Lab 2 - Introduction to Light Microscopy - Wet Mounts

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15 Terms

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Light Microscopes

  • Bright Field Microscope

  • Dark Field Microscope

  • Phase Contrast Microscope

  • Fluorescent Microscope

  • Confocal Laser Scanning Microscope

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Electron Microscopes

  • Scanning Electron Microscope

  • Transmission Electron Microscope

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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