Microscopy and Nanoscopy for 3D tracking and visualization of biological molcules in cellular systems by means of linear and non-linear optical interactions

Research project

Multiphoton introduction [download movie, .mov 6 Mb]

The size of biological molecules operating and interacting in cellular systems varies dramati-cally, from small fatty acids and sugars (∼1 nm = 10-9 m), to macromolecules like proteins (5-10 nm), starches (>1000 nm), and the enormously elongated DNA molecules. In order to elucidate biological functioning in terms of the molecular structures and properties of specific molecules a significative reserch effort is directed to determining the structure and trafficking of specific bio-logical molecules and of the larger structures into which they assemble within living cells.

Fig 1: Multiple fluorescence image taken under two-photon excitation microscopy at 720 nm (Diaspro et al. (1999) MRT, 47: 196). [+zoom]

Our main research activity is focused on inventing new methods and building new instruments for monitoring these structures, and on exploiting many of the exciting new developments in optical microscopy, in terms of imaging and manipulation, spectroscopy and visualization. Optical mi-croscopy is still unique in allowing to explore the 3D space occupied by biological systems - from macromolecules to cells, from tissues to organs - while temporal changes occur within a temporal scale from microseconds to several hours and days. We are currently developing appro-aches down to the nanoscale within a 7D observation window: from 3D to time until spectral in-formation (5D) plus lifetime (6D) and high-order harmonics (7D). 3D is the starting point: from computational issues (from image deconvolution to fuzzy approaches) to architectural solutions (from fast confocal to multiphoton high resolution interactions).

Fig 2: Autofluorescence (green) image as cellular landmark and punctuated bright dots (blu) spectrally selected in the SHG region. This is the first demonstration of backscattered collected SHG from forming vescicles in cells (Diaspro A. et al. (2002) Proc. SPIE, 4622: 24). [+zoom]

In particular, the advent of two-photon excitation (2PE) of fluorescence has led to terrific advances in the study of biological systems down to single molecule imaging. As well, 2PE is particularly relevant for the study of the 3D and dynamic properties of biological molecules within their natural environment, cells or tissues. In particular the advent of 2PE and non linear methods mitigates overall photoblea-ching and phototoxicity problems, opening new perspectives by providing new attractive advan-tages with respect to the companion confocal 3D microscopy. The confinement of the volume of event within a subfemtoliter capacity makes this excitation modality a sort of discrete voya-ger into the intricate and complex world of the biological cell. A two-photon shuttle controlled into the cell like in an earlier Feynamn's prediction. Optical schemes and architectures for 2PE from microscopic level to single molecule imaging are tailored for specific experimental needs. 7D can be achieved on long term imaging and for thick samples by means of multiphoton exci-tation.

The utilization of confocal and 2PE microscopy is coupled to subdiffraction driven pho-toactivation of visible fluorescent proteins. Moreover, new enhanced biocompatible fluorescent molecules are designed and realized. Optical nanoscopy developments, including the realization of nanostructured calibration devices, are oriented to the improvement of a correlative electron-optical microscopy program. The current resolution is diffraction limited within a 100 nm scale and we are moving to macromolecular resolution aiming to achieve image formation on the 30-60 nm scale along the three spatial dimensions. Calibration devices are on the 2.5-5 nm scale.

As part of our research developments we carry out a special program named nanobiorobots for the realization of hybrid polyelectrolyte-biological systems useful for correlative microscopic approaches and for smart drug/sensor delivery in cells.