Bmsc 210 Mid1 p2

- Specimens are visualized because of differences in contrast (density) between specimen and surroundings

• Two sets of lenses form the image
• -Objective lens and ocular lens
• – Total magnification = objective magnification x ocular magnification– Maximum magnification is ~2,000x

the ability to distinguish two adjacent objects as separate and distinct

• -the smallest distance between two distinguishable objects
• -proportional to wavelength of light used and inversly proportional to numerical aperture of lens
• – Limit of resolution for light microscope is about 0.2 um
• -blue light has short wavelenght (increases resolution) red has long (decreases resolution)
• -high NA increases resolution

• • Differential stains separate bacteria into groups
• • The Gram stain is widely used in microbiology

purple

red

• 
• – Invented in 1936 by Frits Zernike
• – Phase ring amplifies differences in the refractive index of cell and surroundings
• – Improves the contrast of a sample without the useof a stain
• – Allows for the visualization of live samples– Resulting image is dark cells on a light background

• 
• – Light reaches the specimen from the sides
• – Light reaching the lens has been scattered byspecimen
• – Image appears light on a dark background
• -Excellent for observing motility

• 
• – Used to visualize specimens that fluoresce
• • Emit light of one color when illuminated with another color of light
• – Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI
• – Widely used in microbial ecology for enumerating bacteria in natural samples

• 
• – Uses a polarizer to create two distinct beamsof polarized light
• – Gives structures such as endospores,vacuoles, and granules a three
• -dimensionalappearance
• – Structures not visible using bright
• -fieldmicroscopy are sometimes visible using DIC

• 
• – A tiny stylus is placed close to a specimen
• – The stylus measures weak repulsive forcesbetween it and the specimen
• – A computer generates an image based on thedata received from the stylus

• 
• – Uses a computerized microscope coupled with a laser source to generate a three
• -dimensional image
• – Computer can focus the laser on single layers of the specimen
• – Different layers can then be compiled for a three dimensional image
• – Resolution is 0.1 um for CSLM

electrons

•  
• – Electromagnets function as lenses
• – System operates in a vacuum
• – High magnification and resolution (0.2 nm)– Enables visualization of structures at the molecular level
• – Specimen must be very thin (20–60 nm) and be stained

• 
• – Specimen is coated with a thin film of heavy metal(e.g., gold)
• – An electron beam scans the object
• – Scattered electrons are collected by a detectorand an image is produced
• – Even very large specimens can be observed
• – Magnification range of 15x–100,000x