Resolving Power

The 5 I’s of Culturing Microbes • Inoculation • Isolation • Incubation • Inspection • Identification 8/18/12 MDufilho 1 Table 4.1 Metric Units of Length 8/18/12 MDufilho 2 Microscopy • General Principles of Microscopy – Wavelength of radiation – Magnification – Resolution – Contrast 8/18/12 MDufilho 3 Figure 4.1 The electromagnetic spectrum 400 nm 700 nm Visible light X UV Infra- Micro- Radio waves and Gamma rays rays light red wave Television Increasing wavelength 10–12m 10–8m 10–4m 100m 103m Crest One wavelength Trough Increasing resolving power 8/18/12 MDufilho 4 Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Specimen Convex Inverted, lens reversed, and enlarged 8/18/12 MDufilho image 5 Principles of Light Microscopy • Magnification occurs in two phases – – The objective lens forms the magnified real image – The real image is projected to the ocular where it is magnified again to form the virtual image • Total magnification of the final image is a product of the separate magnifying powers of the two lenses power of objective x power of ocular = total magnification 8/18/12 MDufilho 6 8/18/12 MDufilho 7 Resolution Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power – Function of wavelength of light that forms the image along with characteristics of objectives • Visible light wavelength is 400 nm–750 nm • Numerical aperture of lens ranges from 0.1 to 1.25 • Oil immersion lens requires the use of oil to prevent refractive loss of light • Shorter wavelength and larger numerical aperture will provide better resolution • Oil immersion objectives resolution is 0.2 μm • Magnification between 40X and 2000X 8/18/12 MDufilho 8 Calculating Resolving Power Formula D = ________wavelength (nm)_________ NA (condenser) + NA (objective) 8/18/12 MDufilho 9 Effect of wavelength on resolution 8/18/12 MDufilho 10 Figure 4.5 The effect of immersion oil on resolution-overview Microscope Microscope objective Lenses objective Refracted light More light rays lost to lens enters lens Immersion oil Glass cover slip Glass cover slip Slide Slide Specimen Light source Light source Without immersion oil With immersion oil 8/18/12 MDufilho 11 Figure 4.3 The limits of resolution of the human eye and of various types of microscopes Diameter Typical bacteria of DNA Ribosomes and archaea Flea Large protozoan Atoms Proteins Viruses Chloroplasts (Euglena) Chicken egg Amino Human red acids Mitochondrion blood cell Scanning tunneling microscope (STM) 0.01 nm–10 nm Transmission electron microscope (TEM) Unaided human eye 0.078 nm–100 µm 200 µm– Scanning electron microscope (SEM) 0.4 nm–1 mm Atomic force Compound light microscope (LM) 8/18/12 microscope (AFM) MDufilho 200 nm–10 mm 12 1 nm–10 nm Contrast • Differences in intensity between two objects, or between an object and background • Important in determining resolution • Staining increases contrast • Use of light that is in phase increases contrast 8/18/12 MDufilho 13 Light Microscopy Bright-field microscopes • Simple – Contain a single magnifying lens – Similar to magnifying glass – Leeuwenhoek used simple microscope to observe microorganisms • Compound – Series of lenses for magnification – Light passes through specimen into objective lens – Oil immersion lens increases resolution – Have one or two ocular lenses – Total magnification (objective lens X ocular lens) – Most have condenser lens (direct light through specimen) – Video on Microscopy 8/18/12 MDufilho 14 8/18/12 MDufilho 15 8/18/12 MDufilho 16 Care and Use of Microscope • Moving microscope from storage • Storing microscope – Short objective in place, center stage – Clean, cord wrapped correctly and covered • Cleaning microscope – Lens paper, swabs and cleaning solution • Learn components • Focusing the microscope on specimen • Use of Oil Immersion lens 8/18/12 MDufilho 17 .

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