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Lecture 3: Special contrast enhancement methods in microscopy Contrast enhancement methods using visible light (VIS) Darkfield microscopy Phase contrast microscopy Polarized light microscopy Differential interference contrast (DIC) microscopy Hoffman modulation contrast microscopy AMM-2012 AMU_HI_HY 1 Darkfield microscopy The oldest, simplest contrast method Image formation of darkfield microscopy: A condenser central stop: block incoming direct light Generate a hollow cone of illumination When no specimen on stage: an oblique illumination with dark background When there is specimen on stage: Oblique rays hit light scattering, granular objects light is scattered away from propagation direction enter objective lens cone of light Form bright dot image on a black background Suitable specimen: unstained, non-light absorbing small particles AMM-2012 AMU_HI_HY 2 Construction of a low magnification, low NA darkfield microscope Condenser of Abbe type Condenser NA > objective lens NA (Cond_NA usually 0.95) Objective lens NA < 0.75 (usually =< 40x) If objective lens NA is too high, ordinary condenser NA cannot match Obj_lens NA AMM-2012 AMU_HI_HY 3 Low Mag, Low NA darkfield microscope can be home-made Darkfield microscope with low mag, ≤ 40x, low NA, < 0.75 does not need complicated optics, can be home-made easily An Abbe condenser with NA 0.95. a swing-in top lens. A central-stop cut from carton board at 22 mm. Tape the stop on the bottom surface of the condenser. This central stop size works for 20x/0.5, 40x/0.75 objective lens condenser central stop size for different Mag/NA obj lens 4x/0.1 10x/0.25 20x/0.4 40x/0.65 8-14 mm 16-18 mm 18-20 mm 20-22 mm AMM-2012 AMU_HI_HY 4 Other types of darkfield microscope Darkfield microscope with high magnification, high NA obj-lens specially made high NA paraboloidal immersion condenser has to be used Dry DF condenser Narrow light cone Paraboloidal oil DF condenser Wide light cone Or objective lens NA is reduced by variable NA iris lens Reflected light darkfield microscope: used for material microscope. AMM-2012 AMU_HI_HY 5 Usage of darkfield microscopy Detecting microbes in rapid diagnosis (field diagnosis) Syphilis Spirochete One of the oldest disease with human AMM-2012 AMU_HI_HY leptospirosis In south east Asia water-born bacterial Borrelia burgdorferi spirochete In north America bacteria spread by tick Lyme disease 6 Phase contrast microscopy Amplitude objects: capable of absorbing light, cause intensity change of visible light Phase objects: Do not interact with light strongly Light passes through, little absorbing little intensity change Diffracted light is slightly delayed by ¼ λ (phase difference) Phase object image formation Original ray (O) and diffracted light (D) reach BFP of objective interfere with each other, resulting particle wave P on image plane P wave has 1/20 λ phase difference and no visible intensity difference compared to original wave O AMM-2012 AMU_HI_HY 7 Development of phase contrast microscope Fritz Zernike, a Dutch physicist found a way to enhance the contrast in 1934. Shift direct light ¼ λ distance ahead to make total ½ phase difference between direct and diffracted light. Constructive or destructive interference occur on image plane. Visible light intensity change on the image. 2. Obj. phase ring Dr. Zernike won Nobel prize to speed up direct ray for Physics in 1953 1. Condenser phase stop surround by a transparent ring to separate direct ray apart Figure Adopted from Zernike AMM-2012 AMU_HI_HY 8 Phase contrast optics Condenser phase annulus (phase-stop) Objective lens Phase plate (phase ring) Phase-stop and phase ring overlay Seen at Objective lens back focal plane (BFP) Condenser turret With phase, DIC optics Thinned Ring with light-attenuation coating Aligned rings PH2: 10-20x objective lens PH3: 40-100x obj. lens AMM-2012 AMU_HI_HY 9 Results of phase contrast Direct light is ½ λ ahead of deviated light with similar intensity. Constructive or destructive interference occur at image plane effectively: D ¼ λ delay No phase contrast D ½ λ delay Constructive interference D ½ λ delay Destructive interference The method of advancing direct light or O-wave is called positive phase contrast in which high RI region looks darker. (Negative phase contrast retard deviated light, in which high RI region looks brighter) AMM-2012 AMU_HI_HY 10 Interpreting phase contrast images Concept of optical path length(OPL) Refractive index n and specimen thickness T together determine: OPL = n.t; Optical thickness difference between two parts of the specimen: ∆OPL (or OPD) = (n2-n1)t Ex. Cell culture: cell thickness 5 µ, n1=1,36, media: n2=1,335 ∆OPL=5.(1.36-1.335)=125 nm (about 1/4 λ of green light) (If not enhanced by phase contrast, 1/4 λ phase change is not visible. If thinner than 5 µ, even phase contrast enhancement is not effective enough) Phase contrast enhance the boundary where refractive index differ sharply In Positive phase contrast: structure with higher RI looks darker. Cell nuclei, nucleoli, mitochondria, ribosomes looks darker than media. Pinocytic vesicles, vacuoles, lipid droplet, looks brighter than media, it is not thinner though The darker region is not necessarily the thick region, it is region with bigger OPL In Negative phase contrast: D-ray is further delayed 1/4 λ, effect is same but opposite AMM-2012 AMU_HI_HY 11 Pros and cons of phase contrast microscopy Advantage: View live cell without staining with high image quality becomes possible, which made a big impact on biomedical research field. Disadvantage: Reduced resolution: Objective cone of light not fully filled, objective aperture not fully utilized: about 10% NA used. Phase halo around large, low spatial frequency structure like entire cells, nuclei. Shade-off: in large, extended structure, intensity is not uniform, increase towards center. Not works well with very thin specimen, poor depth of field and z-resolution. AMM-2012 AMU_HI_HY 12 Polarized Light Microscopy Polarized light and cross-polarization Electromagnetic wave is formed by oscillation of electrical field (and magnetic field) Most light source emit light with electric field vibrating randomly all planes perpendicular to its propagation: they are unpolarized light. If vibration is restricted in one plane, the light is plane polarized light EM wave has both electric and magnetic field Polarized light Only electric field is described in polarization Plane polarized light cannot pass polarizer perpendicular to its vibration direction AMM-2012 AMU_HI_HY 13 Birefringent material for polarized light microscopy Birefringence material (double refraction) Distinct optical axis: axes have different Reflective index Light pass through it subjects to different RI Structure of birefringent material Regular repeated structures: crystalline or paracrystalline Common birefringent material: Anisotropic crystal like quartz, calcite, some plastic polymer Bio-birefringent material: DNA, collagen fiber, muscle fibrils uric acid crystal structure with beta-pleated sheet: amyloid fibrils, Ig-light chain AMM-2012 AMU_HI_HY 14 Birefringent material for polarized light microscope Interaction of light with Birefringence material Rays oblique to axis is divided: Ordinary ray: same speed Extraordinary ray: slowed down. phase retardation. Wave perpendicular to axis E ray slower but same direction as O Rays parallel to optical axis E and O rays are the same AMM-2012 AMU_HI_HY 15 Configuration of microscope • Polarizer: to provide polarized light • Analyzer (another crossed polarizer) • *A 360 degree rotate stage is useful (helps to identify beam direction) • *An accessary retardation plate (λ plate) helps enhance contrast (change interference color) *not necessary but helpful Specimen observation, image formation • When there is no specimen: dark background • When there is birefringent specimen: Light divided, retardated and recombined Interference occurs, visible intensity change Max intensity at a particular rotation angle Newton’s color (polarization color) AMM-2012 AMU_HI_HY 16 Birefringent specimen under polarized light Amyloid fibrils Congo Red staining Brightfield microscopy Dull brick-red color in bright field microscopy AMM-2012 AMU_HI_HY Amyloid fibrils Congo Red staining Polarized light microscopy Apple green birefringence in polarized light microscopy 17 Differential interference contrast microscopy (DIC) Image formation in DIC 7. Analyzer allows elliptical polarized light to pass 6. Obj. Nomarski remove shear, combine two phase changed wavefronts to elliptical polarized light 5. Objective lens accepts parallel ray from specimen, focus them to obj BFP, interaction 4. Sheared rays pass through specimen, diffracted, optical path changed and wavefront distorted. 3. Condenser project sheared rays parallel apart 2. Condenser Nomarski divides beam (shear) The divided rays reach condenser FFP 1. Polarizer produce linear polarized light AMM-2012 AMU_HI_HY PL_MIC + DIC optics 18 How DIC form images of cells DIC Contrast formation A and C: no OPD, no contrast enhancement. B: an apparent change of OPD, contrast enhanced. DIC enhances contrast where the shear of two rays show a big change rate of optical path difference (OPD). AMM-2012 AMU_HI_HY 19 DIC image formation and Introduction of bias retardation Bias retardation translation http://microscopy.fsu.edu/primer/techniques/dic/dicintro.html AMM-2012 AMU_HI_HY 20 DIC and its usage Advantage of DIC High resolution, use full objective aperture; no restriction from condenser Shadow-cast, psudo-3D profile of the structure Good depth of field and z-resolution, optical sectioning is possible View slightly stained specimen is possible Determine orientation of phase gradient Disadvantage of DIC Qualitative, not quantitative, not true thickness but optical path length Birefringent specimen cannot be used Polystyrene plastic cell culture dish, bottle, chamber cannot be used Even the mounting media has to be under scrutiny: not use PVA which a popular component in mounting media AMM-2012 AMU_HI_HY 21 Example of DIC images AMM-2012 AMU_HI_HY 22 DIC VS. Phase contrast (1) Phase contrast: halo effect at edge, less details visible. AMM-2012 AMU_HI_HY DIC image: no halo effect, more intracellular details, sharp edge 23 DIC VS. Phase contrast (2) Phase contrast: thick specimen Poor image quality and depth of field AMM-2012 AMU_HI_HY DIC: perform well in thick specimen Optical section effect 24 DIC VS. Phase contrast (3) Phase contrast: poor z-resolution AMM-2012 AMU_HI_HY DIC: good z-resolution reveals intracellular structures 25 Hoffman modulation contrast Inverted microscope with Hoffman modulation P1: rotatable polarizer P2: polarizer with slit Modulator: 3-zone ND AMM-2012 AMU_HI_HY Re-map phase gradient to intensity gradient 26 Hoffman contrast image formation Polarizer and modulator alignment, adjustment Align p2 with modulator P2 light Micrograph of cell culture by Hoffman contrast P2 dark Contrast control effects of P2 by rotating P1 AMM-2012 AMU_HI_HY 27 Image formation mechanism in Hoffman modulation contrast Does not change phase of light (the P-wave is 1/20 λ delayed measured at BFP, same as phase specimen in ordinary bright field microscopy) The specimen’s phase gradient is re-mapped to different zone (on objective lens’s BFP) Image is formed on image plane with different intensity Phase gradient has direction, rotate specimen help achieve better result when direction of the gradient is unknown AMM-2012 AMU_HI_HY 28 Comment on Hoffman contrast Better resolution than phase contrast. Bypass birefringent restriction from DIC. Cheaper to implement than DIC. Through gradual phase gradient to gradual intensity gradient, an image with 3D relic is formed and depth detection is easier Newer on the market: If want add to existing system, need buy accessories and modify existed objective lens. Extra cost need. http://micro.magnet.fsu.edu/primer/techniques/hoffman/hoffmanintro.html AMM-2012 AMU_HI_HY 29 DIC VS. Phase contrast Vs. Hoffman modulation contrast (1) mechanism All work with phase specimen. All use RI difference (phase difference) between structures and surrounding. Convert RI difference to image amplitude difference for contrasting. Enhance contrast by different mechanisms: DIC Convert deep OPD over pairs of ray shear divided by Nomarski prism into polarization difference, filter away linear polarized rays, and finally produce intensity difference. The shear is small, usually smaller than obj-lens resolution: enhancement is very localized Phase contrast enhance contrast by speed up direct ray to stretch small phase differences existed in the specimen, making the difference big enough for interference, yield intensity difference Hoffman modulation contrast Re-map (modulate) phase gradient into different intensity zones to enhance contrast AMM-2012 AMU_HI_HY 30 DIC VS. Phase contrast Vs. Hoffman modulation contrast (2) Usage Phase contrast Simple, easy to configure, cheap, time-honored Phase halo, not good for too thin, too thick specimen Low resolution: 1/10 of the objective lens NA is used DIC Full resolution of the lens, high image quality, good for both thick and thin specimen Not suitable for birefringent specimen or container with birefringent material. Expensive, difficult to setup and align the optics Hoffman contrast avoid phase halo with higher resolution, bypass birefringent restriction Not widely available, image quality is moderate AMM-2012 AMU_HI_HY 31