Non-linear Optical Microscopy and Spectroscopy for Biomedical Studies
Abstract
This thesis is based on the application of non-linear optical microscopy and spectroscopy
techniques within biomedical research. Non-linear optical microscopy
gives the possibility of exciting fluorophores using near infrared light. This is an
advantage when working with biological tissue, which has low absorption in this
wavelength area, making up an ”open window”for non-invasive three dimensional
imaging. Of particular interest has been the study of fluorescent xenobiotics in
human skin using two-photon fluorescence laser scanning microscopy. The background
is the desire to develop new non-invasive tools to study topical drug
delivery and improve the understanding of mechanisms involved in contact allergy.
In addition, two-photon fluorescence microscopy is a potential tool for
non-invasive skin cancer diagnostics, which also is a topic of this thesis.
In order to acquire quantitative data, two-photon fluorescence microscopy has
been combined with fluorescence correlation spectroscopy (TPFCS). This is to
the best of my knowledge the first time TPFCS has been applied to study the
diffusion and distribution of fluorescent molecules in human skin.By the use of
this method a reactive compound, acting as a contact allergen, has been demonstrated
to bind to proteins in the top epidermal layers of the skin, resulting in a
significantly slower diffusion.
It has been proposed that endogenously formed protoporphyrin IX (PpIX) can be
applied to improve contrast when performing two-photon fluorescence microscopy
for diagnostics of non-melanoma skin cancer. In this thesis, it is demonstrated
that detection of two-photon excited fluorescence of endogenous PpIX in human
skin is not possible. Instead, it is preferable to use a slightly shorter wavelength,
i.e. 710 nm, to induce one-photon anti-Stokes fluorescence. This finding is of
great importance for continued work in the field, bringing non-linear optical microscopy
into the clinics.
Plasmonic noble metal nanoparticles, e.g. gold nanoparticles, have been proposed
as contrast enhancers for several biomedical applications. In this thesis,
gold nanoparticles have been explored with respect to their multiphoton induced
luminescence when combined with non-linear optical microscopy. By investigating
10 nm gold nanoparticles deposited on glass plates, it is here demonstrated
that aggregation and short inter-particle distances are prerequisites in order to
detect multiphoton induced luminescence. Thus detection of single particles in
a biological environment is unlikely, and future work should be undertaken to
explore how the clustering can be controlled in a biological environment to, e.g,
be used as a contrast mechanism.
Parts of work
Paper I: S.Guldbrand, C.Simonsson, M.Goksör, M.Smedh and M.B.Ericson. Two-photon fluorescence correlation spectroscopy combined with measurements of point spread function; investigations made in human skin, Optics Express, 18:15289 – 15302, 2010 ::doi::10.1364/OE.18.015289 Paper II: S.Guldbrand, V.Kirejev, C.Simonsson, M.Goksör, M.Smedh and M.B.Ericson. Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular
diffusion within human skin, European Journal of Pharmaceutics and Biopharmaceutics, accepted in October,2012 ::doi::10.1016/j.ejpb.2012.10.001 Paper III: D.Kantere, S.Guldbrand, J.Paoli, M.Goksör, D.Hanstorp, A.-M.Wennberg, M.Smedh
and M.B.Ericson. Anti-Stokes fluorescence from endogenously formed Protoporphyrin IX – Implications for clinical multiphoton diagnostics,
Journal of Biophotonics, accepted in August 2012 ::doi::10.1002/jbio.201200119 Paper IV: S.Guldbrand, H.Evenbratt, J.Borglin, V.Kirejev and M.B.Ericson. Multiphoton induced luminescence from 10 nm gold nanoparticles – the effect of interparticle distance and aggregation
Unpublished Manuscript
Degree
Doctor of Philosophy
University
Göteborgs universitet. Naturvetenskapliga fakulteten
Institution
Department of Physics ; Institutionen för fysik
Disputation
Onsdagen den 12 december 2012, kl 9:15, Kollektorn MC2, Kemivägen 9
Date of defence
2012-12-12
stina.guldbrand@physics.gu.se
Date
2012-11-21Author
Guldbrand, Stina
Keywords
Two-Photon Excitation Microscopy
Fluorescence Correlation Spectroscopy
Multiphoton Luminescence
Human Skin
Gold nanoparticles
Publication type
Doctoral thesis
ISBN
978-91-628-8599-1
Language
eng