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
• high contrast
• high sensibility
• high specificity
• photobleaching
• photo-toxicity
• bleed-through
• ‘haze’
Fluorescence
The ‘Haze’ problem
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!
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:
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
Fluorescent Microscope
Objective
Arc Lamp
Emission Filter
Ocular
Excitation Filter
Objective
Laser
Emission Pinhole
Excitation Pinhole
PMT
Emission Filter
Excitation Filter
Confocal Microscope
Fundamental elements of a Confocal Microscope
✘Pinhole
- accepts light from one point
out of focus rays miss aperture
- cuts even 90-95% sample light
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
• Pinhole
• Laser
• PMT
PhotoMultiplier Tube
Source: http://micro.magnet.fsu.edu/primer/
Fundamental elements of a Confocal Microscope
• 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/
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
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)
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
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
Real Point Spread Functions (PSF)
Source: I. Usson – TIMC-IMAG, Grenoble (France)
the optical system ‘spread’ my signal
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)
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
PC 12 Cells
Nuclei (DAPI) Golgi (Giantin)Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)
Deconvolution vs. confocal
raw
datadeconvolved
data
PC 12 Cells
Nuclei (DAPI) Golgi (Giantin)Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)
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
HELA cells, Actin, 757 MYCCesare Covino - ALEMBIC (Courtesy of Cristina Sironi – Molecular Genetics Unit )
San Raffaele Scientific Institute (2004)
Nature Methods (2012) Vol 9, Number 7
Chroma’s 2012 advertising campaign
(Chroma’s 2012 Calendar
celebrating 20 years of art and science)