Exploiting optical non-linearities for super-resolution label-free optical microscopy
Creators: name: ZANINI, GIULIA
Publisher: name: Universit� degli studi di Genova
Year: 2019

description: Optical microscopy uniquely provides non-invasive imaging of biological specimens and has
become an essential tool in life sciences. So far, fluorescence microscopy techniques have been
the most widely used due to their ability to visualize the molecules of interest with high
contrast, high specificity, and high spatial and temporal resolution. In the last decades, much
effort was put into the development of advanced super-resolution fluorescence microscopy
techniques to circumvent the diffraction limit and image with unprecedented spatial
resolution. The non-linearities contained in saturation phenomena were exploited for this
purpose, and led to the development of techniques like stimulated emission depletion (STED)
and saturated excitation (SAX) microscopy, which proved to have the potentiality to really
break the diffraction barrier and tune the resolution down to infinitely small focal volumes
(Chapter 1). Despite their well-established benefits, (super-resolution) fluorescence
microscopy techniques rely on the photophysical properties of fluorescent molecules to obtain
the desired contrast and resolution, and the labeling procedures may alter the physical
properties of the specimen and may come at the cost of photobleaching and photodamage
effects.
With the development of ultrashort pulsed laser sources, new types of non-linear optical
interactions became accessible (Chapter 1) and started to acquire a central role in optical
microscopy for label-free imaging, providing novel non-fluorescence-based contrast
mechanisms which purely rely on intrinsic properties of the molecules of interest. In this way,
the invasiveness and the phototoxicity can be reduced, and the degradation of the fluorescence
signal due to photobleaching can be avoided. Moreover, non-linear optical microscopy
techniques allow for three-dimensional imaging due to the intrinsic optical sectioning
capabilities of non-linear phenomena, while the use of longer wavelengths in the near-infrared
part of the spectrum results in lower absorption and scattering and permits to image deeper
inside tissues (Chapter 1). The main drawback of these non-linear optical microscopy
techniques is their relatively poor spatial resolution, especially when using longer excitation
wavelengths, and the fact that the imaging of non-fluorescent species with sub-diffraction
resolution is still a challenging task.
In this framework, this work aims at extending the super-resolution approaches to
label-free microscopy techniques, based on the fact that, in principle, any saturable optical
process between molecular states, not necessarily involving fluorescent transitions, is a
potential candidate for breaking the diffraction barrier.
In particular, non-linear optical processes are here exploited in near-infrared pump-probe
microscopy techniques, such as transient absorption microscopy (TAM) and stimulated Raman scattering (SRS) microscopy (Chapter 3). In these techniques, two synchronized femto/picosecond
pulsed laser beams are used to investigate ultrafast electronic and vibrational
dynamical properties of the sample with high spatial and temporal resolution, high sensitivity
and high chemical specificity. The interaction with the sample is recorded as an intensity
variation of one of the two beams, which is extracted from the background adding a fast
intensity modulation and filtering with a lock-in amplifier. New dynamical and chemical
information can be accessed at the molecular level and in a label-free way, using intrinsic
biomolecules as natural contrast agents.
Absorption-based pump-probe microscopy is here optimized to retrieve structural and
dynamical information from graphene-based samples with high sensitivity, and its
conventional configuration is combined with an additional doughnut-shaped pump beam,
which allows for the reduction of the effective focal volume exploiting transient absorption
saturation. By optimizing the experimental parameters, such as power and temporal overlap of
the saturation beam, single layer graphene deposited on a glass surface can be imaged at the
nanoscale (Chapter 4).
Moreover, the saturation of the vibrational excitation in SRS microscopy is here theoretically
and experimentally assessed, in order to evaluate the applicability of the SAX approach to
achieve isotropic sub-diffraction imaging capabilities (Chapter 5).