Huygens Professional User Guide

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original imageFigure 13. Example of differentSNR values.Same image, differentSNR ratiosTop left: originalimage. Top right:image withSNR=30. Bottomleft image withSNR=15, Bottom:right: image withSNR=5Signal to Noise Ratio (SNR)The SNR should be estimated from the quality of the image. In Figure 13 you find some examplesof recordings where different noise levels were added to an original (restored) image.BlacklevelFigure 14 shows thehistograms of threesynthetic images. Atleft an image homogeneouslyfilled with thevalue 5. At the middlewe applied Poissonnoise as if the imagewas build by a CCDcamera. At the imageon the right a value ofblacklevelFigure 14. Histogram of images with various blacklevelvalues.20 was added to simulate electronic shift. This shift is called ‘blacklevel’. A large blacklevelvalue will reduce the effective dynamic range of your microscope, but will do no harm to thedeconvolution since it is automatically accounted for in the background estimation stage. However,it is also possible that the blacklevel is negative. An image histogram will show a spike onthe left (Clipped at zero values).20 The Huygens Professional User Guide
Sampling densitiesIt is very important forthe quality of a deconvolutionresult that allinformation generatedby the optics of the 1000microscope is capturedin digital form. Itcan be shown that ifthe sampling density ishigher than a certain 100value all informationabout the object is captured.We’ll call thisvalue the critical samplingdistance, correspondingto the10Numerical ApertureNyquist rate. Apartfrom practical problemslike bleaching,acquisition time anddata size there is noobjection at all againstusing a smaller sampling distance than the critical distance, to the contrary.Sampling distance (nm)lateral WFaxial WFaxial confocallateral confocal0.4 0.6 0.8 1 1.2 1.4Figure 15. Critical sampling distance vs. NAThe curves above show the critical sampling distance in axial and lateraldirections for WF and confocal microscopes. The emission wavelength inthe WF case was 500nm; the excitation wavelength in the confocal case isFigure 15 shows the dependency of this critical sampling distance on the NA for specific wavelengths.To apply the plot of Figure 15 to different wavelengths you can simply scale the verticalaxis with the wavelength. Example: you are working with a WF microscope with NA 1.3 atemission wavelength 570nm. From the plot you read that the critical lateral Nyquist samplingdistance at 500nm emission is 95nm, so in your case this becomes 570/500 * 95nm = 108nm.In the confocal case it is the excitation wavelength which determines the Nyquist sample distance.In theory the pinhole plays no role, but larger pinholes strongly attenuate fine structuresat the resolution limit. Therefore, as a rule of thumb, with a common pinhole diameter of 1Airy disk the lateral critical sampling distance may be increased by 50% with negligible loss ofinformation. In cases were the pinhole is much larger, the lateral imaging properties muchresemble those of a WF system and the sampling distance can be set accordingly. We do notrecommend to increase the axial sampling distance appreciably beyond the critical distance.Data acquisition pitfallsRefractive index mismatchA mismatch between the refractive index of the lens immersion medium and specimen embeddingmedium will cause several serious problems:• Geometrical distortion: the fishtank effectObjects will appear elongated in the microscope. Huygens Essential will automaticallyadapt the PSF to this situation, but assumes the image geometry is not corrected.• Spherical aberration (SA)SA will cause the oblique rays to be focussed in a different location than the central rays.The distance in this focal shift is dependent on the depth of the focus in the specimen. If themismatch is large, e.g. when going from oil immersion into a watery medium, the PSF willThe Huygens Professional User Guide 21
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Page 8 and 9: Installation and system requirement
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Page 12 and 13: Deconvolving my first imageFigure 2
Page 14 and 15: Deconvolving my first imageHistogra
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Page 18 and 19: The easy way• Press the Run butto
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Page 28 and 29: d2rd 2=πr 2 dand thus r = ------π
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