Light microscopy BMB 173, Lecture 14, Feb. 21, 2018

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1 Light microscopy

2 The Structural Biology Continuum Next two lectures: Light microscopy Many slides taken from Scott Fraser, Murphy s Fundamentals of light microscopy, Alberts Molecular Biology of the Cell, Cox s Optical Imaging Techniques in Cell Biology

3 Types of light microscopes Magnifying glass Compound Stereo Dark field Phase contrast Polarization Differential interference contrast (Nomarski) Fluorescent Deconvolution Confocal Two-photon (multiphoton) 4Pi STED Total internal reflection FRET PALM/STORM/iPALM Structured illumination

4 Your eye is the first lens 1x f = 250 mm Call this a magnification of 1x Scott Fraser, Murphy Fig. 2-1

5 Magnifying Glass 1x f = 250 mm 5x f=50mm Courtesy Scott Fraser

6 Lenses introduce the possibility of magnification 1/f = 1/u + 1/v M = v/u

7 Murphy Fig. 4-6

8 Murphy Fig. 4-7

9 Real lenses have aberrations and distortions

10 Optical lenses can be either convergent or divergent, so aberrations can be balanced. Note also infinitycorrected optics Murphy color plate 4-1

$diffraction$

11 resolution =.6 * lambda / n sin(theta) Aberration versus diffraction limited Cox Figs

12 Numerical aperture = n sin (theta) Can be increased through oil immersion Snell s law:sin(alpha) /sin(theta) = n air / n glass Fig. 1-20, Cox

13 upright versus inverted microscope upright inverted Murphy color plate 4-1

14 Example bright field image of a cell

stereo microscopy http://www.microscopyu.

15 stereo microscopy

16 Effects of light on living cells UV light breaks covalent bonds, creating free radicals visible light is poorly absorbed IR light is strongly absorbed by carbon bonds, adding heat phototoxicity recognized by cessation of cell motility and organelle movement, filopodia retraction, gelled cytoplasm IR- and UV-blocking filters useful can add ascorbate or succinate to scavenge free radicals, or oxygen-scavenging enzymes such as catalase reduce the concentration of oxygen (slows formation of free radicals)

17 4sin(x) + 1.5sin(x+phi) Im ψ unscat ψ scat ψ tot phi = 0 ψ scat Re Just like a driven oscillator, scattered radiation emerges 90 phase-delayed with respect to unscattered radiation, and is thus almost invisible phi = 180 phi = 90

18 Just like a driven oscillator, scattered radiation emerges 90 phase-delayed with respect to unscattered radiation

19 To generate significant contrast, must use either some stain or some way of detecting phase shifts amplitude contrast phase contrast

Hematoxylin is blue and binds negatively charged species like DNA, RNA, and acidic proteins-marks the nucleus Eosin is pink, is acidic, and binds positively charged structures H&E stained lung tissue

20 Hematoxylin is blue and binds negatively charged species like DNA, RNA, and acidic proteins-marks the nucleus Eosin is pink, is acidic, and binds positively charged structures H&E stained lung tissue sample from an end-stage emphysema patient. RBCs are red, nuclei are blue-purple, and other cellular and extracellular material is pink. en.wikipedia.org/wiki/haematoxylin_and_eosin_stain Tissues are typically fixed, embedded, sectioned, and then stained

21 Contrast agents Stains (eosin, hematoxylin) Enzymatic amplification (peroxidase, alkaline phosphotase, gold/silver enhancement) Bioluminescence (like luciferase) Fluorescent markers (dyes, GFP and relatives, quantum dots)

23 The lenses in real microscopes are usually arranged vertically, so rotate mental image by 90 Now add lots of rays sample lens Back focal plane Image

24 bright field light microscopes use both scattered and unscattered beams sample lens Back focal plane (see diffraction pattern) Image

25 dark field light microscopes block the unscattered beam sample lens Beam stop blocks unscattered beam Back focal plane (see diffraction pattern) Solid lines here should be erased Image

26 Dark field microscope

27 Bright field Dark field

28 phase contrast light microscopes introduce a phase shift between the unscattered and the scattered beams to enhance contrast ψ unscat Im π/2 phase delay from scattering ψ tot π/2 phase advance by Zernike phase plate ψ scat Re

29 Phase contrast microscope Nobel prize in Physics 1953 to Zernike

30 Bright field Dark field Phase contrast

31 Light can be polarized

32 Plane polarization 32

33 Plane polarization 33

34 Plane polarization 34

35 Circular polarization 35

36 Circular polarization 36

37 Plane polarization 37

38 Polarization microscope

39 Example polarization image Kinetoplasts (highly condensed and ordered mitochondrial DNA) Murphy Fig. 9-1

40 Birefringent crystals can split waves into O- and E- waves

41 Differential Interference Microscopy (DIC, or Nomarski ) Source

42 Murphy Fig. 10-5

43 If either the O or E wave is delayed, you get elliptically polarized light

45 DIC reveals gradients in the index of refraction

46 Bright field Phase contrast Differential interference contrast Dark field

48 colorized phase contrast DIC galleries/ dicphasecontrast/ index.html

49 Concept check questions How is the lens of our eye different from the lens of a magnifying glass? What is the (thin) lens equation? How does the magnification of an image depend on the position of the object? What is a virtual image? What is an aberration? Distortion? What are infinity optics? What is an Airy disk? Numerical aperature? How can oil increase the resolution of a light microscope? Does light microscopy damage cells? What is the difference between amplitude and phase contrast? What are some common ways to introduce amplitude contrast into specimens? Compare/contrast bright field, dark field, phase, polarization, and differential interference contrast microscopies. Sketch their internal elements. What looks dark and bright in each case? 49

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Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens. Compound Light Micros

PHARMACEUTICAL MICROBIOLOGY JIGAR SHAH INSTITUTE OF PHARMACY NIRMA UNIVERSITY Observing Microorganisms through a Microscope LIGHT MICROSCOPY: This type of microscope uses visible light to observe specimens.