{[ promptMessage ]}

Bookmark it

{[ promptMessage ]}

Lec7_PhaseContrast_EM_ML

Lec7_PhaseContrast_EM_ML - Phase contrast objects...

Info icon This preview shows pages 1–5. Sign up to view the full content.

View Full Document Right Arrow Icon
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 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 = 0, t ) = E 0 sin ! t + " ( x , y ) [ ] Phase modulated wave (red)
Image of page 1

Info icon This preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
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:
Image of page 2
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
Image of page 3

Info icon This preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
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.
Image of page 4
Image of page 5
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern