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