Differential Interference Contrast (DIC) Verses Dark Field and Phase Contrast Microscopy. E. D. Salmon University of North Carolina at Chapel Hill
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1 Differential Interference Contrast (DIC) Verses Dark Field and Phase Contrast Microscopy E. D. Salmon University of North Carolina at Chapel Hill
2 How Does Contrast in DIC Differ from Phase and Pol?
3 n e n o DIC Phase Pol
4 General References Zernike, Frits How I discovered phase contrast. Science 121: Zernike, F The wave theory of microscope image formation. Strong, J. "Concepts in Classical Optics". W. H. Freeman, San Francisco Murphy, D Fundamentals of Light Microscopy and Electronic Imaging. Wiley-Liss, N.Y. Yuste, R. F. Lanni, A. Konnerth, eds, 2000, Imaging Neurons, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Molecular Expressions, a Microscope Primer at:
5 DIC References Salmon ED VE-DIC light microscopy and the discovery of kinesin. Trends Cell Biol. 5: Salmon, ED and Tran P High-resolution videoenhanced differential interference contrast light microscopy. Methods Cell Biol. 72: Salmon ED, Shaw SL, Waters J, Waterman-Storer CM, Maddox PS, Yeh E, Bloom K A highresolution multimode digital microscope system. Methods Cell Biol. 72: M. Shribak and S. Inoué, Orientation-independent differential interference contrast microscopy, submitted to Applied Optics.
6 First Experiment described by Fritz Zernike in discussion of how he discovered Phase Contrast in 1930 s
7 a. b. Pin Hole IMAGE PLANE Slotted Extender Slot Shimstock Narrow Strip 10X NA =.25 APERTURE PLANE BRIGHT IMAGE OF SMALL OPENING IN CONDENSER IRIS OBJECTIVE LENS ABSORBING SPECIMEN c. Observations CONDENSER IRIS Tiny carbon particles
8 Conclusions: 1. Image is formed by interference of direct (undiffracted) and diffracted (scattered) light. 2. Blocking diffracted light results in uniform illumination of image by direct light 3. Blocking direct light results in darkfield image generated by interference of diffraction orders at image plane. 4. Darkfield image emphasizes higher spatial frequencies like those of edges, but does not accurately reproduce object because of absence of direct light 5. Absorbing objects behave like transparent objects that make λ/2 retardation relative to direct light
9 For Darkfield imaging, specimen is illuminated with a hollow cone of light with: NA cond > NA Obj Objective can have iris diaphragm to limit NA obj and prevent illuminating light from entering objective
10 Stroboscopic Darkfield Imaging of Flagella Motility of Sea Urchin Sperm and Chlamydomonus
11 Phase Contrast Gives Contrast to Structural Detail in Transparent Specimens Brightfield Phase Contrast (NA obj = 1.4)
12 BASIC CONCEPTS Image Plane Undiffracted Illumination Light Back Focal Plane Scattered (Diffracted) Light Plane Wavefront Objective Specimen NAcond = 0
13 Summary: Specimen (thickness, t) nsp nm EXAMPLES: nsp = 1.4 organelle nm = 1.36 cell cytosol t = 1 micron Γ =.04 = 550nm /13 Illuminating Wavefront At Specimen Wavefront just after specimen Γ = t (nsp -nm) Specimen Light (S) Undiffracted Light (U) Diffracted Light = S - U In Dark Phase Contrast At Image Plane Background Undiffracted Light (U), Attenuated, Phase Advanced by λ/4 Specimen Light = U + D Diffracted Light (D)
14 BASIC CONCEPTS Image Plane Phase Advance λ/4 and Attenuate 75% Undiffracted Illumination Light Phase Plate Back Focal Plane Scattered (Diffracted) Light Plane Wavefront Objective Specimen NAcond = 0
15 MICROSCOPE ALIGNMENT FOR PHASE CONTRAST To Increase NA cond illumination, Modern Phase Contrast Uses Annular Ring as Condenser Stop and Phase Ring in Objective Back Focal Plane Phase Plate Ring Annulus Objective Back Focal Plane Objective Lens Specimen Condenser Lens Condenser Turret Illumination light
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17 Depletion of hnuf2 from HeLa cells using sirna.
18 10X Phase Contrast of HeLa Cells: Time- Lapse for 10 hours At 5 min intervals; 1/10 field, 1of 25 fields recorded
19 Induce Anaphase in Early Prometaphase by Overcoming the Spindle Checkpoint Example: Mad2 Antibody Injection into Early Prometaphase Ptk1Cells Julie Canman
20 CCD Camera Analyzer Filter Wheel Camera Mount 1X-2X Optivar Emission Filter Dichroic Mirror 1.25X Mag. Upper DIC Prism 60X Objec tive 1.4 N.A Motorized Stage 1.4 N.A. Cond. Lower DIC Prism Po la rize r Electronic Shutter Field Diaghram 100W Halogen Lamp Ground Glass Filter IR Filter 540nm Filter 100W Hg Arc Lamp IR Filter Electronic Shutter N. D. Filter Wheel Excitation Filter Wheel Iris Diaghram Digitally Enhanced- DIC with Fluorescence In A Multi- Mode Microscope With CCD Detector
21 Cdc20 Persists At Kinetochores Throughout Mitosis and Exhibits Green: GFP-Cdc20 At Kinetochores Red: Phase Contrast Images of PtK1 Tissue Cells
22 A DIC Microscope is a Polarizing Microscope with Condenser an Objective DIC Prisms Analyzer N-S Objective Condenser Compensator DIC Prism Rotatable Stage DIC Prism Polarizer E-W
23 Comparison of Phase Contrast to DIC for Cheek Cell
24 What Are 5 Major Features of A DIC Image? High Resolution VE-DIC Image of Microtubules
25 What Are 6 Major Features of A DIC Image? Contrast is directional: maximum in one direction and minimum in the orthogonal direction Contrast highlights edges; uniform areas have brightness of background In direction of contrast, one edge is brighter, the other darker than the background Each point in object is represented by two overlapping Airy disks in the image, one brighter and one darker than background The Direction of Airy disk separation is the Shear direction and direction of maximum contrast Peak-to-Peak separation of Airy Disks is amount of Shear, typically ½ to 2/3 radius of Airy Disk
26 The DIC Microscope Is a Dual-Beam Interferometer Made with Polarization Optics
27 The Condenser DIC Prism Splits Illumination Light into 2 Divergent Orthogonal Polarized Beams α Prism is Oriented with the Optic Axes at 45 o to Polarizer.Why?
28 A Modified-Wollaston Prism is Used To Place Prism Above Objective or Below Condenser Diaphragm Effective Beam Splitting Plane
29 Nikon s New DIC System Uses One Birefringent Prism Combined With A Glass Wedge (courtisy of Mr. Toshimitsu)
30 Divergent Beams from Condenser Prism Pass through Specimen as Parallel Beams
31 Microscope Alignment For DIC 1. Achieve Koehler illumination 2. Align for Polarization Microscopy: Polarier E- W, Analyzer Crossed 3. Rotate Condenser Turret to Select DIC Prism to Match Objective 4. Use Correct Objective DIC Prism 5. Add Bias Retardation to Brighten Image 6. Adjust Compensation for maximum Contrast of Specimen Detail of Interest
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33 Microscope Alignment for DIC Objective Back Aperture: Full Aperture Illumination
34 Objective Back Aperture: 1/3 Aperture Illumination Poor Condenser Illumination
35 Miss-Matched Prisms, or a Missing DIC Prism Extinction Fringe Not Spread Across Aperture; This is View When Objective or Condenser prism Removed
36 Matched DIC Prisms; Full Objective Aperture Illumination Extinction Fringe Spread Across Aperture
37 Image Intensity for Test Specimen With No Compensation
38 Image Intensity for Test Specimen With Plus Compensation
39 Image Intensity for Test Specimen With Minus Compensation
40 Comparison of DIC Image Intensity for Test Specimen With No, Plus and Minus Compensation
41 Two Types of Compensation Are Used For DIC Microscopes
42 How Intensity Changes With Compensation I sp = I c + I p sin 2 ((Δ comp +Δ sp )/2) I bg = I c + I p sin 2 (Δ comp /2)
43 For Maximum Contrast: 1) Adjust Compensation So One Edge of Specimen Detail is Near Extinction; or 2) Use About λ/10- λ/20 for Video/Digital- Enhanced Contrast
44 Why is shear chosen to be 0.5 to 0.6 of radius of Airy Disk? The Abbe limit of resolution is: r = λ o /(NA obj + NA cond ) = 0.5 λ o /NA obj when NA cond = NA obj For NA obj = 1.4 and λ o = 550 nm: r = 190 nm This resolution limit corresponds to a maximal resolvable spatial frequency: fsmax = 1/r = 5.1 cycles/μm A shear of ~r/2 will give the maximum retardation (and contrast) between the e and o wavefronts at fsmax; let s see how
45 Use Cheek Cells for Contrast and Resolution Test A. 20X/NA =.45 Objective B. 100X/NA = 1.4 DIC Objective
46 Example DIC: Mitosis in Mitotic Newt Lung Cells
47 CCD Camera Analyzer Filter Wheel Camera Mount 1X-2X Optivar Emission Filter Dichroic Mirror 1.25X Mag. Upper DIC Prism 60X Objec tive 1.4 N.A Motorized Stage 1.4 N.A. Cond. Lower DIC Prism Po la rize r Electronic Shutter Field Diaghram 100W Halogen Lamp Ground Glass Filter IR Filter 540nm Filter 100W Hg Arc Lamp IR Filter Electronic Shutter N. D. Filter Wheel Excitation Filter Wheel Iris Diaghram Digitally Enhanced- DIC with Fluorescence In A Multi- Mode Microscope With CCD Detector
48 Fluorescent Images of 200nm bead: 100x/NA=1.4, detector pixel scale =.065 nm No DIC Prism With DIC Prism Peak = 3650 Peak = 2710 (75%)
49 Yeast Digital Imaging System(s) Kerry Bloom Lab, UNC-CH
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54 Pearson et al., 2001, JCB
55 Video-Enhanced Contrast Methods Developed in Early 1980 s by Inoue and Allen Revealed Cellular Structures and Macromolecular Complexes Invisible by Eye or Film
56 Video-Enhanced DIC Microscope System
57 Practical Example: VE-DIC of Isolated Microtubules View by eye Analog Contrast Enhancement Live Image De-focus Slightly; Acquire Background Image and Store into Frame Buffer Subtract Background from Live Image at Video Rates Increase Contrast Digitally
58 Preparations for Motility Assays Slide #1.5 Coverslip 70mm Thick Double- Stick Tape Perfusion Chamber
59 VE-DIC Microtubule Motility Assay for Minus-Kinesin ncd (3.3 rotations/um forward movement)
60 Color DIC with Full Wave (RED) Plate
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