Live Cell Imaging By CLASS Microscopy

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Live Cell Imaging By CLASS Microscopy Rajarshi
Choudhury
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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?

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Different types of microscopy

• Light
• Phase Contrast
• Differential Interference
• Fluorescence
• TEM
• SEM
• Confocal Laser Scanning Microscopy
• Atomic Force Microscopy

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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
1725517260
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How confocal microscope work?
J. Phys. Condens. Matter 19 (2007) 113102
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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.

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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 (?) 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 f. Some of the light remains
undiffracted and some is found in higher
diffracted orders. The value of f can be changed
by changing the frequency of the sound wave the
intensity of the first order spot by changing the
amplitude.
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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.

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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.

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Is there a Confocal microscope in diagnostics
OptiScan FIVE 1 confocal microscope scanhead.
Image courtesy of Optiscan Pty. Ltd. (Victoria,
Australia).
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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
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Thank you.