Deconvolution Microscopy

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Deconvolution Microscopy
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Deconvolution microscopy operates on the
principle of a point spread function (PSF). As
one moves away from focal plane in which an
object lies the light from it will spread in a
predictable manner
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The situation is analogous to what happens in an
SEM, as we move away from the focal plane the
beam spreads in a uniform and predictable manner
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The point spread function must be measured for a
given lens and a specific O.D. immersion oil
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Much like a confocal a deconvolution system
collects images in a Z-stack by repeatedly
sampling the specimen at different focal planes
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The components are much simpler consisting of a
conventional fluorescence microscope, focus
motor, CCD camera and computer with software.
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Processing time for deconvolving images depends
upon the size of the volume. A 32 by 32 by 32
volume takes 40 seconds to process on an Indigo2,
100 MHZ R4000 processor. Thus a 256 by 256 by 256
volume requires about 6 hours of processing
time. Memory RequirementsMemory is an extremely
important factor in determining how large a
volume can be processed. To determine the
required memory, three volumes must be present at
one time the original blurred volume, the 3D PSF
and the deconvolved volume. This leads to the
following table Volume Size Required Memory 512
cubed 3.2 gigabytes 256 cubed 402
megabytes 128 cubed 50 megabytes 64 cubed 6.3
megabytes 32 cubed 785 kilobytes
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The goal is to go from the 2D image stack to
create a 3D confocal-like image
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Removal of the out of focus portion of the image
results in an in-focus section
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Pollen Grain Muscle Cell
Mitotic Cell
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DeltaVison Deconvolution System
Commercial system with integrated CCD, stepper
motor, and image processing station
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DeltaVison Deconvolution System
Schizosaccharomyces
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Advantages of Deconvolution over Confocal
Minimal equipment needed Microscope (with
standard U.V. light source and
filters) Focus Motor CCD Camera Computer and
software No confocal or confocal aperture Light
efficient since the entire image is collected at
once (widefield)
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Disdvantages of Deconvolution over Confocal
Processing time can be considerable (minutes to
hours) Still restricted to monochromatic input
(confocal can handle several different
wavelengths simultaneously) Calculation of
point spread function must be precise
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TPEM Two Photon Microscopy LSCM Laser
Scanning Confocal Microscopy DDM Digital
Deconvolution Microscopy
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Deconvolution techniques can be used in
Widefield fluorescence microscopy Create
in-focus Z-series Confocal and multiphoton
microscopy Clean up and improve a Z
stack Transmission electron microscopy Improve
image contrast Scanning electron
microscopy Achieve increased depth of field
without decreasing resolution
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