Visualizing Visualizing Prokaryote CellsProkaryote Cells

Chapter 3 - BlackChapter 3 - Black

LightLight

Key Words - Key Words -

Visible lightVisible light Ultraviolet lightUltraviolet light ReflectionReflection TransmissionTransmission RefractionRefraction AbsorptionAbsorption

Light MicroscopyLight Microscopy

ResolutionResolution

Resolution is the ability to see two Resolution is the ability to see two images as separate and discrete.images as separate and discrete.

The wavelengths of visible light The wavelengths of visible light from 420 to 620 prevent from 420 to 620 prevent resolution of two points closer resolution of two points closer than 220 nmthan 220 nm

By using the light emitted from an By using the light emitted from an electron it is possible to resolve electron it is possible to resolve two points that are .2nm aparttwo points that are .2nm apart

Specialized MicroscopySpecialized Microscopy

Dark fieldDark field Phase ContrastPhase Contrast Differential interferenceDifferential interference FluorescenceFluorescence ConfocalConfocal DigitalDigital

Digital photomicrosopyDigital photomicrosopy

Camera can be Camera can be used to used to photograph photograph imagesimages

Specimen can be Specimen can be viewed on TV viewed on TV screen as well as screen as well as on computer on computer screenscreen

Classroom MicroscopyClassroom Microscopy

Phase Contrast Phase Contrast MicroscopyMicroscopy Accentuate small Accentuate small

differences in the differences in the refractive index of refractive index of the specimenthe specimen

More detail is More detail is apparent in living apparent in living cellscells

Assist in the Assist in the visualization of cell visualization of cell structure in structure in transparent cellstransparent cells

micro.magnet.fsu.micro.magnet.fsu.edu/.../edu/.../dicphasecomparisdicphasecomparison.htmlon.html

Differential Differential interference interference microscopymicroscopy Produces higher Produces higher

resolutionresolution Depends on a Depends on a

gradientgradient It can produce It can produce

almost a three almost a three dimensional dimensional imageimage

Comparison of Phase Contrast Comparison of Phase Contrast and Differential Interference and Differential Interference ContrastContrast

Fluorescent Fluorescent MicroscopyMicroscopy Organisms such as Organisms such as

Mycobacterium Mycobacterium tubercuolosistubercuolosis and and Treponema pallidum Treponema pallidum are are treated with a fluorochrome treated with a fluorochrome dyedye

Ultra violet light is used to Ultra violet light is used to excite the fluorochromeexcite the fluorochrome molecules and produce a molecules and produce a glowing imageglowing image

Used in clinical workUsed in clinical work Also used with antigens and Also used with antigens and

antibodies to identify the antibodies to identify the presence of molecules on presence of molecules on the surface of a cellthe surface of a cell

Fluorescent antibody Fluorescent antibody stainingstaining Used in immunologyUsed in immunology Fluorescent Fluorescent

antibodies are used antibodies are used to detect antigens to detect antigens on the surface of on the surface of cellscells

In the picture to the In the picture to the right are right are Bacillus Bacillus anthracisanthracis cells cells tagged with a tagged with a fluorescent antibodyfluorescent antibody

Confocal MicroscopyConfocal Microscopy

Utilizes beams of Utilizes beams of ultraviolet light to excite ultraviolet light to excite fluorescent dye molecules. fluorescent dye molecules.

The exciting light is The exciting light is focused on the specimen focused on the specimen with a thin optical fiberwith a thin optical fiber

Resulting fluorescence is Resulting fluorescence is focused through a narrow focused through a narrow apertureaperture

The light is detected and The light is detected and analyzed by a computeranalyzed by a computer

Very sharp focusVery sharp focus For thick specimens an For thick specimens an

image is constructed in image is constructed in layerslayers

Confocal imagesConfocal images

http://www.microscopyu.com/http://www.microscopyu.com/galleries/confocal/galleries/confocal/applecedarrustaeciaandpycnia.htapplecedarrustaeciaandpycnia.htmlml

http://www.microscopyu.com/http://www.microscopyu.com/galleries/confocal/galleries/confocal/chlamydomonas.htmlchlamydomonas.html

Electron MicroscopyElectron Microscopy

Transmission electron microscopy Transmission electron microscopy Scanning electron microscopyScanning electron microscopy

Transmission Electron Transmission Electron MicroscopyMicroscopy Electrons are used Electrons are used

as the source of as the source of lightlight

Produced by a high Produced by a high voltage current voltage current running through a running through a tungsten filamenttungsten filament

www.steve.gb.cowww.steve.gb.com/science/m/science/electron_microscelectron_microscopy.htmlopy.html

Transmission Electron Transmission Electron MicroscopeMicroscope The lenses are The lenses are

electromagneticelectromagnetic They act on the They act on the

negatively charged negatively charged electrons to focus electrons to focus them in a them in a concentrated path concentrated path through the through the specimenspecimen

The image is The image is magnified by magnified by additional lenses and additional lenses and visualized on a visualized on a screenscreen

TEM imagesTEM images

Images produced Images produced display high display high resolutionresolution

Staining with heavy Staining with heavy metals that interact metals that interact with the electronswith the electrons

Gradations of black, Gradations of black, gray, and white gray, and white contrast areas of contrast areas of greater density that greater density that absorb the stainabsorb the stain

Scanning Electron MicroscopyScanning Electron Microscopy

Electrons are Electrons are reflected and reflected and collected off of collected off of the surface of a the surface of a cellcell

SEMSEM

Images show Images show surface contours surface contours

Three Three dimensional dimensional imageimage

Freeze FractureFreeze Fracture

Cells are quickly frozen in liquid nitrogen (196C), Cells are quickly frozen in liquid nitrogen (196C), which immobilizes cell components instantly.which immobilizes cell components instantly.2. Block of frozen cells is fractured. This fracture is 2. Block of frozen cells is fractured. This fracture is irregular and occures along lines of weakness like the irregular and occures along lines of weakness like the plasma membrane or surfaces of organelles.plasma membrane or surfaces of organelles.3. Surface ice is removed by a vacuum (freeze 3. Surface ice is removed by a vacuum (freeze etching)etching)4. A thin layer of carbon is evaporated vertically onto 4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica.the surface to produce a carbon replica.5. Surface is shadowed with a platinum vapor.5. Surface is shadowed with a platinum vapor.6. Organic material is digested away by acid, leaving a 6. Organic material is digested away by acid, leaving a replica.replica.7. Carbon-metal replica is put on a grid and examined 7. Carbon-metal replica is put on a grid and examined by a transmission electron microscope.   by a transmission electron microscope.   

Freeze Fracture and Freeze Fracture and EtchingEtching

Freeze FractureFreeze Fracture

ReferencesReferences

http://micro.magnet.fsu.edu/http://micro.magnet.fsu.edu/primerprimer

http://www.mos.org/sln/SEM/http://www.mos.org/sln/SEM/