investigated as much as qualitative imaging. To explore the quantitative aspects, MPM is here combined with fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging (FLIM) for different biomedical applications. The first part of the thesis (papers 1 and 2) emphasizes the importance of validation and optimization of an experimental MPM set up to develop a systematic methodology to combine MPM with FCS utilizing a single-photon counting method. A practical guideline featuring the theoretical and experimental boundaries to implement two-photon excited FCS in the MPM experimental setup is developed in paper 1. Concentration range, numerical aperture of the objective lens, and laser excitation power were found as prime factors to be optimized to study the diffusion time using MPM-FCS. To extend the applicability of MPM-FCS in biological samples, proof of principle was demonstrated by measuring the viscosity of collagen gel from the diffusion time measurements of Rhodamine B in different water glycerol mixtures (Paper 2). In the final part of the thesis (papers 3 and 4), MPM-FLIM was employed for different biomedical applications. An exploratory study was performed using MPM-FLIM for ex vivo investigations in positive and negative sentinel lymph nodes derived from melanoma patients (Paper 3). MPM-FLIM demonstrates the potential to differentiate atypical cells, healthy lymphocytes, and blood vessels in sentinel lymph nodes along with morphological features and fluorescence lifetime data. Two-photon spectral and FLIM characterization of the complex intrinsic cellular fluorophores such as nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and keratin in keratinocytes were performed to facilitate the non-invasive imaging of epidermal and dermal tissue cultures in vitro (paper 4). This study exposed the importance of keratin signal and should not be neglected when FLIM data is interpreted which needs to be done very carefully in complex biological samples. Taken together, this thesis demonstrates how to adopt MPM in combination with FCS and FLIM highlighting both the methodology development and biomedical applications.
Parts of work
paper 1 Fluorescence correlation spectroscopy combined with multiphoton laser scanning microscopy - a practical guideline. Jeemol James, Jonas Enger, and Marica B. Ericson. Applied Sciences 2021, 11(5). ::doi::10.3390/app11052122Paper 2 Validating multiphoton fluorescence correlation spectroscopy as a tool to measure viscosity dependent molecular diffusion Jeemol James, Jonas Enger, and Marica B. Ericson. [Unpublished manuscript 2022]Paper 3 Report on fluorescence lifetime imaging using multiphoton laser scanning microscopy targeting sentinel lymph node diagnostics. Jeemol James, Despoina Kantere, Jonas Enger, Jan Siarov, Ann Marie Wennberg and Marica B. Ericson. Journal of Biomed Optics 2020, 25(7). ::doi::10.1117/1.JBO.25.7.071204Paper 4 Changing the paradigm of interpreting cellular autofluorescence in context of multiphoton fluorescence lifetime imaging focusing on keratinocytes Monika Malak, Jeemol James, Marica B. Ericson. [Unpublished manuscript 2022]
Doctor of Philosophy
University of Gothenburg. Faculty of Science
Department of Chemistry and Molecular Biology ; Institutionen för kemi och molekylärbiologi
at 14.00 at PJ lecture hall, Institutionen för fysik, Kemigården 1, Göteborg
Date of defence
Laser scanning multiphoton microscopy
intrinsic cellular fluorophores.
fluorescence correlation spectroscopy
fluorescence lifetime imaging