1 Parts of the Microscope Ocular (eyepiece) Interpupillary adjustment Body Arm Coarse adjustment knob Fine focus adjustment knob Stage adjustment knobs Nosepiece Objective lens (4) Mechanical stage Substage condenser Aperture diaphragm control Base with light source Field diaphragm lever Light intensity control Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © Leica Microsystems Inc.

2 The Microscope Key characteristics of a reliable microscope are: Magnification – ability to enlarge objects Resolving power – ability to show detail

3 Total magnification of the final image is a product of the separate magnifying powers of the two lenses objective power ocular power total magnification x = Total Magnification Retina Brain Objective lens Specimen Ocular lens Eye Condenser lens Light source Virtual image Formed by ocular lens Real image formed by objective lens Light rays strike specimen Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

4 The capacity to distinguish or separate two adjacent objects and depends on –The wavelength of light that forms the image along with characteristics of the objectives Resolution (a)(b) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

5 Quantifying Resolution Shorter wavelength and larger numerical aperture will provide better resolution Oil immersion objectives resolution is 0.2 μm Magnification between 40X and 2000X Resolving Power (RP) Wavelength of light in nm 2 X Numerical aperture of objective lens =

6 The Purpose of Oil Air Oil Objective lens Slide Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Magnification & Resolution 0.2 µm 1.0 µm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Not resolvable Resolvable

PROCESS BOX M-1 Name at least two differences between magnification and resolution. MUST BE AT LEAST 2 LINES

Leave space for these! You’ll need 7, but just do one a time Microscope typeImage sketch/descriptionUsed for? Microscope Variations

10 Variations on the Optical Microscope Bright-field – most widely used; specimen is darker than surrounding field; used for live and preserved stained specimens

11 Variations on the Optical Microscope Dark-field – brightly illuminated specimens surrounded by dark field; used for live and unstained specimens

PROCESS BOX M-2 Out of the two, we will mostly be using bright- field as opposed to dark-field. Why do you think this may be? MUST BE AT LEAST 2 LINES

13 Variations on the Optical Microscope Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

14 Fluorescence Microscope Modified microscope with an ultraviolet radiation source and filter. Uses dyes that emit visible light when bombarded with shorter UV rays - fluorescence Useful in diagnosing infections Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © Invitrogen Corporation

15 Scanning Confocal Microscope Uses a laser beam of light to scan the specimen. Integrates images to allow focus on multiple depths or planes.

PROCESS BOX M-3 Although the phase-contrast, fluorescent, and scanning confocal microscope are rather different, what similarities do they share? MUST BE AT LEAST 2 LINES

cilia MAC macro- nucleus pre cv chamber cv fv13 pak mic pak myoneme pi Courtesy Dr. Richard Allen 17 2 Types of Electron Microscopes Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. Viruses (a) Hazelton, PR and Gelderblom, HR Electron microscopy for Rapid diagnosis of Emerging Infectious Agents. Emerging Infectious Diseases 9: mm Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (b)

18 2 Types of Electron Microscopes Scanning electron microscopes (SEM) – provide detailed three- dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

Comparing Microscopes: 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Light Microscope Lamp Electron gun Electron beam Ocular lens Viewing screen Eye Image Transmission Electron Microscope (a)(b) Condenser lens Light rays Specimen Objective lens

20 Electron Microscopy Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds Electron waves are 100,000 times shorter than the waves of visible light Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength Magnification between 5,000X and 1,000,000X

PROCESS BOX M-4 What type of microscope was used to produce the image of the rotavirus to the right? How do you know? MUST BE AT LEAST 2 LINES

Sum it up! What are all the parts of the MS and their functions? – Ms. Lichtenwalner in the back How do you calculate total magnification? – Make up a sample problem! How do you calculate the field of view? – Make up a sample problem! What is the difference between resolution and magnification? How do you use a microscope? Be ready to use one! – Ms. Lichtenwalner in the back How should a microscope look when put away? How does a microscopic image of a specimen compare to the actual orientation of a specimen? What are similarities between light and electron microscopes? Differences? What are the two types of electron microscopes? What types of images do they produce? What are the 5 different “non-electron” microscopes? – What types of images do they produce? – When are they used – BE ABLE TO RECOGNIZE IMAGES!!!