Confocal Laser Scanning Microscopy (CLSM) is one of a series of methods to generate slices from microscopic samples by means of optics. The sample staysintact, and the slicing may be repeated many times. True Confocal Scanning (TCS) isa technique, where only a single, diffraction limited spot is illuminated and observe data time.
The benefit of confocalimagingisa dramaticallyincreasedcontrastbyremovalof out-of-focus haze. Z-sequences of optical slices(3D image stacks) are sources for subsequent render in gas 3D movies.
TCS is also very well compatible with multi-fluorescence imaging, time-lapse imaging,FLIM, FRAP and a whole world of spectral applications.
Wide field (non confocal): all planes have the same amount of light. Illumination and collection of in and out of focus planes (97% Out of focus light considering a 10 μm diameter object).
Confocal (Leica TCS SP8 Confocal Microscope): amount of light decreases away from focus. Pinhole blocks the vast majority of the out of focus light. Only light coming from a slice of a certain thickness is collected
Total Internal Reflection Fluorescence (TIRF) Microscopy
Total internal reflection fluorescence microscopy (TIRF) is a highly sensitive technique to perform functional investigations in living cells.
The high signal-to-noise ratio and a resolution in z direction of usually 70-250 nm above the coverslip/water interface allows to visualize and to analyse vesicles transport and signallingevents, as well as kinetic studies and single molecules detection.
Physical background is the total reflection of a laser beam at the interface of glass and water and the resulting electromagnetic wave, the so-called evanescent field. The energy of the evanescent field decreases exponentially with the distance of the interface coverslip/water and allows exciting fluorochromes.
Wide field fluorescence image of brest carcinoma tumor cells expressing GFP tagged cell adhesion Molecule CD44 that is expressed on the cell membrane.
The same cells imaged in TIRF (Leica Infinity TIRF).
TIRF Applications
•Explore events close to the plasma membrane which are usually hard to observe(e.g. vesicle trafficking, cell adhesion, cell motility)
•Great signal-to-noiseratio:acquirehigh qualityimages with almostno background
•Perfect for live-cell imaging: less photobleaching, less phototoxicity
Ask me for info about these services: Luisa Bracci (cell analysis) (luisa.bracci@unisi.it), Lorenzo Leoncini (tissue analysis) (lorenzo.leoncini@unisi.it)
Electron Microscopy
The Electron Microscopy laboratory, based in the Department of Life Sciences at Siena University, has a strong history in the high resolution microscopic analysis of cell motility complexes, invertebrate ultrastructure and several other biological subjects of biomedical interest. Rapid freezing and cryofracturing protocols have been employed for several years as a valid complement to standard procedures for both Transmission and Scanning Electron Microscopy studies.
The expertise is mainly on electron tomography and image analysis, allowing significant advances in the knowledge of axoneme structure, ciliary trafficking, comparative spermatology and cell functional morphology, as documented in the literature.
The laboratory is fitted with two conventional TEM and a 200 Kv Philips CM 200 transmission electron microscope, with field emission gun, wide angle compustage, cryo holder for the observation of unfixed frozen hydrated samples and a TVIPS 2Kx2K cold CCD camera with EM-Menu4: a dedicated software for automatic image collection and electron tomography. The laboratory is also equipped with a Vitrobot Mark IV ethane-plunging device for preparation of vitrified biological samples. Recently in addition to our 20Kv scanning electron microscope, a FEI Quanta 400 ESEM fitted with EDS X ray probe for element microanalysis. was also installed. These new additions, coupled with our already proven abilities in the preparation and analysis of QF-DE metal replicas of axonemes and other material, establishes our laboratory as one of the most productive in Italy also offering multidisciplinary and well established package of EM services to faculties, as well as to external contractors interested in high resolution ultrastructural charchterization and 3D modeling of biological samples.
Ask me for info about these services: Pietro Lupetti (Electron Microscopy) (pietro.lupetti@unisi.it)
