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Light Microscopes
Bright Field Microscope
Dark Field Microscope
Phase Contrast Microscope
Fluorescent Microscope
Confocal Laser Scanning Microscope
Electron Microscopes
Scanning Electron Microscope
Transmission Electron Microscope
Light vs Electron Microscope
Light Microscope
Radiation Source: Light
Specimen: Can be living or non-living
Maximum Magnification: 1,000-2,000 X
Maximum Resolution: 0.2 um or 200 nm
Electron Microscope
Radiation: Electrons
Specimen: Must be non-living
Maximum Magnification: 500,000 X
Maximum Resolution 10 - 0.5 nm and even 50 pm
Examples:
Microscopic animals: Tardigrade ~500 um
Eukaryotic cells: Amoeba ~10 to 100 um
Bacteria cells: Escherichia Coli ~0.2 um to 10 um
Viruses: ~20-200 nm or 0.02-0.2 um
Brightfield Microscope
Standard scope used in teaching labs
Light source comes from below the specimen
Limitations:
Low contrast with biological samples
Low apparent optical resolution
Naturally, colorless samples are not easily visualized
Enhancements of Bright Field Microscope:
Adjust light source to specimen
Oil immersion
Staining
Staining will kill biological samples
Images with Bright Field Microscope
Appearance of dark organism on light background
Inverted Light Microscope
Light source comes from above the sample and objective lenses are below the sample
Allows for the viewing of larger samples sizes than a regular light microscope
Range of magnification is lower than a traditional light microscope (40x to 400x) versus (40x to 1000x)
Dark Field Microscopy
Light source from above; good for seeing interior organelles
Used to enhance contrast in unstained specimens
Typically better resolution than light field
Limitations:
Low levels of light available so the specimen must be highly illuminated which can cause damage
Images with Dark Field Microscope
Specimen will be light with a dark background
Phase Contrast Microscopy
Provides a 3-D image
Allows for the detailed visualization of in vivo cellular processes, such as, cell division
Result is specimen image of various shades on light background
Images more detailed than bright- and dark-field microscopy images
Images with Phase-Contrast Microscope:
3-D image with a light background
Fluorescent Microscopy
Allows for the visualization of fluorescent proteins/dyes
Dyes often bind to specific targets, allowing for the visualization of cellular structures
DAPI, a blue fluorescent-dye, stains DNA (allows for visualization of nucleus) in eukaryotic cells
Fluorescent dyes/proteins are illuminated with UV radiation
Limitations
UV radiation causes damage to cells
Phototoxicity
Photobleaching
Images with Fluorescent Microscope
Image shown will be the fluorescent protein/dye of the appropriate wavelength set
Several images at different wavelengths are often taken and combined to make a complete image
Confocal Laser-Scanning Microscopy
Allows for 2D and 3D imaging samples
Laser scans through fluorescent samples at different focal planes, providing a series of cross-sections which are stacked to generate detailed images
Can image live or dead cells
Limitations:
Very expensive!
Not user friendly
Can’t scan thick samples
Photobleaching
Images taken with Confocal Laser-Scanning Microscopy
Images will be a series of cross-sections which are combined on a computer to generate a 3-D image
Scanning Electron Microscopy (SEM)
Generates 3D, high resolution images
Uses electrons rather than light to form image
Electrons scatter on the surface of the specimen resulting in the release of signals which are detected by the microscope
The signals detected provide information on the topography of the sample and are used to form the image
Resolution is about 0.5 nm
Limitations:
Can’t view live specimens
Very expensive
samples must be fixed and dehydrated
Only scans surface of specimen
Images from SEM
Generates high resolution 3D images that are black & white
Images can be colored using the microscope software
Transmission Electron Microscopy (TEM)
Generates a 2D image
Uses electrons that pass through the sample (transmit) rather than electrons that refract from the surface of the specimen (SEM)
Provides information about the interior of the specimen
TEM = interior structure
SEM = surface structure
TEM also has much higher resolution, with recent TEMs being able to visualize specimens as small as 50 pm!
Limitations:
Only able to generate 2D image
Expensive!
Sample preparation is quite complex, since the electrons must pass through the sample the sample must be very thin (150nm - 30nm)
Images taken with TEM:
Generate High Resolution 2D images
Images will be black and white but can be colored
Light Microscope Parts
Ocular Lens
Remagnifies the image formed by the objective lens
Body
Transmits the image from the objective lens to the ocular lens using prisms
Arm
Objective Lenses
Primary lenses that magnify the specimen
Stage
Holds the microscope slide in position
Condenser
Focuses light through specimen
Diaphragm
Controls the amount of light entering the condenser
Illuminator
Light Source
Coarse Focusing Knob
Moves the stage up and down to focus the image
Fine focusing knob
Base
Magnification vs. Resolution
Magnification:
Is how much bigger a sample appears to be under the microscope than it is in real life
Resolution:
Is the ability to distinguish between two points on an image - the amount of detail
E.g. if two objects are less than 200 nm apart they are seen as one object
Total Magnification = Objective magnification x Eyepiece magnification
Increasing the magnification does not increase the resolution of the image
Calculating Magnification
Objective Lens X Ocular Lens = Total Magnification
4X (Scanning) x 10X = 40X
10X (low power) x 10X = 100X
40X (Hi dry power) x 10X = 400X
100X (Oil immersion) x 10X = 1000X
Wet Mounts
Purpose:
To observe living microbes
To test motility of an organism
Non-motile (do not move)
Either will appear sessile (no movement on slide), Brownian motion (shaking/vibrating), or may be “moving” due to water movement on the slide
Motile (move)
Organism will move freely on the slide and will exhibit directional movement
To observe the natural size and color of an organism
To observe biotic processes such as cell division