Microscopy Matti Hotokka Department of Physical Chemistry Åbo Akademi University
What s coming Anatomy of a microscope Modes of illumination Practicalities Special applications
Basic microscope Ocular Objective Sample stage Condenser lens Light source http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr99/rnscope.html
Parts of a microscope http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr99/rnscope.html
Modern microscope microscopy.fsu.edu/primer/
Types of microscopes Stereo microscope Preparation microscope http://www.microscopyu.com/articles/stereomicroscopy/stereointro.html Biological microscope Ordinary microscope
Stereo microscopy Magnification typically 10-150 x Three-dimensional image The sample can be almost anything Large working area
Ordinary microscopy Magnification 100-1000 x Two-dimensional Sample on a microscope glass Small working area
Elementary optics Magnification of a lens b M = b f f 1 f 1 Real image
Elementary optics Magnification of a lens m = 1+ d f d For a normally accomodated eye d = 25 cm Imaginary image f 2 f 2
Elementary optics Magnification = Mxm = Optical length = approx. b b Microscope f 1 f 2
Numerical aperture Let be the half-angle of the cone of light that can enter the lens. Let n be the refractive index of the surrounding medium (1 for air, 1.5 for oil). The the numerical aperture NA=n sin
f-number For small numerical apertures f 1 2NA
Typical objectives Magnification NA 4x 0.10 10x 0.25 20x 0.40 100x (dry) 0.95 100x (immersed) 1.25 Dry NA=0.4 Immersion media Water, n=1.33 Glycerin, n=1.47 Oil, n=1.51 Most objectives with magnification 60x or higher are designed to allow immersion in oil. Do not use other immersion media since the lenses in the objective are glued together. The glue may be affected by other liquids. Immersed in oil NA=0.8 http://microscopy.fsu.edu/primer/anatomy/numaperture.html
Resolution Two objects are seen separately if their distance is larger than the Airy radius r = 122. λ Airy 2NA r http://microscopy.fsu.edu/primer/anatomy/numaperture.html
Resolution 1.25 NA r=0.61/na =550 nm 0.65 0.1 Magnification NA m 4x 0.10 3.36 10x 0.25 1.34 20x 0.40 0.84 100x (dry) 0.95 0.35 100x (immersed) 1.25 0.27 m 3.36 http://microscopy.fsu.edu/primer/anatomy/numaperture.html 0.51 0.27
Depth of field The higher the numerical aperture the smaller depth is in focus. Magnification NA m 4x 0.10 55.5 10x 0.25 8.5 20x 0.40 5.8 100x (dry) 0.95 0.19 100x (immersed) 1.25 0.85 Nearest plane in focus Farthest plane in focus
Field number Field of view diameter The diameter of the visible region is (Ø mm) N P = M q Obj N = The field number of the ocular, e.g., 10/20 (magnification/n) M obj = Magnification of the objective, e.g., 40/0.65 (magnification / NA) q = The tubus, usually 1, sometimes 1.25 In the example, diameter of the visible part of the sample is 20/40 mm = 0.5 mm
Methods in microscopy Transmittance illunimation Bright field Dark field Polarization Phase contrast Interference contrast Reflectance illumination A similar list
Bright field Objective Stained sample Stage Condenser
Dark field Light scattered from the sample Objective Unstained sample Stage Condenser Shade Good for samples in water.
Phase contrast Useful for large magnifications (400 x ->) Useful when the sample is colorless or the details are so fine that color does not show in bright field. www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html
Interference contrast DIC Thicker samples than usual can be observed in focus.
Polarization Tartaric acid crystals http://www.leica-microsystems.com
Digital photography The photograph shows approx. 60 % of the field visible in the ocular
Practicalities All knobs that can be turned with fingers without tools may (and should) be turned. Otherwise the grease will harden. Clean Adjust Set Köhler illumination
Clean the objectives Blow the dust away Use clean and dry pressure air Wash the objective Use a solution of 70 % ether, 30 % alcohol Circular motions Inspect visually (use ocular)
Adjust eyepieces Adjust focus Close right eye Adjust carefully focus so that the image is sharp Adjust accomodation Close left eye instead Turn the accomodation ring to make the image sharp
Köhler illumination Maximize the illumination of the sample Use a 10x objective. Focus to the sample. Move the condenser lens to uppermost position. Close the field aperture. Move the condenser lens down a bit to make the edge of the aperture sharp. Move the illuminated dot to the center of the visible field. Open the field aperture until the aperture edge is just outside the visible field.
Special applications Confocal microscopy Fluorescence microscopy Near-field microscopy Image analysis
Confocal microscopy Marvin Minsky 1955 Laser Detector Beamsplitter Pinhole Good depth profile. Crisp images. Http://www.cs.ubc.ca/ladic/overview.html d z Focal plane
Fluorescence microscopy Detector Fluorescence filter Dichroic mirror Hg lamp Objective Excitation filter Sample
Fluorescence microscope
Fluorescence microscopy Topology: shear force; transmission, fluorescence: SNOM. Phase separated polymer sample. Http://physics.nist.gov/Divisions/Div844/facilities/nsom/nsom.html
Near-field microscopy Only intensity is measured. Position of the tip maps the surface Reflection y 10 nm x Transmission
Image analysis