Confocal Microscopy

 Patrizia Vaghi        Amanda Oldani
tel. 0382987548    microscopyCGS-group@unipv.it

Confocal microscopy, thanks to the innovative optical system, eliminates the interference of out-of-focus fluorescence on immunofluorescent sample and allows  three-dimensional reconstruction. The production of the confocal image is achieved by illuminating the sample with a laser beam that is sequentially focused on each individual point of the sample along both the XY and Z plane. The image of the entire object is reconstructed by a subsequent analysis of the individual points scanned through appropriate software.

Equipment

Inverted Leica DMI 6000 microscope  with the following objectives: 63x oil, 40x oil, 20x oil, 25x water for diafanized preparations; The microscope is also equipped with a interfering contrast kit.

Leica TCS SP5 II confocal microscopy system equipped with the following lasers: 405 nm, Ar 458 nm, 476 nm, 488 nm, 514 nm; HeNe 543 and HeNe 633 nm

    • Leica TCS SP8 STED 3X (Stimulated emission depletion) microscope
    is a super-resolution microscopy technique which reducing the area of effective excitation,in combination with a scanning microscope breaking the diffraction limit becomes possible. The instrument is equipped with a muntiband spectral scan head, White Laser and AOBS device that allow the excitation wavelength selection between 470 nm and 670 nm. High-efficiency HyD fluorescence detection system. 3X STED scan head for nanoscopy with a resolution of about 40 nm. Lightning deconvolution system. 3D Reconstruction Software, Colocation, FRAP, FRET. This cell imaging technique is suitable to resolve biomolecular structures such as DNA and DNA-protein complexes, cytoskeleton microtubules, neurofilaments, organelles, such as the endoplasmic reticulum, lysosome, endocytic and exocytic vesicles and mitochondria. Many plasma membrane proteins or membrane associated protein complexes have also been studied by super-resolution fluorescence microscopy..

  • Leica DMi8 S TIRF (total internal reflection fluorescence) microscope uses a technique for selectively imaging fluorescent molecules in an aqueous environment only in one plane in the sample of 100–200 nm thickness. The instrument is equipped with Filter wheel FURA allows switching wavelengths between 20 and 28 ms.

This technique is mainly used on in vivo sample to qualitatively and quantitatively describe the roles different proteins play in exocytosis/endocytosis, to observe the size, of the contact region between a cell and the solid substrate, for tracking cell movements or protein movements inside the cells.

Leica TCS SP8 DLS is an innovative concept to integrate the Light Sheet Microscopy technology into the confocal microscope. This technique is minimally invasive, strongly reduced photo-bleaching and photodamage and has a high acquisition speed. The instrument is equipped with a muntiband spectral scan head. White laser and AOBS devices allow the excitation wavelength selection between 470 nm and 670 nm. Laser Argon 488. High-efficiency HyD fluorescence emission detection system. Lightning deconvolution system. 3D Reconstruction Software, Colocation, FRAP, FRET. DLS module, digital light sheet, which allows the three-dimensional reconstruction even of “in vivo” preparations such as zebrafish, drosophila, c. elegans. The low phototoxicity allows very long observation times of sensitive samples, the high imaging speed allow fast volumetric imaging and imaging of fast processes. This technique is useful in small organisms developmental biology, mostly in embryos and larvae, cleared organs and spheroid. Fast imaging becomes particularly important in dynamic processes such as cell motion, tracking of vesicles, cell lineage and differentiation.