Phase Contrast. Zernike Phase Contrast (PC) Nomarski Differential Interference Contrast (DIC) J Mertz Boston University
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1 Phase Contrast Zernike Phase Contrast (PC) Nomarski Differential Interference Contrast (DIC) J Mertz Boston University
2 Absorption Scattering
3 / n wave-number k 2 n = index of refraction
4 / n wave-number k 2 n = index of refraction
5 / n wave-number k 2 1 z i e 1 e i( k z t) i( nk zt ) z e z zn ( 1) phase shift
6 Object CCD Imaging system Born approximation Field: Intensity: i E e 1i ballistic scattered 2 2 I 1i 1 small What if ballistic field is phaseshifted relative to scattered field? I i i not so small
7 Object CCD x Imaging system x Rule of thumb: x (i.e. need high NA to see small structures)
8 q x x spatial frequency (sample) q k x wave-number (light) 2 k q x (i.e. need high NA to see high spatial frequencies)
9 q x Scattered field x 0 Ballistic field x 0 q x
10 f f 1 1 f2 f2 Object plane maps angle to position Fourier plane Image plane (conjugate to object plane) Ballistic Scattered
11 f f 1 1 f2 f2 Object plane Fourier plane Ballistic light here Image plane (conjugate to object plane)
12 f f 1 1 f2 f2 Object plane Fourier plane Zernike phase contrast Ballistic light here Image plane (conjugate to object plane) 1) Add /2 phase shift to ballistic light in Fourier plane: 2 ii 1 2
13 f f 1 1 f2 f2 Object plane Fourier plane Zernike phase contrast Ballistic light here Image plane (conjugate to object plane) 1) Add /2 phase shift to ballistic light in Fourier plane: 2) Attenuate ballistic light to improve contrast: 2 ii i i 2 similar in magnitude
14 Problem! Illumination must be spatially coherent Object Phase plate Köhler illumination doesn t work!
15 Köhler illumination Solution: Pinhole Incoherent (spatially random phases) Spatially coherent Object Phase plate This solution is not ideal because illumination power is weak
16 Alternative solution: Annular illumination Object Spatially coherent (sort of) Advantages of annular illumination: More illumination throughput Fewer backreflections from optics Better resolution (though difficult to interpret) Annular phase-plate
17 Alternative solution: Phase contrast condenser Phase contrast objective Annular illumination Spatially coherent (sort of) Advantages of annular illumination: More illumination throughput Fewer backreflections from optics Better resolution (though difficult to interpret) Annular phase-plate
18 Cheek cells brightfield phase contrast
19
20
21 Phase Contrast Zernike Phase Contrast (PC) Nomarski Differential Interference Contrast (DIC)
22 Polarizer Wollaston prism Polarizing beamsplitter Nomarski prism 1 mrad
23 In phase, equal amplitude = Linear polarization 45 o In phase, unequal amplitude = Linear polarization Out of phase, equal amplitude = or Circular polarization Elliptic polarization
24 Transparent sample CCD Spatially coherent illumination (polarization 45 o ) Nomarski x f 1m
25 Transparent sample CCD Spatially coherent illumination (polarization 45 o ) Nomarski Polarizer -45 o (analyzer)
26 Scattering sample CCD Spatially coherent illumination (polarization 45 o ) Key condition: in in phase shifted Nomarski Polarizer -45 o (analyzer) Sensitive to phase gradient out x ( x) Problem: how to get bright coherent illumination with a lamp?
27 lamp Polarized Köhler illumination? Scattering sample CCD Nomarski Polarizer +45 o Polarizer -45 o (analyzer)
28 lamp Polarized Köhler illumination? Scattering sample CCD Nomarski Polarizer +45 o Random phases: in in Polarizer -45 o (analyzer) Doesn t work because Köhler illumination not spatially coherent
29 lamp Trick: DIC microscopy Scattering sample CCD Nomarski Polarizer +45 o Pairwise correlated: in in Nomarski Polarizer -45 o (analyzer) Polarized Köhler illumination with Nomarski prism provides bright uniform illumination that is locally pairwise coherent
30 DIC illumination a 1 b 2 c d 3 4 e 5 Köhler illumination (spatially incoherent and unpolarized) + Polarizer 45 o
31 DIC illumination Pairwise coherent a b c d e Köhler illumination (spatially incoherent and unpolarized) + Polarizer 45 o x + Nomarski prism
32 lamp Trick: DIC microscopy Scattering sample CCD Nomarski Polarizer +45 o Pairwise correlated: in in Nomarski Polarizer -45 o (analyzer) Polarized Köhler illumination with Nomarski prism provides bright uniform illumination that is locally pairwise coherent
33 Brightfield Zernike PC Nomarski DIC
34 Very useful in neuroscience
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