Microscopy http://www.microscopyu.com/articles/phasecontrast/phasemicroscopy.html http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html 2005, Dr. Jack Ikeda & Dr. Gail Grabner 9

Nikon Labophot (Question 10) Field lens (collects light) Brightness control dial Nikon Labophot (Question 10) Condenser (focuses light on specimen) 2005, Dr. Jack Ikeda & Dr. Gail Grabner 10

Nikon Labophot (Question 10) Stage Slide holder Stage X/Y-axis travel knobs Nikon Labophot (Question 10) Focus knobs 2005, Dr. Jack Ikeda & Dr. Gail Grabner 11

Eyepiece (2nd level of magnification) Nikon Labophot (Question 10) Nosepiece Objectives (1st level of magnification) Compound Light Microscope Microscope Features Illuminator: light source + collector lens Substage condenser: focuses light on specimen Stage: specimen Objective: 1st level of magnification (10X, 40X, 100X) Nosepiece: to move objective Eyepiece: 2nd level of magnification (10X) ALWAYS MOVE MICROSCOPE BY GRASPING THE STAND AND SUPPORTING THE BASE. 2005, Dr. Jack Ikeda & Dr. Gail Grabner 12

Total magnification = objective (e.g., 10X, 40X, 100X) x eyepiece (e.g., 10X) Magnification (Question 11) e.g., 10X x 10X = 100X 40X x 10X = 400X 100X x 10X = 1000X Real image projected by objective Theoretical limit = 1000X > 1000X: Image enlarged with no additional resolution Virtual image projected by eyepiece Objectives 10X Scanning Find the object (Course focus) 40X High-Dry Focus the object (Fine focus) 100X Oil immersion Fine focus 2005, Dr. Jack Ikeda & Dr. Gail Grabner 13

Wave Phase Relationships (Question 12) Light waves have both phase and amplitude features. A difference in phase between light waves relates to interference. A difference in amplitude relates to brightness. Propagation of Light Some of the light passing through the specimen is undeviated (direct) and some is deviated (diffracted). Deviated light is due to the difference in the refractive index (how light is bent) of specimen as compared to surrounding medium. 2005, Dr. Jack Ikeda & Dr. Gail Grabner 14

Resolution (Question 13) Resolution = the sharpness of an image Resolving power (RP) = smallest distance between two points at which they appear clear and distinct from each other Numerical aperture (NA) = amount of light gathered by the objective RP = 2 NA As and NA,, RP and resolution improves Numerical Aperture (Question 14) Higher numerical aperture means more light reaches objective Larger cone of illumination = Higher NA Without oil (Diffracted light lost) With oil (Diffracted light captured) Immersion oil captures diffracted light (refractive index of glass) NA 2005, Dr. Jack Ikeda & Dr. Gail Grabner 15

Resolution (Question 13) Resolution = the sharpness of an image Resolving power (RP) = smallest distance between two points at which they appear clear and distinct from each other Numerical aperture (NA) = amount of light gathered by the objective RP = 2 NA As and NA,, RP and resolution improves Increased Resolution using Immersion Oil Dry Oil 2005, Dr. Jack Ikeda & Dr. Gail Grabner 16

Aspects of Brightfield Microscopy (Questions 15-16) Relies solely on differences in brightness (wave amplitude) Cells don t contrast well with background Staining enhances contrast Bacteria killed in the process Heat fixing and staining can distort shape Major disadvantage: Can t t view living cells Types of Microscopy (Question 15 and 16) Brightfield: Objects dark against light background; relies on differences of brightness; objects must be stained (positive stain, Gram stain) that kills cells and may produce artifacts Darkfield: Objects light against a dark background; objects need not be stained Phase Contrast: Objects dark against light background; no staining necessary 2005, Dr. Jack Ikeda & Dr. Gail Grabner 17

Darkfield Microscopy (Questions 17-18) Light stop in condenser delivers hollow cone of light to specimen None of the direct light enters the objective (dark background) Only light scattered by specimen enters the objective; Objects light against a dark background Select DF on turret condenser Advantage: View cells too difficult to stain; can help with focusing Disadvantage: use only with 10X and 40X objectives Brightfield vs. Darkfield Brightfield Darkfield 2005, Dr. Jack Ikeda & Dr. Gail Grabner 18

Treponema pallidum in Darkfield Phase Contrast Microscopy (Question 19) Two features of construction distinguish phase contrast from brightfield: 1. Annular diaphragm or plate -- delivers hollow cone of light to condenser 2. Phase plate -- Segregates direct from diffracted light These are matched in diameter. 2005, Dr. Jack Ikeda & Dr. Gail Grabner 19

Features of Phase Contrast (0) (DF) Annular diaphragms in condenser Condenser annular diaphragms Select by rotating condenser turret Ph1: 10X objective Ph3: 40X objective Ph4: 100X objective Diameter of annulus must match diameter of phase plate Image Formation in Phase Contrast (Question 20) Undiffracted (direct) and diffracted light emerging from specimen are segregated Diffracted light is slowed _ of a wavelength (90 ) by specimen Direct light is advanced _ of a wavelength (90 ) at the phase plate Therefore, direct and diffracted light are _ of a wavelength (180 ) out of phase 2005, Dr. Jack Ikeda & Dr. Gail Grabner 20

Phase Shifts (Question 20) Direct light Diffracted light Diffracted light is shifted so that it is _ (or 180 ) out of phase with direct light. This results in destructive interference that the eye perceives as a difference in contrast. Interference of Light (Question 20) Constructive Destructive 2005, Dr. Jack Ikeda & Dr. Gail Grabner 21

Benefit of Phase Contrast (Question 21) Human glial brain tissue cells Major Advantage: Ability to view living cells Better determination of shape and ability to assess motility 2005, Dr. Jack Ikeda & Dr. Gail Grabner 22