三、各种光学显微术 (显微镜附件)
三、各种光学显微术 (显微镜附件)
反射式显微镜的衬度模式 ·明场(BF) Contrast Modes in Reflected Light Microscopy 。暗场(DF) EsL会三>STS LACESSITS 。相位衬度(Phase 兴》 contrast) HYDROLYCUS DROLYCU色 偏振光(Polarized light) ·微分干涉(DIC) (C) Figure 1 2
反射式显微镜的衬度模式 2 • 明场(BF) • 暗场(DF) • 相位衬度(Phase contrast) • 偏振光(Polarized light) • 微分干涉(DIC)
a) Reference wave b) Amplitude Reduced object amplitude Phase c) Retarded object phase Figure 7-2 Effects of amplitude and phase objects on the waveform of light.(a)Reference ray with characteristic amplitude,wavelength,and phase.(b)A pure amplitude object absorbs energy and reduces the amplitude,but does not alter the phase,of an emergent ray.(c)A pure phase object alters velocity and shifts the phase,but not the amplitude,of an emergent ray. ·人眼可以区分振幅变 ·人眼不能区分相位变 化 化 。eg:明场、暗场 。 相位变化->振幅变化 eg:偏光、相衬、微 分干涉
• 人眼可以区分振幅变 化 • eg.: 明场、暗场 • 人眼不能区分相位变 化 • 相位变化->振幅变化 • eg.: 偏光、相衬、微 分干涉 3
明场像的衬度原理 Microscope s 200μm polished etched surface surface groove grain boundary 4
明场像的衬度原理 4
暗场像 试样 曲面反射镜 Tube Lens Oval Mirror 物镜 (a) B A Oval 暗场环形 Mirror 反射镜 Mirror Assembly B Objective 环形光阑 Concave Mirror Specimen 至日镜 5
暗场像 5
Bright-field (BF)and dark-field (DF)are the most commonly used examination modes. BF OBJECTIVE OR.IECT CONDENSER- 一APERTURE一= ANNULAR STOP-IRIS CENTRAL STOP Figure 1.28 (a)Bright-field illumination;and(b)dark-field illumination in transmitted mode.Shaded areas indicate where the light is blocked. Figures from Leng,pages 26,27. 10: 6
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50μm Optical bright field and dark-field metallographic images of a Zircaloy- 4 cladding oxidised in air at 850C(see text section D for signification of regions 1,2,3 and 4) from Nuclear Engineering and Design,Volume 239,Issue 2,February 2009,Pages 244-253
7 Optical bright field and dark-field metallographic images of a Zircaloy- 4 cladding oxidised in air at 850oC (see text section D for signification of regions 1, 2, 3 and 4) from Nuclear Engineering and Design, Volume 239, Issue 2, February 2009, Pages 244–253
相位衬度显微术 a) Reference wave b) Amplitude Reduced object amplitude Phase c) Retarded object phase Figure 7-2 Effects of amplitude and phase objects on the waveform of light.(a)Reference ray with characteristic amplitude,wavelength,and phase.(b)A pure amplitude object absorbs energy and reduces the amplitude,but does not alter the phase,of an emergent ray.(c)A pure phase object alters velocity and shifts the phase,but not the amplitude,of an emergent ray. 8
相位衬度显微术 8
Phase Contrast Imaging of Transparent Thin Specimens (a) (b) (c) △1 2 Nucleus (Thickest Portion) Adherent Cell Cell Edge (Thin) Incident Coverslip Transmitted Light lllumination一 Figure 2 如何将相位差转变为亮度差？ 9
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P wave S wave Incident beam,λ D wave 1 Ad<<A 20 △ (b) B Vectors 2πδ 4π△d S Wave p λ Wave D Wave 10
10 B A d S P S P D Incident beam, 𝜑 = 2𝜋𝛿𝜆 = 4 𝜋 Δ 𝑑 𝜆 d<<