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