Confocal vs. Deconvolution

Transcription

1 Confocal vs. Deconvolution Cesare Covino ALEMBIC Advanced Light and Electron Bio-Imaging Center Istituto Scientifico San Raffaele (Milano)

2 Fluorescence high contrast high sensibility high specificity photobleaching photo-toxicity bleed-through haze

3 The Haze problem

4 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!

5 Confocal = same focus What does CONFOCAL mean? 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:

6 How works a Confocal Microscope The light out of focus coming from the upper and lower planes is removed to obtain OPTICAL SECTIONS Pollen Autofluorescence Arabidopsis root Propidium iodide (cell walls), GAL4-GFP (root meristem)

7 Fluorescent Microscope Confocal Microscope Arc Lamp Laser Excitation Filter Ocular Excitation Pinhole Excitation Filter PMT Objective Objective Emission Filter Emission Filter Emission Pinhole

8 Fundamental elements of a Confocal Microscope Pinhole - accepts light from one point out of focus rays miss aperture - cuts even 90-95% sample light

9 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

10 Fundamental elements of a Confocal Microscope Pinhole Laser PMT PhotoMultiplier Tube Source:

11 Source: cascade of electrons amplifies signal fast response, low quantum yield lower quantun efficiency than CCDs Do not directly generate images! they simply detect light

12 Different kind of Confocal Microscopy 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:

13 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)

14 Galvanometers move the beam through an XY raster scan Source: Source:

15 3D or 2D deconvolution GFAP, DNA, synapsin 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 Credits: David Dunlap

16 Real Point Spread Functions (PSF) the optical system spread my signal Source: I. Usson TIMC-IMAG, Grenoble (France)

17 Concept of deconvolution imaging math 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 Source: I. Usson TIMC-IMAG, Grenoble (France)

18 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

19 PC 12 Cells Nuclei (DAPI) Golgi (Giantin) Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)

20 Deconvolution vs. confocal raw data deconvolved data PC 12 Cells Nuclei (DAPI) Golgi (Giantin) Maria Carla Panzeri, ALEMBIC San Raffaele Scientific Institute (2008)

21 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

22 HELA cells, Actin, 757 MYC Cesare Covino - ALEMBIC (Courtesy of Cristina Sironi Molecular Genetics Unit ) San Raffaele Scientific Institute (2004)

23 Chroma s 2012 advertising campaign (Chroma s 2012 Calendar celebrating 20 years of art and science) Nature Methods (2012) Vol 9, Number 7