Chapter 4. Microscopy, Staining, and Classification. Lecture prepared by Mindy Miller-Kittrell North Carolina State University

Transcription

1 Chapter 4 Microscopy, Staining, and Classification 2012 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell North Carolina State University

2 Microscopy and Staining 2012 Pearson Education Inc. ANIMATION Microscopy and Staining: Overview

3 Table 4.1 Metric Units of Length

4 Microscopy General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast 2012 Pearson Education Inc.

5 Figure 4.1 The electromagnetic spectrum 400 nm 700 nm Visible light Gamma rays m X rays 10 8 m UV light Infrared Microwave Increasing wavelength 10 4 m Crest Radio waves and Television 10 0 m 10 3 m One wavelength Increasing resolving power Trough

6 Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Specimen Convex lens Inverted, reversed, and enlarged image

7 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 Atoms Proteins Viruses Chloroplasts Large protozoan (Euglena) Chicken egg Amino acids Mitochondrion Human red blood cell Scanning tunneling microscope (STM) 0.01 nm 10 nm Transmission electron microscope (TEM) nm 100 µm Unaided human eye 200 µm Scanning electron microscope (SEM) 0.4 nm 1 mm Atomic force microscope (AFM) 1 nm 10 nm Compound light microscope (LM) 200 nm 10 mm

8 Microscopy General Principles of Microscopy 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 2012 Pearson Education Inc.

9 Microscopy Light Microscopy Bright-field microscopes Simple Contain a single magnifying lens Similar to magnifying glass Leeuwenhoek used simple microscope to observe microorganisms 2012 Pearson Education Inc.

10 Microscopy Light Microscopy Bright-field microscopes 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) 2012 Pearson Education Inc.

11 Figure 4.4 A bright-field, compound light microscope-overview Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base Line of vision Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator

12 Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Lenses Microscope objective Refracted light rays lost to lens Glass cover slip More light enters lens Glass cover slip Immersion oil Slide Slide Specimen Without immersion oil Light source With immersion oil Light source

13 Microscopy Light Microscopy Dark-field microscopes Best for observing pale objects Only light rays scattered by specimen enter objective lens Specimen appears light against dark background Increases contrast and enables observation of more details 2012 Pearson Education Inc.

14 Figure 4.6 The light path in a dark-field microscope Objective Light refracted by specimen Light unrefracted by specimen Specimen Condenser Dark-field stop Dark-field stop

15 Microscopy Light Microscopy Phase microscopes Examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining Contrast is created because light waves are out of phase Two types Phase-contrast microscope Differential interference contrast microscope 2012 Pearson Education Inc.

16 Figure 4.7 Principles of phase microscopy-overview Rays in phase Rays out of phase Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. Deviated ray is now 1/2 wavelength out of phase.

17 Figure 4.8 Four kinds of light microscopy-overview Nucleus Bacterium Bright field Dark field Phase contrast Nomarski

18 Microscopy Light Microscopy Fluorescent microscopes Direct UV light source at specimen Specimen radiates energy back as a visible wavelength UV light increases resolution and contrast Some cells are naturally fluorescent; others must be stained Used in immunofluorescence to identify pathogens and to make visible a variety of proteins 2012 Pearson Education Inc.

19 Figure 4.9 Fluorescent microscopy-overview

20 Figure 4.10 Immunofluorescence-overview Antibodies Fluorescent dye Bacterium Antibodies carrying dye Cell-surface antigens Bacterial cell with bound antibodies carrying dye

21 Microscopy Light Microscopy Confocal microscopes Use fluorescent dyes Use UV lasers to illuminate fluorescent chemicals in a single plane Resolution increased because light passes through pinhole aperture Computer constructs 3-D image from digitized images 2012 Pearson Education Inc.

22 Microscopy ANIMATION Light Microscopy 2012 Pearson Education Inc.

23 Microscopy Electron Microscopy Light microscopes cannot resolve structures closer than 200 nm Greater resolving power and magnification Magnifies objects 10,000X to 100,000X Detailed view of bacteria, viruses, ultrastructure, and large atoms Two types Transmission electron microscopes Scanning electron microscopes 2012 Pearson Education Inc.

24 Figure 4.11 A transmission electron microscope (TEM) -overview Light microscope (upside down) Column of transmission electron microscope Lamp Electron gun Condenser lens Specimen Objective lens Condenser lens (magnet) Specimen Objective lens (magnet) Eyepiece Final image seen by eye Projector lens (magnet) Final image on fluorescent screen

25 Figure 4.12 Scanning electron microscope (SEM) Electron gun Magnetic lenses Beam deflector coil Primary electrons Secondary electrons Specimen Specimen holder Scanning circuit Photomultiplier Detector Monitor Vacuum system

26 Figure 4.13 SEM images-overview

27 Microscopy ANIMATION Electron Microscopy 2012 Pearson Education Inc.

28 Microscopy Probe Microscopy Magnifies more than 100,000,000X Two types Scanning tunneling microscopes Atomic force microscopes 2012 Pearson Education Inc.

29 Figure 4.14 Probe microscopy-overview DNA Enzyme