MicroscopyBoot Camp 20092009/08/25Nikitchenko MaximBaktash Babadi

Plan of the lectureBasic properties of lightLight/matter interactionWide-field microscopyScanning microscopyEMUltra-high resolution microscopyDyesPart 1Part 2Part 3

Corpuscular/wave dualismwww.olympusmicro.com

Diffraction

Basic electromagnetic wave propertiesAmplitudeWavelengthFrequencyPhasePolarizationhyperphysics.phy-astr.gsu.edu

Polarization

Light/matter interactionParticles point of view: AbsorptionEmission ScatteringWaves point of view:RefractionReflectionAbsorptionDiffraction (Change of Phase and Polarization)

Optics of a thin lens (1)FocusdFC=2FThin Lens:

Optics of a thin lens (2)Three different scenarios:F2F2FF

Optics of a thin lens (3)fpq

Compound Microscope

Basic optical structure of a microscopeobjectivespecimen

Specimen Illumination SystemPartsSpecimen plane

Condenser

Diaphragm Light Source

Microscope Illumination Conditions:

Critical illuminationThe condenser focuses the light onto the specimen planeFilament image effect

Khler illuminationThe specimen is illuminated homogenously

The specimen and the images of the light source are in different planes

Types of MicroscopyBright 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)

Dark field Microscopyuses the difference in scattering abilitiesblock out the central light rays (leave oblique only)Result: only highly diffractive and scattering structures are seen

Dark-Field example

uses the /4 phase change when light passes through thin structuresSimilar oblique illumination to the Dark Field methodThe specimen diffracts some of the light that passes through it and introduces phase lagging /4A phase difference (/2) is introduced between background and diffracted light (using phase plate) destructive interferencePhase Contrast Microscopy

Phase Contrast MicroscopySuitable for unstained specimensHuman glial cells

Polarization MicroscopyUses polarization property of light and birefringencePolarizer polarizes lightAnalyzer passes only the light with polarization perpendicular to the source lightBirefringent material introduces 2 perpendicularly polarized components, propagating at different speed in the specimen Constructive interference following analyzer is possible only for phase shifted light

Polarized microscopy example

Differential Interference Contrast (DIC) (Nomarski optics) Addon to the polarization microscopyWollaston prism generates 2 || beams, /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

Nomarski optics principlepolarizerBeamSplittershearcondenserspecimenobjectivecombineranalyzerintensity

DIC example

Fluorescent Microscopy (1)FluorescenceEmission light has longer wavelengths than the excitation light: Stokes shift.

Fluorescent Microscopy (2)Types of FluorescenceAuto-Fluorescence (Plants, Fungi, Semiconductors, etc) Fluorescent dyesFluorochromes (Flurescein, Acredine Orange, Eosin, Chlorophyll A, )Genetically coded (GFP, YFP,)

Fluorescent Microscopy (3)The basic task of the fluorescence microscope:Illuminate the specimen with excitation lightSeparate 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

Epi-Fluorescent IlluminationThe emission light does not pass through the specimenThe objective acts as the condenser

Fluorescent Microscopic images (1)Human cortical neuronsHuman brain glioma cells

Fluorescent Microscopic images Fluorescence/DIC combination, cat brain tissue infected with Cryptococcus

Brainbow

Selection of the microscopeIs it thin? (