MicroscopyMicroscopy

1

UNITS OF UNITS OF MEASUREMENTMEASUREMENT

1 m = 1000 mm (millimeters)1 m = 1000 mm (millimeters) 1000 mm = 1 µm (microns)1000 mm = 1 µm (microns)

Bacteria are about 1µm or smallerBacteria are about 1µm or smaller 1000 µm = 1nm (nanometers)1000 µm = 1nm (nanometers)

Viruses are about 1nmViruses are about 1nm 1000 viruses can fit into one bacterium1000 viruses can fit into one bacterium

Protozoa are fairly large single-celled animals. You can see them with the naked eye.

Bacteria are so small, they are measured in µm. They Bacteria are so small, they are measured in µm. They are the smallest things you can see under a are the smallest things you can see under a microscope with the oil immersion lens.microscope with the oil immersion lens.

Viruses are even smaller, so they are measured in nm.Viruses are even smaller, so they are measured in nm.

2

3Figure 4.3

Limits of ResolutionLimits of Resolution

3

VOCABULARY Immersion oil: keeps light from bending and allows lens to be

refracted. Resolution: ability of two lenses to distinguish two points. Parfocal: focused in all lenses. Depth of field: how much of the background is in focus at the

same time that the foreground is in focus.   RESOLUTION: the ability of the lenses to distinguish two

points. A microscope with a resolving power of 0.4 nm can distinguish between two points greater than or equal to 0.4nm. When you go to the eye doctor, you look at the chart (Snellen chart) and read it from 20 feet away. If you can read what a normal sighted person can read from 20 feet away, it is called 20/20 vision. If you can’t read it well, your eyesight has less resolution than normal.

4

TYPES OF MICROSCOPESTYPES OF MICROSCOPES

SIMPLE MICROSCOPESIMPLE MICROSCOPE: Has only : Has only one lens, like an ocular (eyepiece)one lens, like an ocular (eyepiece)

COMPOUND MICROSCOPECOMPOUND MICROSCOPE: More : More than one lens, like an ocular and an than one lens, like an ocular and an objective. An example is the objective. An example is the Brightfield microscope.Brightfield microscope. There are two main types of compound There are two main types of compound

microscopes: Light Microscopes and microscopes: Light Microscopes and Electron Microscopes.Electron Microscopes.

5

SIMPLE MICROSCOPESIMPLE MICROSCOPE

6

COMPOUND MICROSCOPE: COMPOUND MICROSCOPE: One EyepieceOne Eyepiece

7

COMPOUND MICROSCOPE:COMPOUND MICROSCOPE:Two EyepiecesTwo Eyepieces

8

Types of Compound Types of Compound MicroscopesMicroscopes

DissectingDissecting BrightfieldBrightfield DarkfieldDarkfield Phase-contrastPhase-contrast Differential Interference contrastDifferential Interference contrast FluorescenceFluorescence

9

Dissecting Microscope:Dissecting Microscope:For looking at large For looking at large

objectsobjects

10

BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:

For live organisms with no For live organisms with no stainstain

11

BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:

Can also use for stained Can also use for stained tissuestissues

12

DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION

For live organisms with no For live organisms with no stainstain

13

DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION

For fluorescent organisms For fluorescent organisms

14

Brightfield vs DarkfieldBrightfield vs Darkfield

15

PHASE CONTRAST PHASE CONTRAST MICROSCOPYMICROSCOPY

For seeing organelles in live For seeing organelles in live organismsorganisms

16

DIFFERENTIAL INTERFERENCE DIFFERENTIAL INTERFERENCE CONTRASTCONTRAST

For seeing organelles in live organisms in For seeing organelles in live organisms in three dimensionsthree dimensions

17

DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST

18

DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST

19

Cells are Cells are stained with stained with fluorescent fluorescent dyes called dyes called fluorochromes.fluorochromes.

UV Light is shined on the specimen.

Fluorescent Fluorescent substances substances absorb UV absorb UV light and emit light and emit visible light. visible light.

Fluorescence Fluorescence MicroscopyMicroscopy

Figure 3.6b20

FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY

21

FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY

22

Transmission Electron Transmission Electron MicroscopeMicroscope

Much higher resolution than microscopes.

Can only be used on dead cells

23

Transmission Electron Transmission Electron MicroscopeMicroscope

24

Transmission Electron Transmission Electron Microscope:Microscope:

Inside of a Plant CellInside of a Plant Cell

25

Scanning Electron Scanning Electron MicroscopeMicroscope

Very high resolution like a Very high resolution like a Transmission Electron Microscope Transmission Electron Microscope except makes images in three except makes images in three dimensions.dimensions.

26

Scanning Electron Scanning Electron MicroscopeMicroscope

27

Scanning Electron Scanning Electron Microscope:Microscope:

FleaFlea

28

Scanning Electron Scanning Electron Microscope:Microscope:

PollenPollen

29

Scanning Probe Scanning Probe MicroscopeMicroscope

A scan is passed over the specimen, A scan is passed over the specimen, line by line.line by line.

The surface dimensions are recorded The surface dimensions are recorded and sent to a computer, which shows and sent to a computer, which shows the image in false color.the image in false color.

30

Scanning Probe Scanning Probe Microscope:Microscope:

Red Blood CellsRed Blood Cells

31

Scanning Probe Scanning Probe Microscope:Microscope:

ChromosomesChromosomes

32

COMPARISON OF MICROSCOPES

BRIGHTFIELD Dark objects are visible against a bright background.Light reflected off the specimen does not enter the objective lensNot for looking at live cellsMaximum resolution is 0.2µm and maximum magnification is 2000xStains are used on specimens

DARKFIELD Light objects are visible against dark backgroundUsed for live cells, cilia, flagellaEspecially good for spirochetesUses special condenser with an opaque disc that eliminates all light in the center

PHASE-CONTRAST

No staying requiredAccentuates diffraction of the light that passes through a specimenGood for live cells; good contrastMost sensitive; cilia shows upNot three-dimensional

DIFFERENTIAL INTERFERENCE CONTRAST

Uses two beams of lightShows three dimensionsHas a prism to get different colorsGood for live cells (unstained)Best resolution

33

COMPARISON OF MICROSCOPES

FLUORESCENCE Uses ultraviolet lightStained cells with fluorescent dye; energizes electrons and creates visible lightNo live cellsQuick diagnosis of TB and syphilis

TRANSMISSION ELECTRON

Get flat imagesHave vacuum pumps to allow electrons to float betterStain with heavy metal saltsShows sections of cell, revealing organellesRequires an ultramicrotomeBest resolution of all microscopes

SCANNING ELECTRON

Surface view onlyNeeds a vacuumNo live cellsThree-dimensional view

SCANNING PROBE

Physical probe scans the specimenRaster scan: image is cut up into pixels and transmitted to computerNot limited by diffractionSlower in acquiring images Maximum image size is smaller

34

35