confocal vs. deconvolution - scuola di microscopia...3d or 2d deconvolution mathematically remove...

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

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