OPTBIO Results

Over the last years, laser-based techniques developed in fundamental laser sciences, such as optical trapping, harmonic generation, and multiphoton excitation, have found their applications in biophysical research as non-invasive tools to manipulate molecules and to image cells and tissues, with unprecedented resolution and penetration depth.

Third harmonic generation microscopy through the mouse brain. Femtosecond laser pulses at 1200 nm excite the sample (red beam); blue emitted light is detected in backscattering.

The demand and interest from the biomedical community for application of these tools and development of new ones,
to address open scientific issues and questions, is continuously growing. In particular, an increasing request is coming from molecular and cell biology for the visualization and manipulation of single molecules and cells and for the development of tools apt to image biological processes in living animals. A high demand is also rising from medicine, for the characterization of living tissues, and for disease diagnosis and therapy.
OPTBIO aimed at improving laser-based methods in biomedicine and providing access for the biomedical community to
state-of-the-art instruments. OPTBIO pursued three main objectives, allowing the development of innovative workstations and methodologies, spreading from the investigation of single biomolecules and single cells to in-vivo microscopy on living animals to the development of diagnosis tools for human diseases.

The first objective addressed the need of efficient handling and imaging of tiny biological objects such as individual
cells and molecules:

LENS and LLAMS developed advanced platforms combining single molecule and single cell manipulation tools with single molecule fluorescence imaging, working both in vitro and in living cells.
FORTH provided a laser-assisted workstation for subcellular surgery and processing and micro/nano structuring of biological, biocompatible and biodegradable materials using multiphoton polymerization.
ICFO provided plasmonic micro and nano-structures with enhanced optical fields for in-plane optical manipulation and analysis.

SHG image of an intact muscle fiber. The image shows a line scan
along the sarcomeric pattern (horizontal axis) versus time (vertical
axis) during active contraction and subsequent relaxation. The SHG
intensity is mapped using a false colour scale (from red to green).

The second objective was the improvement in capacities for advanced imaging beyond what is commercially available and the development of novel methodologies for the investigation of living cells and animals. Significant achievements have been obtained:

for the development of multiphoton microscopy by LLAMS and ICFO,
for the establishment of microscopy workstations with extend spatial resolution by LLAMS and ICFO,
for the exploration of non-label methodologies by ILC.
FORTH developed a workstation for combined twophoton and second and third harmonic generation microscopy.
LENS and ICFO exploited second harmonic generation to image and measure structural dynamics of living muscleand neuronal cells.
POLIMI designed and developed a first version of a pumpprobe system to perform transient absorption in real-time.

The third objective was the development of optical techniques for in vivo imaging with the final aim of developing tools for medical diagnosis and optical therapy.

POLIMI and LLC developed and potentiated their time-resolved diffuse spectroscopy systems leading to a great increase in dynamic range and reduction in acquisition time.

FORTH has been adapting multispectral capabilities to their existing tomographic device.
LENS coupled two photon imaging to laser-induced lesions to perform in vivo multiphoton nanosurgery in living mice brains.
ICFO used different techniques to image at high resolution the process of nano-neurosurgery on the C. elegans nematode.
VURLC developed a scanning multispectral imaging system.

optbio3

Multi-spectral fluorescence lifetime imaging microscopy setup with non-linear excitation developed
at ILC.

Francesco Pavone

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OPTBIO Results