Lab 1 Brightfield Microscopy

Background

  • Brightfield Microscopy: produces image made from light that is transmitted through a specimen; uses a cone of light onto a specimen

  • What the light rays do when in contact with the specimen:

    • Absorbed by specimen

    • Scattered by organelles/storage granules in specimen

    • refracted due to difference in the refractive index between cytoplasm and water

  • More complex the specimen is (due to organelles/subcellular organelles) the easier it is to see the specimen using bright field microscopy

  • Increased scattered light

  • Light absorbed or scattered → do not enter the objective lens → contrast between specimen (darker) and background (lighter)

  • Diaphragm controls the diameter of beam of light that enters condenser

  • Bacteria and archaea too small to see using bright-field → poor contrast

    • Increasing the contrast would either use a phase contrast or are often stained with dyes

      • Dyes kill cells and often shrink affecting their size and shape as well as arrangement

A. Introduction to Microscopy

  1. Important Concepts in Microscopy

  • Objective lenses: series of lens nearest specimen; magnifies speci

  • men and produces a real image

  • Ocular/eyepiece: magnifies real image by 10X

  • Resolving power:

  • Wavelength of visible light range ~400 to 700 nm (550 nm approx average and often used in calculating resolving power) → only for parallel light rays

  • When oblique and parallel rays illuminate specimen

    • Relationship becomes

  • Numerical aperture (NA): measure of lens’ ability to “capture” light coming from specimen and use it to make image

  • Max resolution using visible light as an illuminating source is 0.2 micrometers → more resolution if used EM

  • With immersion oil, the refraction is decreased and more light rays pass directly into objective to be fully illuminated and results in improved resolution and clearer image

  1. Other terms

  • Working Distance: clearance between specimen and objective lens when object is in focus

  • Shorter the focal length of the objective lens, the shorter the working distance

  • Aberrations: lenses have aberrations; they have 2 categories: spherical and chromatic

    • Spherical aberrations: result from the use of curved surfaces for lenses and are evident even in monochromatic light; many types of spherical aberrations including the curvature of field

    • Chromatic aberrations: result from dispersion and only occur with heterochromatic light

      • Manifested often by change of color of image as the focus is adjusted

      • Every wavelength is differentially refracted; the shortest the most and the longest the least

  • Instead of sharp image with white light, the image is surrounded by colored halos and responsible for the observed change in color of the image as focus is adjusted → high quality lenses are made to reduce this effect

B. Use of the Axio Lab. AI Student Microscope

  • Structures and Functions

    • Locate and learn names and functions of components of microscope (Lab Instructor should highlight these)

  • Calculation of Resolving Power

    • Complete table in PowerPoint and include in lab notebook; all calculations

  • Condenser Height Adjustment

    • Should be pre-centered, but should be few mm below the slide if not set at the appropriate height

    • Adjusting the Condenser Height

      • Place slide/specimen with sharpie mark on stage

      • Rheostat should be fully turned up and the aperture diaphragm lever should be used to adjust light intensity

      • Raise condenser to few mm below the glass slide

      • Get specimen into focus (use 10X objective lens)

      • Close field diaphragm when small circle of light shown

      • Adjust condenser height, raise or lower the condenser adjustment knob until circle is octagon

    • Centering the condenser/cone of light

      • Use condenser alignment screws with aid of instructor to make the circle of light centered

      • Open field diaphragm until circle of light fills the field of view , adjust rheostat/aperture diaphragm lever to lessen glare and optimal resolution of specimen

  • Rotate 40X objective in place and repeat

  • Lamp used at full intensity for all observation

  • If glare results when using low or high dry objectives, attempt to control it by adjusting aperture diaphragm not rheostat

  • Observation of Life in a Drop of Pond Water

    1. Place small drop of pond water on clean glass slide

    2. Spread thin layer of petroleum jelly on palm on hand

    3. Carefully draw edge of coverslip through jelly to create narrow ridge

    4. Lower cover slip, jelly side down onto drop of pond water and press into place

    5. Record observations in lab notebook under 100X, 400X, and 1000X (easy or difficult to see specimens)