What is Fluorescence Imaging? Unlike reflection or absorption imaging. Use molecules called fluorophores: –Upon illumination by light of a specific wavelength they emit light of longer wavelengths. Absorption (Excitation) Spectrum Emission Spectrum Wavelength Stokes Shift
Lens Light Source Digital Camera Emission Filter Excitation Filter Dichromatic Mirror Objective Lens Fluorescence Microscopy
Microscope Slide Specimen Objective Lens Coverslip Immersion Medium Slide
Fluorescence Microscopy Microscope Slide Specimen Coverslip Slide The Object: A collection of fluorophores small enough to be considered point sources of light.
Image Formation The Object (reality) The Image (reality observed) XY XZ XY XZ What Happened?
PSF (Point Spread Function) Point in object Point Spread Function (PSF) in image. –The image is built out of PSFs, not points. Effect of PSF –Features blur together –Measurements are corrupted Deconvolution –Undo effect of PSF
PSF in 3 Dimensions In 3d Widefield, light from out of focus planes is detected in focal planes. In Confocal, pinholes help but there is still some z-blur especially when SA is present. XY XZ WidefieldConfocal XY XZ
The Problem How to make a measured image better represent the real object ? 1)Counteract the PSF – that is put the light that spread (Airy disk) out back to its 2D location 2) Put light into the proper Z plane 3) Reduce the noise This is best accomplished using deconvolution
Imaging Equation Mathematical operation called a convolution. The Microscope is a convolution operator. The inverse of a convolution is deconvolution = Object convolved with the PSF equals the Image ImagePSFObject
Benefits –Can adapt to imperfections in the Microscope. –Double optimization: Object and PSF are modified –Can adapt to changes in refractive index, specimen, environment. –Handles SA well. –No need to measure the PSF. –The only certain benefit of non-blind over blind is that it is faster. Benefits of Blind Deconvolution (Adaptive PSF)
Comparison Original ImageNearest Neighbors Inverse FilterIterative
What effects the shape of PSF?? Lens parameters Modality Specimen Parameters XY XZ Widefield XY XZ Confocal
Spherical Aberration Caused by –Changes in refractive index –Thick specimens –Incorrect cover slip thickness –Worse deeper in the sample Solve by –Match RI –SA correction collar –Use biased PSF and deconvolution
Blind PSF PSF also changes as part of optimization Blind: 2 step process in Autoquant –Spherical Aberration Detection –PSF modified further as part of Deconvolution Determine PSF without measuring it.
Deconvolution with SA Correction max = 23,938 XY XZ
Geometric Distortion With SA PSF changes with depth Actual focus location also changes (PSF Asymmetry) Images look stretched. Scale in Z Many papers on this (Model 2010)
Deconvolution of Confocal Images Yes Confocal images can be deconvolved Confocal images –some blur. –noisy Accurate statistical model is important Poisson model Deconvolution reduces both blur and noise Improves the Signal to Noise Ratio. Multi-Photon, Spinning Disc, Structured Illumination.
Collecting Optical Sections Setting the top and bottom of the scan Use Nyquist sampling –Sampling rate needed to collect all information Don’t break rules for convenience only out of necessity
ImagingApproach First try Widefield + deconvolution –Better for low light levels –Living cells –Proteins Next Confocal + deconvolution –Thick specimens (Tissues) Multiphoton + deconvolution –Very thick specimens
Deconvolution Tips Check the image stack’s metadata before decon. Use the line profile to prove that decon works. Use statistics to show… –Average image intensity remain relatively the same –Max pixel intensity can increase by 100 times.
Deconvolution Tips 99% of the time Blind is best. Severe spherical aberration correction is best done by CGC on the objective. Use an AOI for SAC testing or decon testing. AutoQuant 30 Day trial program: –Use it to your advantage! –Customers receive the entire product to evaluate.