Microbiology - Week 4 - Lecture 2 - Microscopy PPT.pdf

INTRODUCTION TO MICROBIOLOGY III MICROSCOPY Prepared by: Nick Inglis, Ph.D. BMS 100 IMPORTANT: KNOW YOUR MEASUREMENTS! Common Units of Measurement Unit Abbreviation Value 1 centimeter cm 10-2 meter 1 millimeter mm 10-3 meter 1 micrometer μm 10-6 meter 1 nanometer nm 10-9 meter 1...

INTRODUCTION TO MICROBIOLOGY III MICROSCOPY Prepared by: Nick Inglis, Ph.D. BMS 100 IMPORTANT: KNOW YOUR MEASUREMENTS! Common Units of Measurement Unit Abbreviation Value 1 centimeter cm 10-2 meter 1 millimeter mm 10-3 meter 1 micrometer μm 10-6 meter 1 nanometer nm 10-9 meter 1 Angstrom Å 10-10 meter FIRST KEY VARIABLE: THE REFRACTIVE INDEX Air Water Glass/Plastic Lens Air FIRST KEY VARIABLE: THE REFRACTIVE INDEX Air Water Glass/Plastic Lens Air E.G., A PRISM LENSES – FOCAL POINTS (F) AND FOCAL LENGTHS (F) F f THE BRIGHTFIELD MICROSCOPE Ocular (eyepiece) Body assembly Nosepiece Arm Objective lens Light intensity control Mechanical stage Aperture 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) Objective Working distance Slide with specimen 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 0.5 λ 𝑑= 𝑛 sin ϴ COMMON OBJECTIVE LENSES Properties of Objective Lenses OBJECTIVE Property Scanning Low Power High Power Oil Immersion Magnification 4× 10× 40-45× 90-100× Numerical aperture 0.10 0.25 0.55-0.65 1.25-1.4 Approximate focal length(f) 40 mm 16 mm 4 mm 1.8-2.0 mm Working distance 17-20 mm 4-8 mm 0.5-0.7 mm 0.1 mm Approximate resolving power with light of 450 nm(blue light) 2.3 μm 0.9 μm 0.35 μm 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 Objectiv e Specimen Abbé condense r Dark-field stop DARK-FIELD MICROSCOPY T. pɑllidum (a) Source: CDC/Schwartz; Volvox (b) ©McGraw-Hill Education/Stephen Durr, photographer PHASE CONTRAST MICROSCOPY Wave trough Bacterium in a wet mount Wave crest Ray deviated by specimen is ¼ wavelength out of phase. Phase plate Deviated ray is 1/2 wavelength out of phase. Deviated and undeviated rays cancel each other out. Image plane Undeviated light PHASE CONTRAST MICROSCOPY Phase plate Objective Deviated light Specimen Condenser Condenser annulus PHASE CONTRAST MICROSCOPY Wave trough Bacterium in a wet mount Wave crest Ray deviated by specimen is ¼ wavelength out of phase. Phase 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; Micronucleus (b) ©McGraw-Hill Education/James Redfearn, photographer DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPY FLUORESCENCE MICROSCOPY Light Microscopy (what we’ve been talking about!) Condenser Lens Objective/ Ocular Lenses Specimen Long wavelengths FLUORESCENT MICROSCOPES 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 Fluorochrome Uses Acridine orange Stains DNA Diamidino-2-phenyl indole(DAPI) Stains DNA Fluorescein isothiocyanate(FITC) Often attached to DNA probes or to antibodies that bind specific cellular components Tetramethyl rhodamine isothiocyanate(TRITC or rhodamine) 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! Light Focal Plane x Specimen y z THE SOLUTION: CONFOCAL SCANNING LASER MICROSCOPY (CSLM) CREATING Z STACKS RESULT OF A Z STACK Transmission Electron Microscopee Light Microscope Electron gun LIGHT VS ELECTRON MICROSCOPY Lamp Condenser lens Glass Electromagnet Electron beams Light rays Specimen Electromagnet Objective lens Glass Image Ocular lens Glass Eye Electromagnet Viewing screen ELECTRON MICROSCOPY LIGHT VS ELECTRON MICROSCOPY Characteristics of Light and Transmission Electron Microscopes Feature Light Microscope Transmission Electron Microscope Highest practical magnification About 1,000-1,500 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 Glass Electromagnet Source of contrast 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 Adjust current to the magnetic lens Specimen mount Glass slide Metal grid(usually copper) 1 The resolution limit of a human eye is about 0.2mm. THANK YOU! A N D H AV E A L O V E LY D AY !