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Microscopy. Nikitchenko Maxim Baktash Babadi. Boot Camp 2009 2009/08/25. 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. Basic Properties of Light.

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Presentation Transcript
slide1

Microscopy

Nikitchenko Maxim

Baktash Babadi

Boot Camp 2009

2009/08/25

slide2

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

slide3

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Corpuscular/wave dualism

www.olympusmicro.com

slide4

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Diffraction

slide5

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

slide6

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Polarization

slide7

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)

slide8

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

slide9

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

slide10

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

slide11

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Compound Microscope

slide12

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

eyepiece

Basic optical structure of a microscope

specimen

objective

slide13

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Specimen Illumination System

  • Parts
    • Specimen plane
    • Condenser
    • Diaphragm
    • Light Source
slide14

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

slide15

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
slide16

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)
slide17

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

slide18

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Dark-Field example

slide19

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
slide20

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Phase Contrast Microscopy

  • Suitable for unstained specimens

Human glial cells

slide21

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
slide22

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Polarized microscopy example

slide23

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
slide24

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

slide25

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

DIC example

slide26

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.
slide27

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,…)
slide28

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
slide29

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
slide30

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Fluorescent Microscopic images (1)

Human cortical neurons

Human brain glioma cells

slide31

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

slide32

Basic Properties of Light

Thin lenses

Wide-field Microscopy

Fluorescent Microscopy

Microscope selection

Brainbow

slide33

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