Light microscopy Lysmikroskopi MENA3100,OBK, Repetition, exercises and more
1.3Specimen Preparation OK to read 1.4.1Bright-Field and Dark-FieldDefinetly 1.4.2Phase-ContrastCursori 1.4.3Polarized-LightDefinetly 1.4.4NormarskiWe have it … 1.4.5FluorescenceCursori 1.5ConfocalCursori 1.2Instrumentation Yes 1.1Optical Principles Yes
Eyepice Okular Objective M = M 1 × M 2
1000 X
2000 X
X
X
50 m
Magnification vs. resolution Forstørrelse vs. oppløsningsevne
p. 5 Effective magnification Resolution of the eye is around 0.2 mm Resolution limit is about 0.2 µm M eff ≈ 0.2 mm 0.2×10 −3 mm = 1000
The Nobel Prize in Chemistry 2014 was awarded jointly to Eric Betzig, Stefan W. Hell and William E. Moerner "for the development of super-resolved fluorescence microscopy". But:
Diffraction limit the Rayleigh criterion Airy disk
Numerical aperture (NA) NA = nsin R = 0,61 /NA Resolution Oppløsning
Objectives for oil immersion Brightness = (NA)/M 2 Reflective: (NA)/M 4
Conjugate focal planes Köhler system
Chromatic aberration Spherical aberrasjon Astigmatism
CorrectionsObjective Acromate: Chromatic for red and blue, spherical for green Semi-acromate (fluorite) Apochromate: Chromatic for red, green, blue and dark blue, spherical for green and blue
Optical axis Through the centre is not deflected Parallell to the optical axis goes through back focal plane f (Through focal plane before lense becomes parallel optical axis.) f Projected onto a screen Distance one to two focal lengths
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s (= v)s’s’ f ff
Object Real image (Primary image) Real image on retina Virtuell image OBS! Error in figure hm...
All rays originated from the same point are collected here Image plane All rays spread in the same direction are collected here Diffraction plane Fourier plane
Mørkefelt og lysfelt Darkfield and brightfield
Can observe stress/strain
Optical anisotropy Calcite
Optically active crystals in the microscope