confocal vs. deconvolution - scuola di microscopia...3d or 2d deconvolution mathematically remove...
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Confocal vs. Deconvolution
Cesare Covino
ALEMBIC
Advanced Light and Electron Bio-Imaging Center
Istituto Scientifico San Raffaele (Milano)
www.hsr.it/research/alembic
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• high contrast
• high sensibility
• high specificity
• photobleaching
• photo-toxicity
• bleed-through
• ‘haze’
Fluorescence
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The ‘Haze’ problem
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Optical Sectioning in Microscopy
✘eliminate reflections/scatter
• use Koehler illumination
✘reduce illumination volume
• confocal imaging
• Multi-photon fluorescence excitation
• Total Internal Reflection illumination (TIRM/EWM)
• Selective Plane Illumination Microscopy (SPIM)
✘reduce imaging volume
• confocal imaging
✘computational correction
• mathematically deconvolve the Point Spread Function (PSF)
• Structured light illumination
Eliminating haze improves RESOLUTION!
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What does CONFOCAL mean?
✘Confocal = same focus
✘Illumination and observation systems are focused in the same volume element =
optical path is build in such a way the image on the focal plane only collects light from
the focal point
Source:
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How works a Confocal Microscope
The light out of focus coming from the upper and lower planes is removed to obtain
OPTICAL SECTIONS
Arabidopsis rootPropidium iodide (cell walls),
GAL4-GFP (root meristem)
PollenAutofluorescence
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Fluorescent Microscope
Objective
Arc Lamp
Emission Filter
Ocular
Excitation Filter
Objective
Laser
Emission Pinhole
Excitation Pinhole
PMT
Emission Filter
Excitation Filter
Confocal Microscope
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Fundamental elements of a Confocal Microscope
✘Pinhole
- accepts light from one point
out of focus rays miss aperture
- cuts even 90-95% sample light
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Fundamental elements of a Confocal Microscope
• Pinhole
• Laser
✘Lasers emit light of precise
wavelengths
✘- many commercial
fluorescent dyes have been
designed for specific laser
emission lines
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• Pinhole
• Laser
• PMT
PhotoMultiplier Tube
Source: http://micro.magnet.fsu.edu/primer/
Fundamental elements of a Confocal Microscope
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• cascade of electrons amplifies signal
• fast response, low quantum yield
– lower quantun efficiency than CCDs
• Do not directly generate images!
– they simply detect light
Source: http://micro.magnet.fsu.edu/primer/
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Stage-scanning Confocal Microscope
✘only the range of stage movement
limits the size of the image
✘only central area of the objective
is used, so little spherical correction
is necessary
✘slow framerate tipically
Credits:
www.picoquant.com
Different kind of Confocal Microscopy
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Laser Scanning Confocal Microscope
✘scan specimen point-by-point
highly focused laser substitutes illumination aperture
✘opening aperture increases:
intensity (adds out of focus light)
depth of field (loss of confocality)
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Source:
http://micro.magnet.fsu.edu/primer/
Source:
www.bristol.ac.uk/synaptic/info/imaging/imaging_1.htm
Galvanometers move the beam through an XY raster scan
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3D or 2D deconvolution
✘mathematically remove unfocused light
✘often requires a Z series of images
✘must know or calculate the point spread function (Airy disk)
✘requires several minutes to calculate
GFAP,
DNA,
synapsin
Credits: David Dunlap
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Real Point Spread Functions (PSF)
Source: I. Usson – TIMC-IMAG, Grenoble (France)
the optical system ‘spread’ my signal
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Concept of deconvolution
✘ optically, the point spread function distorts the recorded image
✘ mathematically, the inverse point spread function reverses the effect
the optical convolution with the PSF is compensated by mathematically convolving with the inverse PSF
→ imaging
→ math
Source: I. Usson – TIMC-IMAG, Grenoble (France)
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Algorithms
✘ 2D or Deblurring
• Remove blur
Nearest neighbor, multi-neighbor
✘ 3D or image restoration
• Determine point-source origins of light
– Inverse filters
– Constrained interative
– Statistical iterative
– Blind deconvolution
Source: HUYGENS Software
http://www.svi.nl
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PC 12 Cells
Nuclei (DAPI) Golgi (Giantin)Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)
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Deconvolution vs. confocal
raw
datadeconvolved
data
PC 12 Cells
Nuclei (DAPI) Golgi (Giantin)Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)
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Deconvolution vs. confocal
wide-field microscopy
+ CCD
+ deconvolution• contrast exceeding confocal
CCD’s more sensitive than
PMT’s
• broad wavelength selection
• lower light exposure
• requires computation
Laser Scanning Confocal
(PMTs)
– immediate image
– more limited wavelength
selection
– high light exposure,
photobleaching
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HELA cells, Actin, 757 MYCCesare Covino - ALEMBIC (Courtesy of Cristina Sironi – Molecular Genetics Unit )
San Raffaele Scientific Institute (2004)
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Nature Methods (2012) Vol 9, Number 7
Chroma’s 2012 advertising campaign
(Chroma’s 2012 Calendar
celebrating 20 years of art and science)