Live Cell Imaging By CLASS Microscopy Rajarshi Choudhury Microscopy Why we use microscope? Better magnification Better resolution Determination of ultra structure What are the factors that determine the resolution of a point in microscope? d 0.5 n sin Different types of microscopy • • • • • • • • Light Phase Contrast Differential Interference Fluorescence TEM SEM Confocal Laser Scanning Microscopy Atomic Force Microscopy Confocal light absorption and scattering spectroscopic microscopy (CLASS) monitors organelles in live cells with no exogenous labels Irving Itzkan, Le Qiu, Hui Fang, Munir M. Zaman, Edward Vitkin, Ionita C. Ghiran, Saira Salahuddin,Mark Modell, Charlotte Andersson, Lauren M. Kimerer, Patsy B. Cipolloni, Kee-Hak Lim, Steven D. Freedman, Irving Bigio, Benjamin P. Sachs, Eugene B. Hanlon, and Lev T. Perelman PNAS October 30, 2007 vol. 104 no. 44 17255–17260 How confocal microscope work? J. Phys.: Condens. Matter 19 (2007) 113102 What determines resolution in CLSM? • Like a conventional optical microscope, the resolution of a confocal microscope is limited by diffraction of light. The image of an ideal point viewed through a circular aperture is blurred, and the diffracted image is known as an Airy disc. The size of the Airy disc depends on the wavelength of the laser source and the numerical aperture of the objective lens This Airy disc limits the maximum resolution of the microscope in the sample plane due to the Rayleigh criterion, which states that two Airy discs must be separated by at least their radius in order to be resolved. For the optical setup of most commercially available confocal microscopes this limit is about 200 nm. Scanning speed Schematic of an acousto-optic deflector. The deflector consists of a crystal of a material such as Tellurium oxide (blue) to which is bonded a transducer, consisting of a layer of piezoelectric material (yellow) across which an RF voltage is applied. Sound waves propagate across the crystal and (ideally) are absorbed on the other side (brown). A laser beam (orange) incident at the Bragg angle (θ) is efficiently diffracted by the grating formed by the sound waves. The first order diffracted spot contains about 80% of the incident light and is deflected by an angle φ. Some of the light remains undiffracted and some is found in higher diffracted orders. The value of φ can be changed by changing the frequency of the sound wave; the intensity of the first order spot by changing the amplitude. Where is the hurdle? • Its works in dead cells. • Cells have to be labeled with external flurophore. • Only cell lines or biological samples with lesser thickness can be used. • Image contrast is good, but photo-bleaching of the labeled image are profound. • ‘Size determination’- can be sometimes misleading. Uses • NON invasive. • In vitro monitoring of development of embryo prior to implant. • Use in neurodegenerative diagnostics. • Drug discovery and cell biology. • Organelle drift in real time. Is there a Confocal microscope in diagnostics OptiScan FIVE 1 confocal microscope scanhead. Image courtesy of Optiscan Pty. Ltd. (Victoria, Australia). Early osteoarthritic changes and chondrocyte transplant success in sheep. (A) Noninvasive in vivo imaging of chondrocytes and lacunae in articular cartilage. (B) In a sheep model of cartilage damage, dramatic changes are observed at the site of tissue injury. (C) Following matrix-induced autologous chondrocyte implantation (MACI), there is a dramatic increase in the number of chondrocytes. (D) Assessement of the efficacy of cartilage repair techniques such as MACI. Image courtesy of Drs. Chris Jones and Brett Kirk, University of Western Australia, Perth, Western Australia Thank you….