Few-cycle laser forreal-timenanomedicineresearchRodrigo Alberto de Jesus FerreiraMestrado Integrado em Engenharia FísicaDepartamento de Física e Astronomia2018

OrientadorProf. Dr. Helder Crespo, Docente, Departamento de Física e Astronomia daFCUP

CoorientadorDr. Jana B. Nieder, Head of Nanophotonics Department and Group Leader ofthe Ultrafast Bio- and Nanophotonics group, INL - International IberianNanotechnology Laboratory, INL

Todas as correções determinadas

pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, / /

Universidade do Porto

Master’s Thesis

Few-cycle laser for real-timenanomedicine research

Author:

Rodrigo Ferreira

Supervisor:

Prof. Dr. Helder Crespo

Co-supervisor:

Dr. Jana B. Nieder

A thesis submitted in fulfilment of the requirements

for the degree of Master of Science

at the

Faculdade de Ciencias

Departamento de Fısica e Astronomia

July 2019

“ Normality is a paved road: It’s comfortable to walk, but no flowers grow on it. ”

Vincent van Gogh

Acknowledgements

During the last year, I have had the opportunity to meet and work with wonderful

people and this thesis would not have been possible without their help and support. I have

learned a lot with all of them.

I was lucky enough to have not only one, but two amazing supervisors. I am very

grateful to Prof. Dr. Helder Crespo and Dr. Jana B. Nieder for all their guidance and

patience.

I was also fortunate to work in two different places, Porto and Braga. I would like

to thank everyone from University of Porto, in particular from Femtolab (IFIMUP-IN

and DFA-FCUP): Ana Vieira da Silva, Miguel Canhota, Miguel Miranda and Francisco

Carpinteiro, and from Sphere Ultrafast Photonics: Rosa Romero and Paulo Guerreiro.

It was also a pleasure to work with everyone in the Ultrafast Bio- and Nanophotonics

group at INL in Braga. Especially Dr. Christian Maibohm, for his help with the femtosec-

ond laser experiments and FLIM data analysis, Dr. Oscar Silvestre, for the preparation

of live cells and great support performing MP-FLIM experiments, Ricardo Adao and Dr.

Oleksandr Savchuk for their help with the microscope control software and Matlab analysis

toolbox.

Last but not least, I want to thank my family and friends for all the support given not

only during this thesis, but also throughout the university years.

UNIVERSIDADE DO PORTO

Abstract

Faculdade de Ciencias

Departamento de Fısica e Astronomia

Master of Science

Few-cycle laser for real-time nanomedicine research

by Rodrigo Ferreira

A few-cycle Ti:sapphire laser oscillator, pumped by a DPSS CW laser at 532 nm, was

built at the Femtolab (IFIMUP-IN and DFA-FCUP) and was characterized using state-of-

the-art patented technology (d-scan, from Sphere Ultrafast Photonics). This system is ca-

pable of delivering ultrashort laser pulses with 5 fs in duration and with an ultra-broadband

spectrum. It was then used to, in collaboration with INL, develop an application in the

field of nanomedicine cancer research using a Fluorescence Lifetime Imaging Microscopy

(FLIM) method. The broadband laser spectrum enabled the simultaneous excitation of

endogenous markers (NADH/NADPH) in living cells that indicate the metabolic state of

the cells while being exposed to innovative anti-cancer therapeutic drugs and/or nanodrug

delivery systems. Here, the well known anticancer drug Doxorubicin (DOX) and a commer-

cially available liposomal nanoformulation Doxilr encapsulating this drug, were studied

in vitro using HeLa cancer cells.

UNIVERSIDADE DO PORTO

Resumo

Faculdade de Ciencias

Departamento de Fısica e Astronomia

Mestrado em Engenharia Fısica

Few-cycle laser for real-time nanomedicine research

por Rodrigo Ferreira

Um laser de Titanio-safira de poucos ciclos, bombeado por um laser DPSS CW a 532

nm, foi contruıdo no Femtolab (IFIMUP-IN e DFA-FCUP) e caracterizado usando uma tec-

nologia patenteada (d-scan, Sphere Ultrafast Photonics). Este sistema e capaz de fornecer

pulsos de laser ultracurtos com duracao de 5 fs e com um espectro de banda ultra-larga. Foi

entao utilizado para, em colaboracao com o INL, desenvolver uma aplicacao no campo da

investigacao nanomedica do cancro utilizando o metodo de Fluorescence Lifetime Imaging

Microscopy (FLIM). O espectro do laser de banda larga permitiu a excitacao simultanea

de marcadores endogenos e marcadores (NADH / NADPH) em celulas vivas que indicam

o estado metabolico das celulas enquanto sao expostas a drogas terapeuticas anti-cancro

inovadoras e/ou sistemas de entrega de nanodrogas. Aqui, o bem conhecido farmaco anti-

cancro Doxorubicina (DOX) e uma nanoformulacao lipossomal comercialmente disponıvel,

Doxilr a encapsular este farmaco, foram estudados em celulas cancerıgenas HeLa in vitro.

Contents

Acknowledgements v

Abstract vii

Resumo ix

Contents xi

List of Figures xiii

Abbreviations xv

1 Introduction 1

1.1 Aim and Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Theoretical Background 3

2.1 Ultrashort Pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.1 Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.1.1 Spectral Phase . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1.2 Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.1.2.1 Dispersion Compensation . . . . . . . . . . . . . . . . . . . 5

2.1.3 Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.1.4 Second-Harmonic Generation . . . . . . . . . . . . . . . . . . . . . . 7

2.2 Femtosecond Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.2.1 Gain Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.2.2 Cavity Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.2.3 Standard Femtosecond Lasers . . . . . . . . . . . . . . . . . . . . . . 9

2.2.4 Few-Cycle Femtosecond Lasers . . . . . . . . . . . . . . . . . . . . . 9

2.3 Nanomedicine Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3.1 Nano Drug Delivery Systems . . . . . . . . . . . . . . . . . . . . . . 10

2.3.2 Fluorescence Lifetime Imaging Microscopy . . . . . . . . . . . . . . . 12

2.3.2.1 Time-Correlated Single Photon Counting . . . . . . . . . . 13

3 Few-Cycle KLM Laser 15

3.1 Basic Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.2 Cavity Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

xi

xii Few-cycle laser for real-time nanomedicine research

4 MP-FLIM Experiments 19

4.1 Experimental Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

4.2 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4.3 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5 Conclusions 31

Bibliography 33

List of Figures

2.1 Ultrashort pulse and associated spectrum. . . . . . . . . . . . . . . . . . . . 3

2.2 Grating compressor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.3 Prism compressor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.4 Simplified schematic of a chirped mirror. . . . . . . . . . . . . . . . . . . . . 6

2.5 Second harmonic generation. . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.6 Ti:sapphire absortpion/emission spectrum. . . . . . . . . . . . . . . . . . . . 8

2.7 Spectra Physics Tsunamir spectrum. . . . . . . . . . . . . . . . . . . . . . 9

2.8 Overview of typical nanostructures proposed for drug delivery systems. . . . 11

2.9 TCSPC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.1 Few-cycle Ti:sapphire laser schematic. . . . . . . . . . . . . . . . . . . . . . 15

3.2 Cavity Stability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.3 Laser spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.1 Normalized spectra of the 5 fs laser system with absorption spectra of dyes. 20

4.2 Experimental Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

4.3 Laser spectrum (left) and retrieved laser pulse (right). . . . . . . . . . . . . 22

4.4 Experimental d-scan (left) and retrieved d-scan (right). . . . . . . . . . . . 22

4.5 Fluorescence intensity (left) and confocal image (right) of HeLa cell incu-bated with free DOX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

4.6 Fluorescence intensity image of HeLa cell incubated with free DOX. . . . . 25

4.7 Fluorescence intensity and lifetime of HeLa cell over incubation time. . . . . 26

4.8 Fluorescence lifetime and respective histograms of components 1 and 2. . . 27

4.9 Fluorescence intensity image of HeLa cell. . . . . . . . . . . . . . . . . . . . 28

4.10 Fluorescence lifetime of components 1 and 2. . . . . . . . . . . . . . . . . . 28

4.11 Fluorescence lifetime and respective histograms of components 1 and 2. . . 29

4.12 Fluorescence intensity image of a 10 x 10 µm2 area. . . . . . . . . . . . . . 29

4.13 Fluorescence intensity image of a 40 x 40 µm2 area. . . . . . . . . . . . . . 30

xiii

Abbreviations

AOM Acousto-Optic Modulator

APD Avalanche Photodiode

ATP Adenosine Triphosphate

CEP Carrier-Envelope Phase

CW Continuous Wave

DCM Double Chirped Mirror

DNA Deoxyribonucleic Acid

DPSS Diode-Pumped Solid-State

FDA Food and Drug Administration

FLIM Fluorescence Lifetime Imaging Microscopy

GD Group Delay

GDD Group Delay Dispersion

IR Infrared

KLM Kerr-Lens Mode-Locking

LSM Laser Scanning Microscope

MEM Modified Eagle’s Medium

MP Multi-Photon

ML Mode-Locking

NADH Reduced Nicotinamide Adenine Dinucleotide

NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate

NIR Near-Infrared

xv

xvi Few-cycle laser for real-time nanomedicine research

NP Nanoparticle

PMT Photomultiplier Tube

ROI Region of Interest

ROS Reactive Oxygen Species

SHG Second-Harmonic Generation

TCSPC Time-correlated Single Photon Counting

TEM Transverse Electromagnetic

TOD Third-Order Dispersion

UV Ultraviolet

Chapter 1

Introduction

1.1 Aim and Outline

This thesis is the result of a joint master research project between University of Porto,

Sphere Ultrafast Photonics and International Iberian Nanotechnology Laboratory - INL.

The aim was to build an ultrabroadband laser capable of performing nonlinear microscopy

in the field of nanomedicine cancer research. University of Porto (IFIMUP-IN and DFA-

FCUP) and Sphere Ultrafast Photonics had the experience in the field of optics and few-

cycle lasers, while INL had the expertise regarding bioimaging and nonlinear microscopy.

The work presented in this thesis has been performed both in Porto and Braga.

This master’s thesis is organized as follows: The second chapter reviews the theoretical

background of all the concepts necessary to have a good understanding of the the work done,

from ultrashort pulses to nanomedicine; chapter three presents the few-cycle Ti:sapphire

laser built at Femtolab (University of Porto) and its unique features; chapter four gives a

detailed explanation of the campaign at INL, namely the experimental technique used, d-

scan compression and analysis of bioimaging measurements. Finally, chapter five discusses

the conclusions drawn from the all the work performed.

1

Chapter 2

Theoretical Background

2.1 Ultrashort Pulses

Ultrashort pulses play a very important role in numerous fields of science and technology.

There is no commonly accepted definition of “ultrashort”, but one usually applies this

term if the pulse duration is in the range of femtoseconds. The invention of the laser [1]

and later the development of mode-locked lasers [2] has made this kind of pulses become a

reality.

Figure 2.1: Ultrashort pulse and associated spectrum. Adapted from Jung et al. [3].

2.1.1 Description

As shown in Fig. 2.1, the time and spectral domain of an ultrashort pulse are related

quantities and one can represent them through the Fourier transform relations

3

4 Few-cycle laser for real-time nanomedicine research

U(ω) = F{U(t)} =

∫U(t)e−iωtdt (2.1)

U(t) = F−1{U(ω)} =

∫U(ω)eiωtdω (2.2)

The time duration and spectral width of a pulse are also related by the following

universal inequality:

∆t∆ω ≥ 1

2(2.3)

This classical-physics relationship, which leads to the quantum-mechanical time–energy

uncertainty principle, has several important consequences in the field of ultrashort light

pulses. The product of this quantities (known as the time-bandwidth product) is often

used to indicate how close a pulse is to the transform-limit, that is, for a given spectral

width there is a lower limit for the pulse duration. One could sum up all this by saying

that the shorter a pulse is, the wider its spectrum will be.

2.1.1.1 Spectral Phase

By expanding the spectral phase, φ(ω), in a Taylor series around the pulse central fre-

quency, ω0, one obtains

φ(ω) = φ0 + φ′0(ω − ω0) +1

2!φ′′0(ω − ω0)

2 +1

3!φ′′′0 (ω − ω0)

3 + ... (2.4)

where

φ0 = φ(ω0), φ′0 =

∂φ(ω)

∂ω|ω=ω0 , φ

′′0 =

∂2φ(ω)

∂ω2|ω=ω0 , φ

′′′0 =

∂3φ(ω)

∂ω3|ω=ω0 (2.5)

etc. The first term (φ0) is the absolute phase, the propagation phase added to all frequen-

cies and also called the carrier-envelope phase. The second term (φ′0) is the group delay

(GD), which is the entire pulse delay when in a dispersive medium compared to a pulse

propagating in free space. The quadratic term of the expansion is the first that leads to a

deformation of the pulse envelope. It is usually called group delay dispersion (GDD) and

it is a frequency-dependent group delay of the different spectral components of the pulse.

For simplicity, higher order terms in the phase expansion, which correspond to third-,

fourth- and fifth-order dispersion effects, are neglected; yet, the higher order terms (along

with the overall gain bandwidth) are eventually the limiting factor for the shortening

mechanism of the pulse duration.

2. Theoretical Background 5

2.1.2 Dispersion

The broad bandwidth of such pulses originates considerable technical challenges. During

propagation in a dispersive media, for instance glasses or gases (i.e., air), the carrier and

the envelope travel at different speeds, and longer wavelengths will usually travel faster.

This changes the shape of the electric field of the pulse and, as seen before, two quantities

are introduced, the group delay (GD) and the group-delay dispersion (GDD). For this topic

of discussion one will primarily focus on GDD, since it’s the first term (not GD) to cause

non negligible distortion to a pulse [4].

2.1.2.1 Dispersion Compensation

A solution to this problem is the use of certain methods and materials to introduce negative

dispersion. The rapid progress in pulse-width reduction is mainly based on improvements

in dispersion compensation [5, 6].

Grating and prism compressors

Figure 2.2: Grating compressor.

Introduction of negative dispersion was first tried using diffraction gratings (see Fig.

2.2) [7] to spatially separate different spectral components, making them travel different

distances and recombining them later. The dispersion can be tuned easily by changing the

distance between the two gratings. This technique led to compression of pulses down to

30 fs [8] and due to the high amount of negative dispersion that can be obtained this way,

it is still extensively used today.

Prisms can also be used for this purpose [9] (see Fig. 2.3). Depending on the geometry

and material properties of the prisms, it is possible to have both a positive or negative

dispersion (using a set of lenses, one can change the sign of the dispersion). Losses are

6 Few-cycle laser for real-time nanomedicine research

usually lower compared to gratings, and to take advantage of that, they can be used at

Brewster’s angle. But there is a downside, the light has to pass through glass and that

causes intrinsic dispersion and limits the amount of total negative dispersion achievable.

More importantly, the residual third-order dispersion (TOD) limits the minimum pulse

duration to about 10 fs even when using low dispersion fused silica prisms [10].

Figure 2.3: Prism compressor.

Chirped Mirrors

Chirped mirrors are also a common component for dispersion compensation [5, 11]. A

chirped mirror is a kind of dielectric dispersive mirror with a spatial variation of the layer

thickness values. As shown in 2.4, by varying the layers thickness different wavelengths pen-

etrate different depths into the multilayer structure (longer wavelengths experience larger

group delay dispersion than shorter wavelengths) allowing the the shorter wavelengths to

”catch up” to the longer wavelengths. A double chirped mirror (DCM) employs an anti-

reflection coating and an extra layer structure [12] that act as an impedance matching from

air to the chirped structure itself. DCM are also commonly used in pairs, The use of these

mirror pairs has resulted in 5 fs pulses with octave spanning spectra [13].

Figure 2.4: Simplified schematic of a chirped mirror.

2.1.3 Generation

Ultrashort pulses are usually achieved using mode-locked lasers. When a laser operates in

continuous wave (CW), multiple longitudinal modes with random phases can oscillate in

the cavity simultaneously. Therefore, the modes are randomly interfering with one another,

2. Theoretical Background 7

and the laser output in time is a noisy continuous intensity, which is periodic in the cavity

round trip time.

A laser is said to be mode-locked if many longitudinal modes are oscillating together

with a well defined phase relation between them, as opposed to CW operation, where the

phases are random. This phase relation causes the modes to constructively interfere only

within a short period of time, while destructively interfering at all other times, forming a

pulse with a high peak intensity. Eventually, mode-locking (ML) reaches a steady state

(due to limiting mechanisms, such as dispersion, gain bandwidth, etc.), producing a stable

pulse that circulates in the resonator.

Mode-locking techniques are either active, where an external modulation inside the

cavity enforces pulsed operation, or passive, where the preference for pulsed operation is

introduced by an additional intra-cavity nonlinear response.

Most modern ultrashort lasers work using the Kerr lens mode-locking (KLM) method.

KLM is a powerful technique for ultrashort pulse generation from a variety of solid-state

lasers [3, 14–16]. In this technique, the active material behaves as a virtual lens, which

together with the cavity alignment enables creating an intensity-dependent loss mechanism,

CW is suppressed and ML is enhanced. This is usually called soft aperturing, in contrast

to actually using a (real) hard apperture to introduce losses on the CW mode.

2.1.4 Second-Harmonic Generation

Second-harmonic generation (SHG) was the first nonlinear optical effect to be observed

after lasers became available. In this process, an input (pump) wave passes through a

crystal material (that exhibits a χ2 nonlinearity) generating another wave with twice the

optical frequency in the medium, as one can see in Fig. 2.5. In most cases, the pump wave

is delivered in the form of a laser beam, and the second-harmonic wave is generated in the

form of a beam propagating in the same direction.

Figure 2.5: Second harmonic generation.

8 Few-cycle laser for real-time nanomedicine research

2.2 Femtosecond Lasers

2.2.1 Gain Media

Although many gain media can produce ultrashort pulses, the Ti:sapphire crystal is by far

the ultimate material of choice for current state-of-the-art ultrashort laser systems. The

use of Titanium-doped sapphire as a laser medium was first studied by Moulton in 1982

[17].

The Ti:sapphire crystal emission spectrum in the near infra-red (NIR) regime (see Fig.

2.6), which spans nearly an octave in frequency, can produce ultrashort pulses down to the

sub-two cycle regime [18–22]. Several other features such as good thermal conductivity,

mechanical strength and high-average-power levels make Ti:sapphire the most used gain

medium for ultrashort lasers.

Figure 2.6: Ti:sapphire absortpion/emission spectrum. Adapted from Ti:sapphiredatasheet (Eksma Optics).

2.2.2 Cavity Stability

The study and control of a laser cavity stability and a critical alignment are very important

to assure one obtains a pulse with the desired features [23]. A widely used approach toward

an understanding and optimization of the self-focusing dynamics in a KLM laser is based

on the ABCD matrix formalism analysis. This results in valuable design guidelines [24].

A typical design criteria for KLM oscillators is to favor the mode-locking operation

compared with the CW operation. This can be acomplished by designing the laser cavity

to be more stable for the ML mode, to have a higher gain on ML the mode, or both.

2. Theoretical Background 9

The cavity stability can be studied by considering the round-trip propagation: if the

cavity is stable, the beam will not diverge as the number of round-trips grows. In order to

avoid losses, one usually places the gain medium at Brewster’s angle.

2.2.3 Standard Femtosecond Lasers

The excellent mechanical properties and cavity compactness of the Ti:sapphire oscillator

renders it perfect for commercial production.

The pulse duration of this lasers is usually between 70 fs and 200 fs (they are considered

”long pulse” lasers in this thesis). In Fig. 2.7, the spectrum of a commercial laser < 100

fs (Tsunami, Spectra Physics) is shown in dependence of the pump power. Mode-locking

operation can be achieved using the KLM method. However, some laser systems like

Spectra Physics Tsunamir use an acousto-optic modulator (AOM) that ensures a stable

frequency for mode-locked operation by giving a constant ”kick” to the system ensuring

that one pulse has a preference over the random modes of the laser. This technique is

known as regenerative mode-locking, but the actual short-pulse generation is due to the

nonlinear effects induced in the Ti:sapphire crystal due to the high peak powers, not the

acousto-optic modulator.

Figure 2.7: Spectra Physics Tsunamir spectrum for various pump inputs. Adaptedfrom Tsunamir series datasheet.

The aim with these sources is to make the system operate with minimal user inter-

vention (output power and wavelength can be adjusted manually), since the whole mode-

locking process is not trivial.

2.2.4 Few-Cycle Femtosecond Lasers

The type of lasers mentioned above are essential in numerous applications. However, there

is still need for improvement of pulse parameters. Especially the increase of the spectral

10 Few-cycle laser for real-time nanomedicine research

bandwidth and consequently the reduction of the pulse duration of ultrashort pulses to

only a few optical cycles is of great interest for many current research and development

applications.

Over time, remarkable progress has been achieved in ultrashort pulse generation, which

resulted in pulses as short as 5 fs [13]. Beyond pulse duration, few-cycle lasers have inu-

merous advantages over longer pulse commercially available lasers. The broadband spectra

allows overlap with various absorbers while longer pulse lasers have to be tuned to match

specific absorption spectra, increasing the hability for nonlinear excitation. Furthermore,

high peak power (with reduced average power) puts them a step further.

With only two intracavity elements (Ti:sapphire and wedges) and a KLM working prin-

ciple, one has a better management of intracavity dispersion compared to a laser working

on an acousto-optic modulator, which is very thick.

2.3 Nanomedicine Research

Nanomedicine is the application of nanotechnology to diagnosis and treatment of diseases.

It deals with the interactions of nanomaterials (surfaces, particles, etc.) or analytical

nanodevices with “living” human material (cells, tissue, body fluids). It is an extremely

large field and has been gaining a great relevance over last decades, ranging from in vivo

and in vitro diagnostics to therapy including regenerative medicine and targeted delivery.

Nanotechnology has allowed a great impact on the development of new diagnosis and

treatment strategies in the medicine field, namely through progresses on drug delivery

systems for cancer treatment.

Cancer is one of the main causes of mortality worldwide. Research based on nanomedicine

has been focusing on the development of new treatments for this disease.

2.3.1 Nano Drug Delivery Systems

Nano drug delivery systems are nanoparticles in which one or more therapeutic agents

can be encapsulated, aiming to deliver it in the right place and time at an optimal dose,

inside the body. A nanoparticle (NP) is defined as the smallest unit (10−9 meters) that

can still behave as a whole entity in terms of properties and transport. The overall drug

consumption and side-effects may be lowered significantly by depositing the therapeutic

agent in the affected region only and in no higher dose than needed. Targeted drug delivery

is intended to reduce the side effects of drugs with simultaneous decrease in consumption

2. Theoretical Background 11

and treatment expenses. This can potentially be achieved by molecular targeting of nano-

engineered devices [25]. A benefit of using nanoscale formulations for medical technologies

is that smaller devices are less invasive and can possibly be implanted inside the body, plus

biochemical reaction times are much shorter. The efficacy of nano drug delivery is largely

based upon efficient encapsulation of the drugs, successful delivery of the drugs to the

targeted region of the body, and successful release of the drug from the nanoformulation.

There are several types of nanocarriers available, with distinct physical and chemical

properties such as shape, surface chemistry and size. Fig. 2.8 presents an overview of the

various nanostructures that exist today.

Figure 2.8: Overview of typical nanostructures proposed for drug delivery systems.Adapted from [26].

Although many studies have been made on this topic, only a few nanotechnology-based

therapeutic agents were approved by the Food and Drug Administration (FDA). The first

nano drug delivery system approved (in 1995) was Doxilr, which consists on Doxorubicin

drug encapsulated inside a pegylated liposome. This drug works in part by interfering with

the function of DNA.

Liposomal formulations and polymer-based systems are found in high abundance among

the approved nano drug delivery systems, and their efficacy was demonstrated in decreasing

the side effects of cancer treatments, improving the distribution of the drug and attenuating

drug clearance [27, 28].

Liposomes are the most commonly studied nanocarriers for drug delivery applications

and consist in spherical, closed colloidal structures that are composed of lipid bilayers

that surround an aqueous space. Due to the demonstrated advantages of liposomes, such

as reduced toxicity, increase in intracellular drug delivery (since the liposome can fuse

with the cell’s external bilayer and hence more efficiently deliver the drug), stabilization

of therapeutic compounds, and also improvement of distribution of compounds to target

sites in vivo, they are one of the most studied structures for drug delivery systems [29]

12 Few-cycle laser for real-time nanomedicine research

Intense research on liposomes has been performed during the last decades for drug de-

livery systems, but their use is still below expectations. There are still some issues that

need to be solved, such as scalability of the manufacturing process, reliability and repro-

ducibility of the results, highly complex fabrication processes, long term stability issues

and above all, the difficulty of fully understanding the interactions with living creatures

in general, and humans in particular. A lot of effort is being made to understand and use

this nanotechnology.

2.3.2 Fluorescence Lifetime Imaging Microscopy

The understanding of drug delivery systems requires analytical tools that ideally allow to

visualize the process of action. Since their broad introduction in the early 1990s, confo-

cal [30] and multi-photon laser scanning microscopes [31] have initiated a breakthrough

in biomedical imaging. Scanning multi-photon microscopy belongs to the family of flu-

orescence based microscope techniques and has become an integral part of bioimaging.

Fluorescence lifetime imaging microscopy (FLIM) is one of these fluorescence based mi-

croscopy techniques.

FLIM produces an image based on the differences in the excited state decay rate from a

fluorescent sample. Thus, FLIM is a fluorescence imaging technique where the contrast is

based on the fluorescence lifetime of fluorophores rather than their fluorescence intensity or

emission spectra. The fluorescence lifetime is defined as the average time that a molecule

remains in an excited state prior to returning to the ground state by emitting a photon.

The resulting contrast allows to distinguish different emitters which may not be resolved

in intensity images. The lifetimes of fluorophores used in cell imaging are typically of the

order of a few ns.

Multi-photon (MP) excitation is particularly useful when performing this kind of imag-

ing. MP excitation relies on the ability of fluorophores to absorb two photons at a wave-

length that is about twice the required wavelength for single-photon excitation. A high

power pulsed laser with very short pulse width is esssential to perform this type of excita-

tion. The high photon density in the focus of the miscroscope objective leads to a certain

probability that a fluorophore absorbs two photons quasi simultaneously. Since the spec-

tral separation of excitation and emission signals is considerable, background free imaging

can be easily achieved. Other benefits include increased depth penetration, reduced pho-

tobleaching and reduced sample damage.

2. Theoretical Background 13

Fluorescence techniques have found broad application in microscopy of live specimens

because they are extremely sensitive and deliver information about biochemical interactions

on the molecular scale. Apart from the fact that samples may be labelled with fluorophores,

fluorescence techniques are noninvasive and nondestructive, and thus can be applied to live

specimens.

Therefore, FLIM is a powerful tool in cell biology and bioimaging applications as it

allows to distinguish various fluorophores by their differences in fluorescence decay prop-

erties.

2.3.2.1 Time-Correlated Single Photon Counting

Time-correlated single photon counting (TCSPC) [32, 33] is used to determine the fluores-

cence lifetime mentioned above. The principle is shown in Fig. 2.9.

Figure 2.9: Measurement of start-stop times (left) and associated histogram (right) intime-resolved fluorescence measurement with TCSPC. Adapted from [34].

More specifically, TCSPC records times at which individual photons are detected by a

fast single-photon detector (typically a Avalanche Photodiode (APD) or a Photomultiplier

Tube (PMT)) with respect to the excitation laser pulse. The recordings are repeated for

multiple laser pulses and after enough recorded events, one is able to build a histogram (see

Fig. 2.9) of the number of events across all of these recorded time points. This histogram

can then be fit to an exponential function that contains the exponential lifetime decay

function of interest, and the lifetime parameter can accordingly be extracted. The recorded

fluorescence decay histogram obeys Poisson statistics which is considered in determining

goodness of fit during fitting.

Chapter 3

Few-Cycle KLM Laser

3.1 Basic Design

This chapter presents a full description of the few-cycle laser oscillator built by the student

during this project in the Femtolab (IFIMUP-IN and DFA-FCUP) and used in this thesis.

The laser is a prism-less Ti:sapphire laser designed for soft aperture Kerr-lens mode-locking

(KLM), its schematic setup is illustrated in Fig. 3.1 and is based on the design in [35].

Figure 3.1: Few-cycle Ti:sapphire laser schematic. Optical setup - PM: pump mirrors,BD: beam dump, L: focusing lens, DCM1-DCM4: double chirped mirrors, OC: output

coupler, P: BaF2 plate, W1/W2: CaF2 wedges, RM: rear mirror.

Usually, Ti:sapphire lasers show good performance for repetition rates ranging from 80

to 100 MHz. This means that the cavity must be long enough to enable this frequency

values, since for a cavity of length L, Frep is given by c/2L.

15

16 Few-cycle laser for real-time nanomedicine research

The laser crystal is pumped by a DPSS CW laser at 532 nm (Spectra Physics Millennia

Pro). The pump beam is focused by a lens through one of the mirrors of the sub cavity

to the crystal. This is a 2.2mm long Ti:sapphire crystal, with Brewster angle cut and ab-

sorption of 67% at 532 nm, placed between two concave mirrors (M1, M2). The remaining

intracavity flat mirrors (DCM1-DCM4) are broadband double chirped mirrors with high

reflectivity between 600 and 1200 nm. The BaF2 plate is also placed in Brewster angle and

the pair of CaF2 wedges (W1,W2) is used to enable fine tuning the intracavity dispersion.

3.2 Cavity Stability

As mentioned earlier, it is crucial to manage the dispersion created in the cavity. In this

case, only group delay dispersion (GDD) is considered and it is introduced when the beam

passes through the crystal and due to the air path that it must travel. The pair of CaF2

wedges and the BaF2 plate also introduce positive GDD.

For intracavity dispersion management (at 800 nm), the double-chirped mirrors used

have nominal GDD of -60 fs2 and the sub-cavity mirrors have nominal GDD of -50 fs2.

This yields negative GDD with tolerance.

The pump lens and the second sub-cavity mirror are placed in positioning stages (XY

axis). This allows adjusting the pump beam (movable lens) and the sub-cavity mirror so

as to maximize the cavity stability.

As mentioned above, this laser is designed for soft aperture Kerr-lens mode-locking

(KLM) and getting it into mode-locking operation is not trivial. First, one needs to adjust

the distance from the second intracavity mirror (M2) to the crystal until a power reduction

of about 30% (can be more) with respect to the CW mode is obtained. Then, it is necessary

to disturb the cavity (changing the position of the rear mirror) to induce mode-coupling

and mode-locking.

3. Few-Cycle KLM Laser 17

Figure 3.2: Cavity stability as a function of distance L2 (above) and mode size insidethe crystal (below).

The cavity stability was calculated with the use of gaussian beam propagation for the

TEM00 mode using the different cavity elements. With this technique, one is able to

simulate the stability function with the ABCD matrix formalism. In Fig. 3.2 the stability

diagram for the cavity is shown as a function of the position of the mirror M2 (top) [from

this, the stable range is identified with the stability parameter being between -1 and 1,

therefore the mirror M2 position should be chosen to set the L2 distance to values between

37.8 and 39 mm] as well as the beam radius as a function of the position inside the crystal

(bottom). This gives ideal CW mode operation.

The optical output spectrum of the custom-built few-cycle KLM laser is shown in Fig.

3.3. It covers nearly one optical octave (spanning from 600nm up to about 1100 nm),

supporting a Fourier-limited pulse duration of 5 fs.

18 Few-cycle laser for real-time nanomedicine research

Figure 3.3: Laser spectrum.

Together with an average output power of about 100mW this feature makes the laser

the ideal light source for few-cycle pulse experiments, delivering an enormous spectral

bandwidth with considerable power. At a pulse repetition rate of 80MHz the pulse energy

is approximately 1.25 nJ.

Chapter 4

MP-FLIM Experiments

4.1 Experimental Technique

Usually a single relatively narrowband excitation wavelength is used in multi-photon mi-

croscopy. Image strategies have revolved around selecting one single excitation wavelength

most common to the chromophores on the sample or alternatively using several excitation

wavelengths and compiling a sequence of images. These methods have some drawbacks

- not all chromophores are efficiently excited and individual cell constituents cannot be

tracked simultaneously, rendering fast processes at the cellular level impossible to track

and the longer exposure during multiple scans can furthermore lead to photo toxicity.

Efficient simultaneous excitation of multiple different chromophores is therefore a highly

desired capability in the field of bioimaging, and several techniques have been implemented

to reach this goal [36–39].

The technique presented in this thesis enables simultaneous tracking of multiple fluo-

rophores in the red, green and blue spectral range within a single scan and with a single

detector (see Fig. 4.1). Since one is dealing with two-photon excitation, the spectra of the

fluorophores is not straightforward and is different from one-photon excitation [40].

19

20 Few-cycle laser for real-time nanomedicine research

Figure 4.1: Normalized spectra of the 5 fs laser system with absorption spectra of dyes.

In Fig. 4.1, two-photon absorption spectra of the fluorescent label Hoechst 33342, the

auto-fluorescent endogenous compounds NAD(P)H and the fluorescent anti-cancer drug

Doxorubicin (DOX) are shown together with the few-cycle laser spectra.

Data with Hoechst 33342 was acquired but is not shown in this thesis. Even so, the

spectra of Hoechst 33342 is included to show a reference of a well know spectra of a

fluorophore with blue excitation.

This experiment was performed using the ultra-broadband 5 fs custom-built Ti:sapphire

laser system presented in the previous chapter.

A key aspect is that this study makes use of complete pulse characterization (ampli-

tude and phase) directly at the focus of the scanning objective, i.e. at the sample position

and not at an arbitrary point in the beam path, using a dispersion scan (d-scan) sys-

tem. The d-scan pulse characterization technique relies on measuring the spectrum of a

nonlinear signal, such as second-harmonic generation (SHG), as a function of dispersion

applied to a single beam. It requires only a pulse compressor (usually already in place),

a nonlinear medium, and an optical spectrometer, yet enables precise measurements down

to single-cycle durations. Besides its single-beam implementation and intrinsic few-cycle

measurement capabilities [41, 42], the d-scan provides the complete temporal intensity pro-

file of the pulses, which should bring the obtained results close to theoretical predictions

of signal intensity dependence on pulse length.

4.2 Setup

The few-cycle laser system was coupled to a custom-built inverted multi-photon microscope

(see Fig. 4.2) equipped with a single detector and was guided into the microscope by

4. MP-FLIM Experiments 21

steering mirrors and, in order to overfil the back aperture of the objective, the laser beam

was expanded using a set of lenses. Sample scanning is performed with combined micro

and nano stages. Signal collection is done in epi-fluorescence configuration through the

microscope objective and detected with a PMT. Signal counting is done with a TCSPC

module that determines the photon arrival times of each detected photon.

Figure 4.2: Schematic of the custom-built multiphoton FLIM setup used: The laserbeam is guided through the d-scan compressor into a custom-built microscope based on

an inverted microscope platform by steering mirrors.

For multi-photon imaging on cells, a 1.4 NA oil objective (100x, WD 0.17 mm, CFI

Plan Fluor, Nikon) collected the emitted photons. A ND 0.4 filter was used combined

with an emission filter (FF01-680/SP-25 Blocking edge Brightliner, Semrock) used in the

filter block and 2x BG39 bandpass filters in series. An additional bandpass filter (Semrock

FF01-439/154-25) was used. This is done since the short wavelength tail of the broadband

laser is not stopped by the 680 SPX filter. Additionally, it allows the detection of the full

two-photon emission spectra of NAD(P)H compounds and partly detection of the DOX

emission spectrum (only the short wavelength tail).

The sample was placed in a combined micro and nano-positioning stages (NanoLPS200,

MadCityLabs), controlled by a software developed to device control (Labview). Signal was

collected in EPI/reflection mode through the objective and detected with a Photomultiplier

Tube (PMT) (H7422P-40 Hamamatsu, 5 mm dia. active area, sensitive spectral range 300-

720 nm) combined with a transfer lens. A single channel TCSPC module (Becker and Hickl,

Berlin – SPC-130, max count rate 10MHz) was used to determine the arrival time of each

photon detected event, resulting in the photon arrival time distribution in each pixel of the

images taken.

22 Few-cycle laser for real-time nanomedicine research

After acquisition, a detailed analysis was performed using a MATLABr toolbox de-

signed by the Ultrafast Bio- and Nanophotonic group at INL.

One-photon microscopy was performed using a LSM 780 Zeiss confocal microscope. A

488nm CW laser was used for one-photon excitation. Proper filter sets were used to remove

leakage of the excitation into the detection path.

The d-scan compressor was used to optimize and control the dispersion of the laser

pulse (see Figs. 4.3 and 4.4) which results in a pulse with 10 fs of duration. Prisms

can be used to perform this but limit pulse durations to approximately 10-30 fs due to

residual third-order dispersion (TOD). A more sophisticated solution was used in this case

and consists in using chirped mirrors and wedges, since prisms do not allow independent

adjustment of GDD and TOD, and one always ends up with too much residual TOD, which

cannot be neglected for the case of ultra-broadband pulses.

Figure 4.3: Laser spectrum (left) and retrieved laser pulse (right). The duration islarger than the Fourier-limit of the spectrum (approximately 5 fs) due to some residual

third-order dispersion, seen as a tilt in the d-scan trace below.

Figure 4.4: Experimental d-scan (left) and retrieved d-scan (right).

4. MP-FLIM Experiments 23

Fig. 4.4 shows the measured and retrieved dispersion scans from the custom-built

few-cycle laser.

4.3 Sample Preparation

HeLa cells were used to perform this study. The cells were routinely cultured with 10mL of

MEM cell medium supplemented with 10% HyClone Fetal Clone III Serum and 1% Peni-

cillin/Streptomycin solution. Cell culture techniques were performed under laminar flow

hood and all cultures were then kept in an incubator at 37◦C in a humidified atmosphere

of 5% CO2. Three different sample types were prepared - cells without any drug, cells with

free DOX (500 µM), and cells with Doxilr nanoparticles (20 µM).

4.4 Results

Fluorescence lifetime of autofluorescent NAD(P)H compounds of living cells, before and

after the administration of the anti-cancer drug DOX, was studied in this thesis in order

to assess differences in cellular metabolic activity. NADH and NADPH are intracellular

compounds associated with the cell ATP energy production and are two major players in

cell metabolism, as they participate as electron carriers in a wide range of redox reactions.

Considering the spectral similarity of NADH and NADPH, analysis based on fluorescence

intensity is not enough to distinguish between the contribution of each of these compounds,

as they absorb wavelengths around 340nm and emit at 460nm. In order to excite them,

one would need to expose the cells to UV radiation, but this might induce some changes in

the mitochondria. Via two-photon excitation, NAD(P)H compounds can be excited with

near IR light, instead of UV radiation.

The analysis of the NAD(P)H signals are able to reveal the fraction of bound to un-

bound compounds – which can then be used to test the therapeutic efficacy of drugs in

cellular metabolism. Additionally, fluorescence lifetime analysis might be able to identify

the lifetimes of DOX and possible presence of the drug in the cell. The delivery of the

drug into the cells was performed in two different ways, with a nanocarrier system (Doxilr

formulation) and without (free DOX).

The multi-photon FLIM technique was used to image the cells. Multi-photon excita-

tion at IR wavelength range was used to excite the endogenous NAD(P)H and free DOX,

using a femtosecond laser pulse. Then, the photon arrival time was measured in correlation

24 Few-cycle laser for real-time nanomedicine research

with the laser excitation pulses. A multi-exponential fitting procedure [43] was then used

to interpret the data in terms of fluorescent lifetime and the two most dominant fluores-

cence lifetime contributions per pixel in each MP-FLIM data set were analyzed in more

detail. Fig. 4.5 presents a two-photon fluorescence intensity image together with a one-

photon fluorescence intensity image. The used excitation lasers were the described ultra

broadband few-cycle femtosecond laser and a 488 nm CW laser, respectively. While in the

case of two-photon excitation the resulting multi-photon signal could stem from either the

NAD(P)H compound and the fluorescent DOX molecules, in contrast the signal collected

after one-photon excitation with 488 nm laser solely stems from DOX. The image taken

with the confocal microscope shows the typical nuclear localization of the drug (with a

z cross section of the cell on the top and right side). The comparison between the two-

photon and one-photon experiment shall help distinguish the fluorescence contributions

from NAD(P)H compounds, which are only excited when using the few-cycle femtosecond

laser, while in the case of one-photon excitation in the blue wavelength range solely the

DOX molecules are efficiently excited. It can be observed that the DOX fluorescence signal

stems predominantly from the cell nucleus for a HeLa cell incubated for 3h50min with free

DOX. Interestingly the average fluorescence lifetime across the cell is homogenous, areas

that are expected to have a higher abundance of DOX (nucleus) and NAD(P)H compounds

(mainly in the cytomplasm regions, close to mitochondria) cannot be distinguished by the

average fluorescence lifetime parameter. Fig. 4.6 presents the same cell, zoomed in a 10 x

10 µm2 ROI area, along with the fluorescence lifetime image.

Figure 4.5: Comparison of the two-photon fluorescence intensity image (5min of scantime, 0.03s of acquisition time, 40 x 40 µm, 100 x 100 pixels) (left) versus one-photonfluorescence intensity image (confocal) taken with a LSM 780 Zeiss confocal microscope

(right) of HeLa cell incubated for 3h50min with free DOX.

4. MP-FLIM Experiments 25

Figure 4.6: Fluorescence intensity image (17min of scan time, 0.4s of acquisition time, 50x 50 pixels) (left) and average fluorescence lifetimes per pixel (right) of HeLa cell incubated

with free DOX.

The image on the left in Fig. 4.6 has its contrast based on the average fluorescence

intensity emitted by NAD(P)H compounds and/or DOX, while in the image on the right,

the contrast is based on the determined average fluorescence lifetime of the compounds. In

this case, as the NAD(P)H compounds are spectrally identical, fluorescence lifetime-based

imaging allows to have more information on the contributions of each compound.

The experiments performed consisted of a nonlinear bioimaging of live cells in order to

track the uptake of the anti-cancer drug DOX.

For the study of drug uptake without a nanocarrier system (free DOX), a single cell

was followed over a period of several hours and it was imaged during drug treatment. The

imaging of such cell (see Fig. 4.7) shows the nucleus and the mitochondria in terms of

fluorescence intensity and respective determined average fluorescence lifetime. The first

measurement was taken 3h50min after free DOX administration. The second measure-

ment was taken after 4h of free DOX administration and the last measurement was taken

6h30min after free DOX administration.

26 Few-cycle laser for real-time nanomedicine research

Figure 4.7: Fluorescence microscopy data taken on HeLa cells incubated with free DOXwith ultrabroadband excitation and single channel detection equipped with TCSPC elec-tronics. Fluorescence intensity images (17min of scan time, 0.4s of acquisition time, 50x 50 pixels) (top) and average fluorescence lifetimes per pixel (bottom) over free DOX

incubation time. The incubation times are given on the top of each image.

The presented data allows us to have a general overview of the effect of free DOX on

the autofluorescent NAD(P)H compounds. The analysis of lifetimes offers the possibility

to distinguish one compound from the other. The lifetimes shown in Fig. 4.7 correspond

to a global overview and in some pixels, more than one lifetime component is detected. To

overcome this, a detailed analysis was performed in order to separate the two components.

Fig. 4.8 shows the fluorescence lifetime images of the two most relevant decay times

determined via the multi-exponential fitting procedure and respective histograms.

4. MP-FLIM Experiments 27

Figure 4.8: Fluorescence microscopy data taken on HeLa cells incubated with free DOXwith ultrabroadband excitation and single channel detection equipped with TCSPC elec-tronics. Average fluorescence lifetimes per pixel (top) and fluorescence lifetimes (bottom)associated to the most dominant fluorescence decay contributions, referred to as ’compo-

nent 1’ and ’component 2’. The incubation times are given on the top of each image.

Analyzing the results, one can clearly distinguish two components with different life-

times. Component 1 is the one that more contributes to the overall obtained lifetimes

with values ranging from 0 up to about 1.5 ns. However, there is a second component

whose presence was pratically negligible in the first measurement (3h50min), but becomes

more visible over incubation time. The lifetime range of this second component is much

wider than for component 1 and ranges from 1 to 4 ns. This longer fluorescence lifetimes,

which were not observed before, might be a response of the cell associated with the pres-

ence of DOX (e.g. a changed equilibrium between protein-bound and unbound NAD(P)H

compounds in cells with affected metabolic activity).

For the study of drug delivery with a nanocarrier system (Doxilr formulation), a single

cell (see Fig. 4.9) was followed over incubation time (see Fig. 4.10).

28 Few-cycle laser for real-time nanomedicine research

Figure 4.9: Fluorescence microscopy data taken on HeLa cell before addition of Doxilr

with ultrabroadband excitation and single channel detection equipped with TCSPC elec-tronics. (3min of scan time, 0.1s of acquisition time, 40 x 40 µm, 40 x 40 pixels).

A similar approach was used, both component 1 and 2 were considered in the analysis

(see Figs. 4.10 and 4.11) and the two components become clearly distinguishable over

incubation time (0h, 24h and 48h). With this formulation, it takes more time for the drug

to act.

Figure 4.10: Fluorescence microscopy data taken on HeLa cells incubated with Doxilr

with ultrabroadband excitation and single channel detection equipped with TCSPC elec-tronics. Fluorescence lifetime images associated to the most dominant decay componentsidentified in a multi-exponential decay fit model. Top row represents component 1 andbottom row represents component 2 fluorescence decay times. The incubation times are

given on the top of each image.

4. MP-FLIM Experiments 29

Figure 4.11: Fluorescence lifetimes associated to the most dominant fluorescence decaycontributions, referred to as ’component 1’ and ’component 2’. The incubation times are

given on the top of each image.

Component 1 contributes more to the overall lifetimes with values ranging from 0 to 1.5

ns. The second component was practically absent before drug administration but becomes

more present over incubation time. Lifetimes of component 2 are also much wider than

component 1 and range from 0 to 4 ns. This increase might have a similar explanation to

the free DOX uptake. The longer fluorescence lifetimes observed over incubation time may

be due to the presence of DOX in the cell, that affects the cell metabolism.

During the imaging of the cell shown in Fig. 4.5, a nonlinear phenomenon was observed.

Fig. 4.12 presents a 10 x 10 µm image of this cell showing the nucleus and mitochondria.

Figure 4.12: Fluorescence intensity image of a 10 x 10 µm2 area (17min of scan time,0.4s of acquisition time, 50 x 50 pixels) of HeLa cell incubated with free DOX.

Two hours after the imaging of this cell, a new image was taken (see Fig. 4.13), but

this time the whole cell in a 40 x 40 µm area was analyzed. In a worst case scenario,

one would expect to observe photobleaching in the previously scanned area (10 x 10 µm).

However, the opposite effect happened, as presented in Fig. 4.13

30 Few-cycle laser for real-time nanomedicine research

Figure 4.13: Fluorescence intensity image of a 40 x 40 µm2 area (5min of scan time,0.03s of acquisition time, 100 x 100 pixels).

The area not previously scanned has very low fluorescence intensity compared to the

50 x 50 pixels square scanned two hours earlier on the top right area of the image. It is

also important to highlight that the first image was taken with 0.4 seconds of acquisition

time per pixel and the second with 0.03 seconds of acquisition time. This sudden increase

of fluorescence intensity is associated to a phenomenon often called hyperfluorescence [44–

46]. This phenomenon has been observed before [45] and only occurs in live cells, which

means the cells in this experiment were still biologically active and hence surviving laser

irradiation. This phenomenon is dependent on the power of the laser, which can unset a

reaction that generates reactive oxygen species (ROS), and to some extent to what part of

the cell one is scanning. Different areas of the cell have lower unset threshold which makes

hyperfluorescence appear.

Chapter 5

Conclusions

This thesis describes the experiments performed in a collaborative research project between

University of Porto, Sphere Ultrafast Photonics and INL in frame of a master project.

During the course of this work, a few-cycle Ti:sapphire laser oscillator was built at

Femtolab (University of Porto). The laser is very robust and that is mainly due to a good

control and critical alignment of cavity stability and intracavity dispersion. It is capable

of delivering pulses of about 5 fs with an ultrabroadband spectrum ranging from 600 up

to almost 1200 nm (one octave), which makes it the ideal tool for simultaneous tracking

of multiple fluorophores.

The laser was then moved to INL to perform bioimaging of live cells. It showed ap-

propriate performance in a real laboratory environment for a proof of concept system.

The measurements obtained were clearly benefited from the use of a few-cycle laser and

d-scan compression technique. Many advantages arise from the use of these technologies in

comparison to commercially available femtosecond lasers - reduced photobleaching, faster

acquisition times, minimal thermal effects and free backgroung images are among the most

important.

Cellular bioenergetics were followed measuring autofluorescence of NAD(P)H com-

pounds in live cells after addition of anti-cancer drug DOX, using an inverted MP-FLIM

setup. Two distinct lifetime components were detected, that are associated with free and

bound-to-enzyme forms of NAD(P)H and/or DOX, with lifetime values between 0 and

1.5 ns and between 1 and 4 ns, respectively. The results allow us to confirm a possible

biological response of the anti-cancer drug DOX in the analyzed cells as a time dependent

variation of fluorescence lifetime parameters is observed in the described multi-component

analysis.

31

32 Few-cycle laser for real-time nanomedicine research

The experiments show that the technique can be used to study the effect of DOX on

cellular bioenergetics of cells, when encapsulated in a nano drug delivery system (Doxilr),

and the promising results that were obtained throughout this thesis support that.

A nonlinear phenomenon, called hyperfluorescence (the sudden increase of fluorescence

intensity in live cells), was observed and showed that the cells were still in good health, at

least after one complete scan.

There is still plenty of work to do in order to optimize the laser system setup and

perform systematic analysis of the time-dependent cellular responses after exposure to the

free drug or drug delivery system to identify the differences between the drug administration

and efficacy. However, a clear fluorescent lifetime profile of DOX was not obvious in the

data, further comprehensive multicomponent analysis might be necessary to separate those

form the NAD(P)H lifetimes. One suggested experiment is to use dedicated bandpass filters

in the detection path to discriminate signals from either the two possible emitters.

Overall, this system ability to study anti-cancer drug delivery systems seems to be very

promising since it might elucidate about the specific metabolic pathways involved in both

cancer resistance and treatment efficacy.

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