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Microscopy Boot Camp 2009 2009/08/25 Nikitchenko Maxim Baktash Babadi.

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Presentation on theme: "Microscopy Boot Camp 2009 2009/08/25 Nikitchenko Maxim Baktash Babadi."— Presentation transcript:

1 Microscopy Boot Camp 2009 2009/08/25 Nikitchenko Maxim Baktash Babadi

2 Plan of the lecture Basic properties of light Light/matter interaction Wide-field microscopy Scanning microscopy EM Ultra-high resolution microscopy Dyes Part 1 Part 2 Part 3

3 Corpuscular/wave dualism www.olympusmicro.com Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

4 Diffraction Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

5 Basic electromagnetic wave properties Amplitude Wavelength Frequency Phase Polarization hyperphysics.phy-astr.gsu.edu Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

6 Polarization Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

7 Light/matter interaction Particles point of view: Absorption Emission Scattering Waves point of view: Refraction Reflection Absorption Diffraction (Change of Phase and Polarization) Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

8 Optics of a thin lens (1) Focus d F FCCF C=2F Thin Lens: Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

9 Optics of a thin lens (2) Three different scenarios: F 2F F F F F F Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

10 Optics of a thin lens (3) f pq FCCF Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

11 Compound Microscope Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

12 Basic optical structure of a microscope objective specimen Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection eyepiece

13 Specimen Illumination System Parts –Specimen plane –Condenser –Diaphragm –Light Source Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

14 Microscope Illumination Conditions: Critical illumination –The condenser focuses the light onto the specimen plane Filament image effect Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

15 Köhler illumination The specimen is illuminated homogenously The specimen and the images of the light source are in different planes Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

16 Types of Microscopy Bright Field (absorption) Dark Field (scattering) Phase-contrast (phase change) Polarization (scattering by birefringent specimen) Differential interference contrast (DIC) (gradients of optical thickness) Fluorescent (frequency change as a result of absorption/emission by fluorophores) Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

17 Dark field Microscopy uses the difference in scattering abilities block out the central light rays (leave oblique only) Result: only highly diffractive and scattering structures are seen Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

18 Dark-Field example Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

19 uses the λ/4 phase change when light passes through thin structures Similar oblique illumination to the Dark Field method The specimen diffracts some of the light that passes through it and introduces phase lagging λ/4 A phase difference (λ/2) is introduced between background and diffracted light (using phase plate) → destructive interference Phase Contrast Microscopy Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

20 Phase Contrast Microscopy Suitable for unstained specimens Human glial cells Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

21 Polarization Microscopy Uses polarization property of light and birefringence Polarizer polarizes light Analyzer passes only the light with polarization perpendicular to the source light Birefringent material introduces 2 perpendicularly polarized components, propagating at different speed in the specimen → Δφ Constructive interference following analyzer is possible only for phase shifted light Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

22 Polarized microscopy example Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

23 Differential Interference Contrast (DIC) (Nomarski optics) Addon to the polarization microscopy Wollaston prism generates 2 || beams, π/4 polarized to polarizer and laterally displaced (this is the difference to polarization microsc., endowing optical density gradient sensitivity) The rest is similar to pol. Micr. (except for 2 nd Wollaston prism) Result: good for edge detection Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

24 Nomarski optics principle polarizer Beam Splitter shear condenser specimen objective combiner analyzer intensity Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

25 DIC example Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

26 Fluorescent Microscopy (1) Fluorescence –Emission light has longer wavelengths than the excitation light: Stokes shift. Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

27 Fluorescent Microscopy (2) Types of Fluorescence –Auto-Fluorescence (Plants, Fungi, Semiconductors, etc) – Fluorescent dyes Fluorochromes (Flurescein, Acredine Orange, Eosin, Chlorophyll A, … ) Genetically coded (GFP, YFP,…) Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

28 Fluorescent Microscopy (3) The basic task of the fluorescence microscope: –Illuminate the specimen with excitation light –Separate the much weaker emission light from the brighter excitation light. –Only allow the emission light to reach the eye or other detector. –The background is dark, the fluorescent objects are bright Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

29 Epi-Fluorescent Illumination The emission light does not pass through the specimen The objective acts as the condenser Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

30 Fluorescent Microscopic images (1) Human cortical neurons Human brain glioma cells Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

31 Fluorescent Microscopic images Fluorescence/DIC combination, cat brain tissue infected with Cryptococcus Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

32 Brainbow Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection

33 Selection of the microscope Is it thin? (<50 microns) Is it reflective? e.g. gold, silver Is it fluorescent? Confocal Epi-fluorecence Is it fluorescent? Is it colored, densely contrasted or stained? Is it transparent? Bright field Phase contrast, DIC Is it reflective? e.g. gold, Silver Is it birefringent? Polarization Dark field no yes Basic Properties of LightThin lensesWide-field MicroscopyFluorescent MicroscopyMicroscope selection Rubbi, C.P., 1994

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