Microbiology 2.2_ Microscopy (editing in progress).pptx

INTRODUCTION TO MICROBIOLOGY III MICROSCOPY Prepared by: Nick Inglis, Ph.D. BMS 100 IMPORTANT: KNOW YOUR MEASUREMENTS! Un it 1 centimete r1 millimete r1 micromete r1 nanomete r1 Angstro m Common Units of Measurement Abbrevia tion c m m m μ m n m Å Val ue 10-2 meter 10-3 meter 10-6 meter 10-9...

INTRODUCTION TO MICROBIOLOGY III MICROSCOPY Prepared by: Nick Inglis, Ph.D. BMS 100 IMPORTANT: KNOW YOUR MEASUREMENTS! Un it 1 centimete r1 millimete r1 micromete r1 nanomete r1 Angstro m Common Units of Measurement Abbrevia tion c m m m μ m n m Å Val ue 10-2 meter 10-3 meter 10-6 meter 10-9 meter 10-10 meter FIRST KEY VARIABLE: THE REFRACTIVE INDEX Ai r Wat er Glass/Plastic Lens Ai r FIRST KEY VARIABLE: THE REFRACTIVE INDEX Ai r Wat er Glass/Plastic Lens Ai r E.G., A PRISM LENSES – FOCAL POINTS (F) AND FOCAL LENGTHS (F) F f THE BRIGHTFIELD MICROSCOPE Ocular (eyepiec e) Body assembly Nosepiec e Ar m Objective lens Light intensity control Mechanical Aperturestage diaphragm control Substage condenser Coarse focus adjustment knob Fine focus adjustment knob Base with light source Stage adjustment knobs RESOLUTION NUMERICAL APERTURE (N SIN ϴ) Objectiv e Working distance Slide with specime n ᶿ ᶿ THINK ABOUT THE MATH • The refractive index of air is 1.0 • The formula for resolution is n sin ϴ • Consider the limits of theta OIL IMMERSION OBJECTIVES Slide Air Oil Cover glass THE ABBÉ EQUATION COMMON OBJECTIVE LENSES Properties of Objective Lenses OBJECTI VE Prope rty Magnificati on Numerical aperture Approximate focal length(f) Working distance Approximate resolving power with light of 450 nm(blue light) Scanni ng 4× Low Power 10× High Power 40-45× Oil Immersio n 90-100× 0.1 0 40 mm 17-20 mm 2.3 μm 0.2 5 16 mm 4-8 mm 0.9 μm 0.550.65 4 mm 0.5-0.7 mm 0.35 μm 1.251.4 1.8-2.0 mm 0.1 mm 0.18 μm ANOTHER COMPLICATION!! • Most microbes we will be looking at are not pigmented! • Dark-field microscopes • Phase-contrast microscopes • Differential interference contrast (DIC) microscopes DARK-FIELD MICROSCOPY Objecti ve Abbé conden ser Specim en Dark-field stop DARK-FIELD MICROSCOPY T. pɑllidu m (a) Source: CDC/Schwartz; Volvox (b) ©McGraw-Hill Education/Stephen Durr, photographer PHASE CONTRAST MICROSCOPY Wave troug h Bacterium in a wet mount Wave crest Ray deviated by specimen is ¼ wavelength out of phase. Phas e plate Deviated ray is 1/2 wavelength out of phase. Deviated and undeviated rays cancel each other out. Image plane PHASE CONTRAST MICROSCOP Y Undeviate d light Phase plate Objectiv e Deviate d light Specime n Condens er Condens er annulus PHASE CONTRAST MICROSCOPY Wave troug h Bacterium in a wet mount Wave crest Ray deviated by specimen is ¼ wavelength out of phase. Phas e plate Deviated ray is 1/2 wavelength out of phase. Deviated and undeviated rays cancel each other out. PHASE CONTRAST MICROSCOPY Macronucleus Pɑrɑmecium sp. An amoeba (a) ©McGraw-Hill Education/Stephen Durr, photographer; Micronucleu s (b) ©McGraw-Hill Education/James Redfearn, photographer DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY FLUORESCENCE MICROSCOPY Light Microscopy (what we’ve been talking about!) Condens er Lens Objective/ Ocular Lenses Specime n Long wavelengths FLUORESCENT MICROSCOPE S Exciter filter (removes long wavelengths) Barrier filter (blocks ultraviolet radiation but allows visible light through) Dichromatic mirror (reflects short wavelengths; transmits longer wavelengths) Mercury arc lamp Short wavelengths Long wavelengths Fluorochrome-stained specimen (absorbs short-wavelength radiation and emits longer-wavelength light) COMMON FLUOROCHROMES Commonly Used Fluorochromes Fluorochr ome Acridine orange Diamidino-2phenyl indole(DAPI) Fluorescein isothiocyanate(F ITC) Tetramethyl rhodamine isothiocyanate(TRI TC or rhodamine) Us es Stains DNA Stains DNA Often attached to DNA probes or to antibodies that bind specific cellular components Often attached to antibodies that bind specific cellular components FLUORESCENCE MICROSCOPY GREEN FLUORESCENT PROTEIN PROBLEM! MOST THINGS WE ARE LOOKING AT ARE 3D, NOT FLAT! Ligh t Foca l Plan e x y z Spe cim en THE SOLUTION: CONFOCAL SCANNING LASER MICROSCOPY (CSLM) CREATING Z STACKS RESULT OF A Z STACK Transmission Electron Microscopee Light Microscope LIGHT VS ELECTRON MICROSCOPY Electron gun Lamp Condenser lens Glass Electromagne t Electron beams Ligh t rays Specimen Electromagne t Objective lens Glass Imag e Ocular lens Glass Eye Electromagne t Viewing screen ELECTRON MICROSCOPY LIGHT VS ELECTRON MICROSCOPY Feature Characteristics of Light and Transmission Electron Microscopes Light Microscope Highest practical magnification About 1,000-1,500 Transmission Electron Microscope Over 100,000 Best resolution1 0.2 μm 0.2 nm Radiation source Visible light Electron beam Medium of travel Air High vacuum Type of lens Electromagnet Source of contrast Gla ss Differential light absorption Scattering of electrons Focusing mechanism Adjust lens position mechanically Adjust current to the magnetic lens Method of changing magnification Switch the objective lens or eyepiece Glass slide Adjust current to the magnetic lens Specimen mount 1 The resolution limit of a human eye Metal grid(usually copper)