Simple Microscope

• Objective magnification• Working Distance• Eyepiece magnification

http://micro.magnet.fsu.edu/

Illumination (Bright Field)• Simple mirror (historical microscope)• Critical illumination• Koehler Illumination

Summary the field of view should be (reasonably) evenly illuminated the illuminating train should be able to fully illuminate the aperture of an objective of NA = 1.0 the light source should be focused in the object in critical illumination, the light bulb is the light source in Köhler illumination, the light source is an iris diaphragm attached to the illuminator (the field stop) the condenser iris is adjusted for each objective

Staining• Staining is a biochemical technique of

adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound.

• Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the

aid of different microscopes.

Dark Field microscopy

bright Dark

Poor-man`s dark field

Dark Field

DIC (Differential Interference Contrast)Wollaston Prism

Optical Path Length (OPL) = n • t

OPL difference = 2*pi*delta/lambda

delta=(n2 - n1) • t

`Nomarski`

Phase Contrast• Converts phase change to Amplitude change

http://micro.magnet.fsu.edu/primer/techniques/phasecontrast/phaseindex.html

Phase Contrast

Phase Contrast• Converts phase change to Amplitude change

• Converts phase change to Amplitude change

φ(x,y) < < 1

PSF(kx,ky) is the Point spread function (PSF)

Without PC optics

With PC optics

Furhter Contrast Enhancement in Phase Contrast Microscopy

• Select part of illumination

Reduce the size of the fat arrow

Proper Choice of the phase shift reverses contrast