WP 5: Measurement Methods for Fluorescence Tagged Cells
State of the art in the field
New methodologies for non-invasive in vivo visualization of specific molecular targets inside cells have been developed in recent years, made possible by new photoluminescent materials, such as nano-particles and of extremely sensitive detectors, have opened up new diagnostic capabilities for biotechnology. These techniques are based on the detection of fluorescent signals emitted from molecules or from nanoparticles embedded in the material and offer advantages over more common analysis tests (PCR, ELISA) that base their results on the behaviour of fluorescent molecules in chemical-physical conditions that are sometimes extreme (temperature, solvents etc.). The applicability of the in vivo diagnostic techniques depends moreover strongly from the development of materials biocompatible and able to emit light in the near visible where the constituents of the tissues-cells (water, lipids, hemoglobin etc.) do not absorb, allowing analysis of target not only on the surface. UNI-TO can provide an exchange of knowledge for the culture of cells (both adherent and in suspension, including fibroblasts, human MSCs, lymphocytes and hemopoietic precursors) and for their analysis/sorting (flow cytometry, confocal microscopy, molecular characterization).
Movement beyond the current state of art
The development of the ability of carry out quantitative studies, rather than simple comparison of repeated analyses, or to intercompare results in different labs will bring major benefits to the medical research and diagnostic communities. This WP aims to improve the capability for measuring the fluorescent spectrum of small numbers of tags in a reproducible way, enabling characterisation of low intensity signals, minimal fluctuations in such intensities (correlation spectroscopy) and the lifetime of few (or even single) fluorophores (FLIM Fluorescent lifetime imaging). Improving the reliability of the diagnostic techniques based on fluorescent measurements to enable them to be used extensively in the medical research needs the metrological validation of the fluorescent substances and the development of reference materials with spectral behaviour established with respect to the physical and chemical variables typical of the biological conditions to be applied. This will be done through a collaborative exercise amongst NMIs focusing on metrology of fluorescent substances and validating those suitable to be employed for medical diagnostics and evaluation of cell cultures in regenerative medicine. The results of the proposed project will lead to the production of improved fluorescent particles for instrument verification and cell labeling. The research will contribute to the development of novel fluorescence techniques, such as the use of pulsed radiation sources, fast fluorometers and low photon measurement, which are not yet fully commercialised.
Scientific tasks
Identification of molecular and nanocomposites biomarkers and definition of key parameters suitable for specific applications, production and finding of the materials
Identification of molecular and nanocomposites biomarkers and definition of key parameters suitable for specific applications Establish influence of parameters affecting fluorescent spectra, for example: thermal, spectral, environmental behaviour, chemical-physical interaction, quantum efficiency, lifetime, stability (photobleaching), geometrical shape, functionalization, concentration of molecules and nanocomposites. Identification and production of materials, free from drawbacks like toxicity, blinking and agglomeration inside cells. Investigation of all-Silicon Q-dots (bio-compatible); techniques of size reduction and study of functionalisation techniques basing on silicon chemistry.
Development of the measurement techniques
Study of measurement methodology for the metrological characterization of the properties of materials defined in task 1. Examine the influence of material and instrument functionality on spectral sensitivity, quantum efficiency, lifetime, stability (photobleaching) measurements in static and in fast dynamic conditions, size selection and geometrical shape, functionalization, concentration measurements Development of traceable characterization techniques, quantify thermal, environmental and chemical-physical interactions establish quantum efficiency, lifetime, stability (photobleaching) measurements in static and in fast dynamic conditions developing novel schemes of fast fluorometers and optical fibres fluorometers in VIS and NIR carry out size measurements and geometrical shape developing a novel scheme for geometrical shape measurement referred to angle standards for “big” nanoparticles Investigate the influence of geometry for the fluorescence measurements and the traceability of fluorescence measurements to SI units using goniospectrofluorometer instruments
Uncertainty analysis
Inter-laboratory comparison for selected parameters Development of an uncertainty budget associated for the measurements of the parameters, studied Validation of the claimed uncertainty budgets.
Definition of the characterization protocols of reference materials
Documentation of characterisation protocols Produce a plan for certifying and producing reference standards, engage interest of potential distributors.
Technical risks Effect of passivation on fluorescence properties and possible instability (over time and in living tissues) may limit the range of possible reference materials; FWHM of the emission and achievability of efficient NIR emitters may limit sensitivity of instruments and require research.
Expected outputs
Identification of molecular and nanocomposites biomarkers and definition of key parameters suitable for specific applications Development of measurement methodologies for chemical and physical validation of nanoparticles and molecules suitable as biomarkers Development of new nanocomposites Certification of chemical and physical properties of nanoparticles and molecules suitable as biomarkers Development of reference materials to be used as biomarkers in diagnostic systems of imaging
Selected references
[1] R. P. Haugland, The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Chicago: Invitrogen Molecular Probes, 2005[2] M. Bruchez, Jr., M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Semiconductor Nanocrystals as fluorescent Biological Labels, Science, 218: 2013-2016, 1998.
[3] X. Gao, L. Yang, J. A. Petros, F. F. Marshall, J. W. Simons, and S. Nie, In vivo Molecular and Cellular Imaging with Quantum Dots, Curr. Opin. Biotech., 16: 63-72, 2005.
[4] P. S. Dittrich and P. Schwille, Photobleaching and Stabilization of Fluorophores used for Single Molecule Analysis with One- and Two-Photon Excitation, Appl. Phys. B, 73: 829-837, 2001.
[5] Direct writing of a protein micro-array: lab-on-a-chip for multipurpose sensing; Massimiliano Rocchia, Stefano Borini, Andrea Mario Rossi, Mosè Rossi, and Sabato D' Auria. Proc. SPIE 6444, 644405 (2007)
[6] Writing 3D protein nanopatterns onto a silicon nanosponge; Stefano Borini, Mosè Rossi, Sabato D' Auria, and Andrea Mario Rossi. LAB ON A CHIP 5 (10): 1048-1052 2005
For more information: Dr. M. Sassi
