Lec7_PhaseContrast_EM_ML - Phase contrast objects...

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1 Biophysical Methods Slide 1 Phase Contrast and EM Phase contrast objects consisting of brighter and darker spots are amplitude objects. Image objective condenser bright spot dim spot low intensity high intensity Focal plane =transform plane object Biophysical Methods Slide 2 Phase Contrast and EM In unstained biological objects light absorption is usually very weak but a cell has features with different refractive index, different optical density. Depending on their optical thickness they generate phase shifts of the light waves. Image objective condenser phase object Small Phase shift Large phase shift focal plane =transform plane E ( z = 0, t ) = E 0 sin ! t , = 2 " f = 2 " c # Incoming wave (black) -1.0 -0.5 0.0 0.5 1.0 6 5 4 3 2 1 0 ! E ( x , y , z = t ) = E 0 sin t + " ( x , y ) [ ] Phase modulated wave (red)
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2 Biophysical Methods Slide 3 Phase Contrast and EM Since the phase shifts are small, we can approximate: -1.0 -0.5 0.0 0.5 1.0 6 5 4 3 2 1 0 ! The phase modulated wave (red) E ( x , y , z = 0, t ) = E 0 sin ! t + " ( x , y ) [ ] sin( + # ) = sin $ cos + cos $ sin E 0 sin % t + & ( x , y ) [ ] = E 0 sin t [ ] cos ( x , y ) [ ]+ E 0 cos t [ ] sin ( x , y ) [ ] cos ( x , y ) [ ] " 1, sin ( x , y ) [ ] " ( x , y ) E 0 sin t + ( x , y ) [ ]= E 0 sin t E 0 ( x , y )cos t [ ] independent of object dependent on object Our eyes as well as our cameras only see light intensities. How to make the phase change visible? Using trigonometric relations and some tricks Use the sine sum formula Biophysical Methods Slide 4 Phase Contrast and EM phase object To get the phase advance of the undisturbed light we insert a phase plate with an indentation such that the unscattered light gets π /2 less phase delay Image objective condenser Small Phase shift Large phase shift Focal plane =transform plane -1.0 -0.5 0.0 0.5 1.0 6 5 4 3 2 1 0 ! E 0 sin t + ( x , y ) E 0 sin t E 0 ( x , y )cos t [ ] independent of object (unscattered) dependent on object (scattered) The trick is to shift the undisturbed light by a phase π /2 or λ /4 We give it a phase advance such that the scattered wave is delayed by π /2:
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3 phase object Image objective condenser Small Phase shift Large phase shift Focal plane =transform plane -1.0 -0.5 0.0 0.5 1.0 6 5 4 3 2 1 0 ! E 0 sin ! t + " ( x , y ) [ ]= E 0 sin t [ ]+ E 0 ( x , y )cos t [ ] independent of object (unscattered) dependent on object (scattered) E 0 sin t + # ( x , y ) [ ] = E 0 sin t [ ]+ E 0 ( x , y )cos t + $ 2 % & ( ) * = E 0 sin t [ E 0 ( x , y ) cos t + cos $ 2 , sin t + sin $ 2 % & ( ) * = E 0 sin t [ ] , E 0 ( x , y )sin t [ ] = E 0 1 , ( x , y ) [ ] sin t [ ] Thus we have converted the phase contrast into amplitude contrast Biophysical Methods Slide 6 Phase Contrast and EM To get brighter images, we use a broad light source illuminating an annular diaphragm in the back focal plane of the condenser. The annular diaphragm may be viewed as a ring-shaped light source instead of the point source. Accordingly the phase plate is also ring-shaped
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4 Biophysical Methods Slide 7 Phase Contrast and EM Phase contrast has been invented by Zernicke and has been awarded the Nobel Prize in 1953. It is widely used to look at cells under the light microscope but is also of great importance in electron microscopy.
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This note was uploaded on 03/29/2009 for the course A&EP 470 taught by Professor Lindau during the Fall '08 term at Cornell University (Engineering School).

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Lec7_PhaseContrast_EM_ML - Phase contrast objects...

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