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Live Cell Imaging By CLASS Microscopy
Rajarshi Choudhury
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?
n sin 
Different types of microscopy
Phase Contrast
Differential Interference
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.
• NON invasive.
• In vitro monitoring of development of embryo prior to
• 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….