Microscopy: The instrument

Compound Light Microscope

Compound Light Microscopy

(Resolution)

• The ability of lenses to distinguish two points.
• Shorter wave lengths of light provide greater resolution.
• blue filters increase resolution as blue light has the shortest wavelength of visible light.
• a microscope with a resolving power of 0.4nm can distinguish two points if they are at least 0.4nm apart.

Compound Light Microscope

(Refractive Index)

• Is a measure of the ligh-bending ability of a medium.
• The light may bend in air so much that is misses the small high-magnification lens
• immersion oil is used to prevent this

• Dark abjects are visible against a bright background.
• Light reflected off the specimen does not enter the objective lens.

Darkfield Illuminaton

• Light objects are visible against a dark background
• Light reflected off the specimen enters the objective lens.

• Accentuates diffractionof the light that passes through a specimen
• Provides good resolution

Diffential Interference Contrast Microscopy

DIC

• Accentuates diffraction of the light that passes through a specimen; uses two beams of light.
• makes optical illusion.
• makes specimen look three dimmensional.
• used when specimen is colorless and/or dont want to stain it.
• also to do more detailed internal study.

• Uses UV light.
• Flourescent substances absorb UV light and emit visible light.
• Cells may be stained with flourescent dyes(fluorochromes).
• used to track living organisms (highlights)

• Cells are stained w/ floorochrome dyes
• Short wavelength (blue) light is used to excite the dyes.
• The light illuminates each plane in a specimen to produce a three-dimensional image, up to 100um deep.
• reconstruct three-dimensional images.
• are actual images.

Two-Photon Microscopy

• Cells are staines w/ fluorochrome dyes
• 2 photons of long-wavelength (red) light are used to excite the dyes. (red light is less damaging compare to blue light)
• Used to study organisms attached to a surface. up to 1mm deep.
• little less resolution

Scanning Acoustic Microscopy

SAM

• Measures sound waves that are reflected back from an object.
• Used to study cells attached to surfaced
• resolution 1um
• good to look for biofilms
• to visualize molecules (DNA)

• Uses diamonds
• provides 3-d images
• good resolution

Electron Microscopy

• Uses electrons instead of light
• The shorter wavelength of electrons the greater resolution.
• nothing is alive 
• it uses magnets to focus electron bean 
• there are 2 types

Transmission Electron Microscopy

TEM

• Ultrathin sections of specimens
• electron passes thru specimen, then an electromagnetic lens, to a screen or film
• specimens may be stained w/ a heavy-metal salt
• used to observe tiny stuff inside cells or molecules
• resolution 2.5nm

Scanning Electron Microscopy

SEM

• electron gun produces a beam of electrons that scans the surface of a whole specimen
• secondary electrons emitted from the specimen produce image
• it sprays gold on specimen (gold is dense so it produces good images)
• resolutuon 20nm or 1,000-10,000x

Sanned-Probe Microscopy

STM

• uses a metal probe to scan specimen
• resolution 1/100 of an atom

Atomic Force Microscopy

AFM

• uses a metal and diamond probe inserted into the specimen
• produces 3D images

• Acid dye and basic dye
• simple staining
• gram stain,gram posite, gram negative
• gram stain vs acid fast stain
• capsule stain, endorspore stain, flagella stain

Staining:

• Methylene blue
• Crystal Violet
• Saffranin Red

• used to distinguish between bacteria

* gram stain and acid-fast stain

• Differentiate regions 

• Require multiple stains 

Whats the color of Gram-Positive cells when stained with Crystal violet,mordant (iodine),decolorizing agent (alcohol-acetone), and counterstain (Safranin)?

Whats the color of Gram-Negative cells when stained with Crystal violet,mordant (iodine),decolorizing agent (alcohol-acetone), and counterstain (Safranin)?

• Crystal violet,mordant (iodine) result in purple.
• decolorizing agent (alcohol-acetone) result in Colorless
• counterstain (Safranin) result in Red

One that differentiates bacteria into distinctive groups

binds strongly only to bacteria that have waxy material in the cell wall is not decolorized by acid-alcohol Ex:

• Mycobacterium
• Nocardia

used to distinguish parts of cells in microrganisms.

Capsule Stain

Endospore Stain

Flagella Stain

• used to demonstrate the presence of capsules
• capsules don't accept most satins
• appear unstained
• stand out against contrasting background

• used to detect presence of endospores in bacteria
• primary stain machita green with heat
• decolorazes cells red
• endospores turn green when machita green with heat is used
• endospores turn red/pink when counterstain safranin is used

• used to demonstrate the presence of flagella
• mordant is used until it becomes visible
• then is stained with carbolfuchism

• Simple (methylene blue,carbolfuschsin,crystal violet and safranin)
• Differential (Gram, Acid-fast)
• Special (Negative, Endospore and Flagella)