Three-dimensional fluorescence microscopy in two-photon excitation regime

Abstract

Inonrlaboratory,witbinthefiameworkofa given under Werent excitation conditions. We have national INFM project, we have realised a two-photon acquired images of a solution of fluoresoeine (Me& excitation fluorescence microscope part of a multipurpose Germany) at dBeimt excitation intensities ( h m 7.6 to architechxe inclnding also Metime imaging and 57.8 mW) at a wavelength of 800 nm and a pulse width of fluorescence correlation spectroscopy modules. The core about 130 fs in order check the qwJratic de-pendence of of the architectnre are a mode-locked TLSapphire laser fluorescence on the intensity of the excitation some, that (Tsunami 3960, Spectra Physics Inc., CA) pumped by a form the basis for the application of TPE in microscopy. bidmower (5W. 532nm) laser W e n n i a V. SDeetra PCy& Inc., 'CA) and an ultracompact scanning -head, Nikon PCM2W (Nikon Instr., Italy). Three-dimensional confocal images of tlnomphores excitable in W and -d.8ra; I BLUE region will be, shown. We are c m t l y characterising the architecture in terms of point-spreadhction and biological system viability. \ I a u : Many interesting and oulstanding biological experiments that involve microscopic imaging are defeated by photobleaching of the fluorescent label and phototoxicity effects. Tbis sort of problems is particnliuly serious when there is the need for three-dimensional and temwral imaging c o n p~ to the use of w excitable fluomcGmes [l]. The advent of two-photon excitation (TPE) laser Scanning microscopy [2,3] deviates the previonsly mentioned concmns opening new avennes to the application of mirroscopic techniques to the slndy of biological systems and related phenomena and providing attractive advantages over classical confocal microscopy. In P E , a two-photon single entity collides in a single qnantnm event with a fluoresent molecule allowing infmred excitation instead of W-BLLE one. The TPE non-linear effect, combined with the sharp focnsing of a microscope objective lens, is responsible of the very high localisation of excitation within the sample resulting in a 3D confocal-like effect. In OUT laboratory, within the huework of a national project of the National Jnstitnte of Physics of the Malter 0, we have realised a multi-photon excitation fluorescence microscope part of a mdtipurpose architecture including also lifetime imagiog and fluorescence correlation spectroscopy modules [41, Fig.1. As core of the I L ----------I Fig. 1. Overall arohitecave We have made the very same cheek for a solution of DAPI in water, at an excitation wavelength of 746 xnn and pulse ow TPE width of 70 f k We are also c m architecture in tams of point spread M o n (PSF). To this end we use subresolution fluorescent beads as in the case of single photon confocal ~croscopy [5]. TPE microscopy has been used with a wide range of fluorescent molecules like DAPI, Fm-2, Indo-1, DiOC6(3), Flwresxine, Texas red, usually excitable in a single photon range &om UV to green. We are also performing cell viability experiments. .. archi6ecm a mode-locked T~:s&&-gs...