Microscopy

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

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. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Corpuscular/wave dualism www.olympusmicro.com

4. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Diffraction

5. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Basic electromagnetic wave properties • Amplitude • Wavelength • Frequency • Phase • Polarization hyperphysics.phy-astr.gsu.edu

6. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Polarization

7. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Light/matter interaction • Particles point of view: Absorption Emission Scattering • Waves point of view: Refraction Reflection Absorption Diffraction (Change of Phase and Polarization)

8. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection C F F C Optics of a thin lens (1) Focus d F Thin Lens: C=2F

9. Thin lenses Basic Properties of Light Wide-field Microscopy Fluorescent Microscopy Microscope selection 2F F F 2F 2F F F 2F Optics of a thin lens (2) • Three different scenarios: 2F F 2F F

10. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection C F F C Optics of a thin lens (3) f p q

11. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Compound Microscope

12. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection eyepiece Basic optical structure of a microscope specimen objective

13. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Specimen Illumination System • Parts • Specimen plane • Condenser • Diaphragm • Light Source

14. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Microscope Illumination Conditions: • Critical illumination • The condenser focuses the light onto the specimen plane Filament image effect

15. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Köhler illumination • The specimen is illuminated homogenously • The specimen and the images of the light source are in different planes

16. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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)

17. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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

18. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Dark-Field example

19. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Phase Contrast Microscopy • 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

20. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Phase Contrast Microscopy • Suitable for unstained specimens Human glial cells

21. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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

22. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Polarized microscopy example

23. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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 2nd Wollaston prism) • Result: good for edge detection

24. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Nomarski optics principle polarizer shear specimen combiner intensity Beam Splitter condenser objective analyzer

25. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection DIC example

26. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Fluorescent Microscopy (1) • Fluorescence • Emission light has longer wavelengths than the excitation light: Stokes shift.

27. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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,…)

28. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection 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

29. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Epi-Fluorescent Illumination • The emission light does not pass through the specimen • The objective acts as the condenser

30. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Fluorescent Microscopic images (1) Human cortical neurons Human brain glioma cells

31. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Fluorescent Microscopic images Fluorescence/DIC combination, cat brain tissue infected with Cryptococcus

32. Basic Properties of Light Thin lenses Wide-field Microscopy Fluorescent Microscopy Microscope selection Brainbow

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