Microscopy Flashcards
Robert Hooke
was the first to describe microbes
- first use of microscope - 1664
- Illustrated the fruiting structures of molds
Who was the first to describe bacteria and described them as Wee Animacules?
Antoni van Leeuwenhoek
Ferdinand Cohn
Founded the field of bacteriology and discovered bacterial endospores
Light microscopes
used to look at intact cells under relatively low magnification
the ability to distinguish two adjacent objects as separate and distinct and is is determined by the wavelength of light used
Resolution
Limit of resolution for light microscope is about
0.2 μm
Why are specimens are visualized on bright-field scope?
because of differences in contrast (density) between them and surroundings
Two sets of lenses form the image
objective lens and ocular lens
Equation total magnification is a product of the magnification of the two sets of lenses
Objective magnification X ocular magnification
Maximum magnification is
1500X
An easy way to improve contrast
Staining
What is staining?
Dyes are organic compounds that have different affinities for specific cellular materials
Examples of common stains are methylene blue, safranin, and crystal violet
Recall how to stain
From lab/pcuicture
The Gram stain
differential stain and mostly used in lab.
Gram-positive bacteria appear ? and gram-negative bacteria appear ? after staining
+ purple
- red
Phase-Contrast Microscopy
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 use of a stain
- Allows for the visualization of live samples
- Resulting image is dark cells on a light background
Dark-Field Microscopy
Light reaches the specimen from the sides
- Light reaching the lens has been scattered by specimen
- Image appears light on a dark background
- Excellent for observing motility
Fluorescence Microscopy
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
Differential Interference Contrast (DIC) Microscopy
Uses a polarizer to create two distinct beams of polarized light
- Gives structures such as endospores, vacuoles, and granules a three-dimensional appearance
- Structures not visible using bright-field microscopy are sometimes visible using DIC
Atomic Force Microscopy (AFM)
A tiny stylus is placed close to a specimen The stylus measures weak repulsive forces between it and the specimenA computer generates an image based on the data received from the stylus
Confocal Scanning Laser Microscopy (CSLM)
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 0.1μm for CSLM
Two types of electron microscopes
Transmission Electron Microscopes (TEM)
Scanning Electron Microscopes (SEM)
Transmission Electron Microscopy (TEM)
electrons of the high voltage beam form the image of the specimen
- Electromagnets function as lensesSystem operates in a vacuum
- High magnification and resolution (0.2 nm)
- Enables the visualization of structures at the molecular level
- Specimen must be very thin (20 –60 nm) and be stained (permanganate or heavy metal salts
Scanning Electron Microscopy (SEM)
produces images by detecting low energy secondary electrons which are emitted from the surface of the specimen due to excitation by the primary electron beam
- Specimen is coated with a thin film of heavy metal (e.g., gold)
- An electron beam scans the objectScattered electrons are collected by a detector and an image is produced
- Even very large specimens can be observed
- Magnification range of 15X – 100,000X
