MicroscopyMicroscopy

UNITS OF UNITS OF MEASUREMENTMEASUREMENT

1mm = 1000µm1mm = 1000µm 1µm = 101µm = 10-3-3mm (convert mm to µm by multiplying by 1000 mm (convert mm to µm by multiplying by 1000

= 3 zeros)= 3 zeros) Bacteria are about 1µm or smallerBacteria are about 1µm or smaller

1nm = 101nm = 10-6-6mm (convert nm to mm by dividing by 1000000 mm (convert nm to mm by dividing by 1000000 = six zeros)= six zeros) Viruses are about 1nmViruses are about 1nm

1000nm = 1µm1000nm = 1µm 1000 viruses can fit into one bacterium1000 viruses can fit into one bacterium

0.001µm = 1 nm0.001µm = 1 nm

Bacteria are so small, they are measured in µm.Bacteria are so small, they are measured in µm. Viruses are even smaller, so they are measured in nm.Viruses are even smaller, so they are measured in nm.

VOCABULARYVOCABULARY Immersion oilImmersion oil: keeps light from bending and : keeps light from bending and

allows lens to be refracted.allows lens to be refracted. ResolutionResolution: ability of two lenses to distinguish : ability of two lenses to distinguish

two points.two points. ParfocalParfocal: focused in all lenses.: focused in all lenses. Depth of fieldDepth of field: how much of the background is : how much of the background is

in focus at the same time that the foreground is in focus at the same time that the foreground is in focus.in focus.

Refractive IndexRefractive Index: a measure of the light-: a measure of the light-bending ability of a mediumbending ability of a medium

Numerical apertureNumerical aperture: numerical aperture : numerical aperture increases as depth of field decreases.increases as depth of field decreases.

Resolution powerResolution power: limits the useful : limits the useful magnification of the microscope resolving power.magnification of the microscope resolving power.

RESOLUTIONRESOLUTION

The ability of the lenses to The ability of the lenses to distinguish two points. distinguish two points.

RESOLVING POWERRESOLVING POWER

The distance between two closely The distance between two closely adjacent objects where the objects adjacent objects where the objects still appear separate and distinct. still appear separate and distinct. The shorter the distance (the smaller The shorter the distance (the smaller the number), the better the resolving the number), the better the resolving power (the sharper the image). power (the sharper the image).

RESOLVING POWERRESOLVING POWER

To calculate the resolving power (to To calculate the resolving power (to see how close two objects can be so see how close two objects can be so you can still see them): you can still see them):

D = distance (smaller number is better)D = distance (smaller number is better) 0.61 = a constant number, does not change0.61 = a constant number, does not change NAobj = numerical aperture of the objective NAobj = numerical aperture of the objective

(larger number causes D to decrease, which (larger number causes D to decrease, which is better)is better)

λλ = (lambda): the wavelength (nm) of the = (lambda): the wavelength (nm) of the light going through the microscope. Convert light going through the microscope. Convert nm to mm by dividing by 1000000 (six zeros)nm to mm by dividing by 1000000 (six zeros)

D = 0.61 λ / NAobjD = 0.61 λ / NAobj

Resolving PowerResolving Power

2 objects 2 objects resolvedresolved

2 objects not 2 objects not resolvedresolved

Same 2 objects resolved Same 2 objects resolved with with better optical better optical instrumentinstrument

                                                                                                           

Resolving power limits Resolving power limits for several optical for several optical

instruments:instruments:Optical InstrumentOptical Instrument Resolving Resolving

PowerPower

Human eyeHuman eye0.2 0.2 millimeters millimeters (mm) (mm)

Light microscopeLight microscope0.25 0.25 micrometers micrometers (µm)(µm)

Scanning electron Scanning electron microscope (SEM)microscope (SEM)

5-10 5-10 nanometers nanometers (nm)(nm)

Transmission electron Transmission electron microscope (TEM)microscope (TEM)

0.5 0.5 nanometers nanometers (nm)(nm)

PRISMPRISM

This is a triangular device that This is a triangular device that breaks up light into its various breaks up light into its various wavelengths so you can see all the wavelengths so you can see all the colors of the rainbow (the visible colors of the rainbow (the visible spectrum). spectrum).

The visible spectrum of colors starts The visible spectrum of colors starts with violet (350nm), and goes on to with violet (350nm), and goes on to indigo, blue, green (550nm), yellow, indigo, blue, green (550nm), yellow, orange, red (700nm). orange, red (700nm).

Sample ProblemSample Problem

When we want to observe the color When we want to observe the color green (550nm) under an oil-immersion green (550nm) under an oil-immersion objective lens of a microscope, where objective lens of a microscope, where the NAobj is 1.25, the resolution is as the NAobj is 1.25, the resolution is as follows:follows:

D = (0.61)(0.000550) / 1.25D = (0.61)(0.000550) / 1.25 D = 0.0002684 mm D = 0.0002684 mm convert this convert this

to microns (µm) by multiplying by 1000to microns (µm) by multiplying by 1000 D = 0.27 µmD = 0.27 µm

Sample ProblemSample Problem

The NAobj for the high-dry (400x) The NAobj for the high-dry (400x) lens is 0.65lens is 0.65

What is the resolving power (D) of What is the resolving power (D) of this objective when viewing a this objective when viewing a wavelength of 550nm?wavelength of 550nm?

D = 0.61 λ / NAobjD = 0.61 λ / NAobj D = (0.61)(0.000550) / 0.65D = (0.61)(0.000550) / 0.65 D = 5.1615mmD = 5.1615mm D = 0.52µmD = 0.52µm

ConclusionsConclusions

Therefore, we can see an organism such Therefore, we can see an organism such as as E. coliE. coli, which is 2µm long and 1µm , which is 2µm long and 1µm wide because it is larger than the wide because it is larger than the resolving power. However, we could not resolving power. However, we could not see see Haemophilus influenzaHaemophilus influenza, which is , which is 0.2µm long because it is smaller than our 0.2µm long because it is smaller than our resolving power.resolving power.

Therefore, the resolving power limits the Therefore, the resolving power limits the useful magnification of the microscope.useful magnification of the microscope.

Resolution determines the magnification.Resolution determines the magnification.

REFRACTIONREFRACTION Refraction is the Refraction is the

bending of light caused bending of light caused by the surrounding by the surrounding medium. medium.

N = Refraction Index of N = Refraction Index of the medium the medium surrounding the lenssurrounding the lens

Air: N= 1Air: N= 1 Glass: N = 1.5Glass: N = 1.5 Immersion Oil: N = Immersion Oil: N =

1.51 (about the same as 1.51 (about the same as glass)glass)

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.

SIMPLE MICROSCOPESIMPLE MICROSCOPE

COMPOUND MICROSCOPE: COMPOUND MICROSCOPE: One EyepieceOne Eyepiece

COMPOUND MICROSCOPE:COMPOUND MICROSCOPE:Two EyepiecesTwo Eyepieces

Types of Compound Types of Compound MicroscopesMicroscopes

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

Dissecting MicroscopeDissecting Microscope

BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:

No StainNo Stain

BRIGHTFIELD BRIGHTFIELD ILLUMINATION:ILLUMINATION:

With StainWith Stain

DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION

DARKFIELD DARKFIELD ILLUMINATIONILLUMINATION

Brightfield vs DarkfieldBrightfield vs Darkfield

PHASE CONTRAST PHASE CONTRAST MICROSCOPYMICROSCOPY

DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST

DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST

DIFFERENTIAL DIFFERENTIAL INTERFERENCE CONTRASTINTERFERENCE CONTRAST

Uses UV light.Uses UV light. Fluorescent Fluorescent

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

Cells may be Cells may be stained with stained with fluorescent fluorescent dyes dyes (fluorochromes(fluorochromes).).

Fluorescence Fluorescence MicroscopyMicroscopy

Figure 3.6b

FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY

FLUORESCENCE FLUORESCENCE MICROSCOPYMICROSCOPY

Transmission Electron Transmission Electron MicroscopeMicroscope

Transmission Electron Transmission Electron MicroscopeMicroscope

Transmission Electron Transmission Electron Microscope:Microscope:

Inside of a Plant CellInside of a Plant Cell

Scanning Electron Scanning Electron MicroscopeMicroscope

Scanning Electron Scanning Electron Microscope:Microscope:

FleaFlea

Scanning Electron Scanning Electron Microscope:Microscope:

PollenPollen

Scanning Probe Scanning Probe Microscope:Microscope:

Red Blood CellsRed Blood Cells

Scanning Probe Scanning Probe Microscope:Microscope:

ChromosomesChromosomes

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

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