Lec4_Microscopy_ML - The main limitation is blurred...

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Biophysical Methods Slide 1 Microscopy-2 The main limitation is blurred out-of-focus light from thick objects Object Image Objective Eye piece CCD camera Biophysical Methods Slide 2 Microscopy-2 Confocal microscopy Schematic of a Laser scanning fluorescence microscope. A laser of power P ! is reflected by a dichroic mirror and focused at the center (u=0) of a fluorescent sample of thickness t, Fluorescence emission from the sample is collected by the objective lens and transmitted to the image plane. Image plane photons passing through the detector aperture of radius v d are detected and used to form an image. For simplicity, the scanning system is not shown in the diagram Ideal confocality is achieved for pinholes approaching zero diameter but then allso the in-focus light is blocked out because of the PSF
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Biophysical Methods Slide 3 Microscopy-2 The in-focus point spread function in generalized optical units is: J1 - first order Bessel function In the object space this corresponds to: v = k ! NA ! r = n ! sin " k = 2 # / $ h 2 = 2 J 1 v [ ] v ! " # $ 2 In the image space these are: v I = k ! ! r / M M = magnification = n ! sin k = 2 # / " Biophysical Methods Slide 4 Microscopy-2 In the object space the PSF is three-dimensional. This is the shape of the intensity profile of an excitation point light source as it appears in the object space. v = k ! ! r u = k ! 2 ! z / n = n ! sin k = 2 # /
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Biophysical Methods Slide 5 Microscopy-2 Confocal microscopy The radius of the detector aperture = v d Ideal confocal optics are obtained at v d =0 paraxial approximation for PSF (valid for (<0.8 and NA<1.25): PSF: h 2 u , v [ ] = 2 ! d J 0 v [ ] exp iu 2 / 2 ( ) 0 1 " 2 The in focus airy disc ( u =0): h 2 0, v [ ]= 2 J 1 v [ ] v # $ % & ( 2 , 1st minimum at v=1.22 ! ou (=3.83 ou) longitudinal: h 2 u ,0 [ ] = 4sin u /4 ( ) u # $ & 2 , 1st zero at u=4 ! ou Biophysical Methods Slide 6 Signal and background The signal: The intensity in the image plane from a fluorescent point source is distributed as the airy pattern but is scaled by various factors: I s v I [ ]= q Q kNA ( ) 2 4 " N f I 0 h 2 0,0 [ ] ( ) h 2 0, v I [ ] h 2 [0,0]=1 q =fraction of fluorescence reaching the image plane =absorption cross section Q =quantum efficiency for fluorescence N f =number of fluorophores I o =peak excitation intensity The detected signal is the intensity integrated over detector pinhole: S v d # q QN f I 0 1 $ J 0 2 v d [ ] $ J 1 2 v d [ ] ( ) =detection efficiency
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Biophysical Methods Slide 7 Microscopy-2 PINHOLE SIZE AND DETECTED INTENSITY Ideal confocal properties require v d ! 0.5 ou. However, with such a small aperture >90% of the in-focus-signal is rejected.
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Lec4_Microscopy_ML - The main limitation is blurred...

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