Ottimizzazione dell’efficienza di un microscopio confocale con eccitazione a due fotoni per misure di FLIM e FRET su cellule tumorali


Funded by: Ente Cassa di Risparmio di Firenze  
Calls: Ente Cassa di Risparmio di Firenze
Start date: 2010-01-01  End date: 2011-09-30
Total Budget: EUR 20.000,00  INO share of the total budget: EUR 20.000,00
Scientific manager: Quercioli Franco   and for INO is: Quercioli Franco

Organization/Institution/Company main assignee: CNR – Istituto Nazionale di Ottica (INO)

other Organization/Institution/Company involved:
Università degli studi Insubria Varese-Como

other INO’s people involved:

Abstract: Introduction
For about ten years at the Department of Anatomy, Histology and Forensic Medicine of the University of Florence, it is active a biophotonics laboratory of the National Institute of Optics of CNR. It is equipped for performing measurements on biological materials with advanced microscopic techniques.
Lately new techniques were developed, like FLIM (Fluorescence Lifetime Imaging Microscopy) and FRET (Fluorescence Resonance Energy Transfer), with which they can be acquired morphological as well as functional images of biological material in culture.
A fluorescent molecule (donor) who is in its excited state may, instead of emitting a photon, transfer its excitation energy to another fluorophore (acceptor) in its close proximity (1-10 nm). This interaction process is denoted by the term FRET.
Exploiting this process, it is therefore possible to detect whether two biological molecules, appropriately labelled, one with a donor fluorophore and the other with an acceptor one, interact with each other. All this by using an optical technique which is inherently lacking of spatial resolution (≈ 500 nanometers).
The presence of FRET can be detected in two ways: by measuring the fluorescence intensity at the emission wavelength of the acceptor (which in the absence of FRET is zero), or by measuring the reduction in lifetime of the donor fluorescence.
In the presence of FRET in fact, the fluorescence lifetime decreases because, in addition to the normal process of de-excitation by the emission of a photon, another way is available via FRET.
The measure of the fluorescence lifetime is possible through the FLIM technique, with which “images” can be obtained, where at each pixel is associated a fluorescence decay curve.
FLIM is therefore a very powerful technique of optical micro-spectroscopy because it yields functional images. The lifetime is indeed a parameter which is very sensitive to the microenvironment in which the biological molecule under study is located.
The aim of this research was the optimization of these techniques for efficiency and selectivity of excitation and fluorescent emission collection, both for single and two-photon excitation.
Research activity
The contribution of the Ente Cassa was used mostly for the purchase of a “Cavity Dumper” (APE GmbH, Berlin, Germany), which is an acousto-optic switch to be placed inside the LASER cavity of the oscillator mentioned above.
This device serves to reduce the repetition rate of the pulse train (from 1:20 to 1: 5000) while increasing the peak power of each pulse.
The purpose is two-fold:
1) To allow, using the FLIM technique, the measurement of fluorescence decay curves with a longer time interval. The original repetition frequency of the Ti: Sapphire MIRA 900F is in fact 76 MHz, corresponding to a time period of 13 ns. Slower decay curves are therefore, in this case, difficult to measure.
2) The increase of the peak power of the pulses increases the efficiency of the nonlinear conversion either for a direct two-photon excitation of the sample as for the generation of supercontinuum light based on the use of microstructured optical fibers.
the second step of the research activity was devoted to the optimization of single-photon excitation, either by making the nonlinear conversion process more efficient in order to obtain a broader supercontinuum spectrum and a greater power and by improving the following excitation wavelength selection stage.
Some non-commercial microstructured fibers, made by various research groups we work with, were used, and supercontinuum radiation from around 300 nm in the UV to about 1300 nm in the NIR was obtained.
For the single-photon excitation wavelength selection both interference filters, and a custom-made monochromator with a special axial design, were used.
A water immersion bath thermostat with circulation pump (Falc Instruments srl, Treviglio (BG)) was used for the maintenance of vital conditions for the biological material under observation.
Achieved objectives
We made a monochromator with an axial symmetry using a non-commercial, custom-designed optical element, characterized by a strong longitudinal chromatic aberration. The broad-spectrum supercontinuum radiation output of the microstructured optical fiber is collected and focused by the monochromator onto the fiber input to the confocal scanning head. The selection of the excitation wavelength is carried out by simply moving the optical element in the axial direction.
The complex beta1 integrin / hERG1 channel is involved in the proliferation, motility and invasiveness of tumor cells. Its molecular characterization is therefore particularly relevant.
With FLIM-FRET techniques, using fluorescent proteins integrin-YFP and CFP-hERG1 as a pair of acceptor / donor fluorochromes, it was shown that these interact directly to form a complex on the plasma membrane of HEK cells.
The Cx43 (Connessina43) and CDOs (CAM-related / down regulated by oncogenes) are two proteins involved in the process of myogenesis.
With FLIM-FRET techniques and single-photon excitation, using the fluorophores Alexa 488 (CDOs) and Alexa 568 (Cx43) as donor / acceptor pair, we proved the direct interaction of these two proteins in C2C12 myoblast cells undergoing differentiation.
Using this same pair of fluorophores we proved the direct interaction between amyloid fibrils Sup35NM with ganglioside GM1 (Alexa488 – GM1; Alexa568 – Sup35NM).
We succeeded in conveying the excitation supercontinuum radiation within individual cells, by using an optical fiber with terminations of nanometric dimensions, and handled with a micromanipulator.
We were able to generate supercontinuum light in the blue / UV spectrum down to 300 nm with the use of non-commercial microstructured optical fibers and exciting higher propagation orders.

INO’s Experiments/Theoretical Study correlated:
Development of a combined Raman and multiphoton microscopy platform

The Scientific Results:
1) Supercontinuum source tuned by an on-axis monochromator for fluorescence lifetime imaging
2) Dermal matrix scaffold engineered with adult mesenchymal stem cells and platelet-rich plasma as a potential tool for tissue repair and regeneration
3) Microscopia confocale con luce bianca coerente
4) Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1
5) Fiber optic nanoprobes for biological sensing
6) Neuronal rat cell imaging using a new UV-extended supercontinuum source