Microscopy

Magnification

• the human eye is unable to see objects smaller than 0.1 mm (100 mm)
• the compound light microscope bends light waves (using a dual lens system, consisting of an ocular and objective lens) to enlarge the image of a small object so that we can observe its structure

• ability to distinguish two objects as distinct or separate
• ability to see the fine detail of a viewed object
• the compound light microscope increases resolution (microscope can resolve two objects as close together as 0.2 mm)

• dyes or stains are used (e.g., methylene blue) to enhance our ability to tell things apart by their relative lightness or darkness (cheek cell wet mount)

• your microscope does not need extensive refocusing when switching between objective lenses - specimen remains in focus at different magnifications

• ocular lens - usually 10X
• body tube
• arm - to carry scope
• revolving nosepiece - holds objective lenses
• objective lenses - 10X, 43/45X, 90X (oil immersion)
• TOTAL MAGNIFICATION = OCULAR X OBJECTIVE
• stage with clips to hold slide
• substage condensor - concentrates light on specimen - helps increase resolution
• iris diaphragm - controls amount of light hitting specimen - opening up the diaphragm can help increase resolution, but closing down the diaphragm can help increase contrast - an adjustment may have to be made for each slide that�s looked at
• coarse and fine adjustment - to change working distance, focus
• substage lamp - in drawers, or use tensor lamp with mirror; may come with a blue filter to enhance resolution

• only clean ocular, objective lenses with lens paper, nothing else
• start with lowest objective lens first; move lens toward slide with coarse adjustment, focus on specimen with fine adjustment; view specimen with lowest power first, then move to 43/45 objective - notice what happens to working distance when you move from 10X to 43X objective
• be very careful when moving coarse adjustment - can slam lens onto slide, break slide, and worse, ruin objective lens; ONLY USE THE FINE ADJUSTMENT WHEN WORKING WITH THE HIGH DRY (43-45x) AND OIL IMMERSION OBJECTIVES!
• can adjust light level with iris diaphragm
• try to look through ocular with one eye, leave the other eye open
• DO NOT USE OIL IMMERSION OBJECTIVE WITHOUT IMMERSION OIL
• Immersion oil has the same refractive index as glass, so that no light is lost to scattering between the light source and the slide - need all the light you can get at high magnifications, to improve resolution
• PROPER USE OF OIL IMMERSION OBJECTIVE: swing nosepiece so that no objectives are pointed at slide; place 1 drop of immersion oil on center of slide; swing immersion objective into place, sliding it through the oil on slide; when finished, CLEAN SLIDE AND OBJECTIVE LENS THOROUGHLY WITH LENS PAPER - DO NOT LEAVE OIL ON SLIDES OR LENSES!

• make a wet mount of 2 crossed threads plus a human hair; view slide at 10X and 43 or 45X - what happens to the depth of field as magnification increases? Is it harder or easier to tell the order of the threads/hair?

• slide of the letter "e" - what happens to the "e" when viewed under the scope? Practice using stage controls with this slide

•  cheek epithelial cells - look for nucleus, cell membrane - any other structures visible?

1. measure the diameter of the field of view at 10X with short rulers clipped onto stage diameter ~ 1600 mm (1.6 mm)

What is the diameter of field of view for 45X objective?

    d.    Calculate field of view diameter for 90X objective (178 mm)
    e.    knowing the diameter of field of view at a particular magnification, we can estimate the
           sizes of cells by estimating how much of the distance along an imaginary "ruler" in the
           field of view a cell takes up
 
example 1: If the length of a cell = ½ the distance across the field of view at 45X, then
the diameter of the cell = 178 mm (1/2 (356 mm))

example 2: If we estimate that it would take 10 cells to fill an imaginary line across the
field of view at 90X, then the diameter of one cell = 18 mm (1/10 (178 mm))

ASSIGNMENT: estimate the diameter of the following cell types:
    1.    white blood cell (fig. 39-3) - use blood smear slide
    2.    simple squamous epithelium cell (fig. 7-1) - length
 

    9.    CELL MITOSIS - use slide of whitefish blastula; be able to recognize each phase,
            know events in each phase, e.g., when do chromosomes becomevisible, when does
            DNA replicate, etc.

Mitosis - process by which somatic cells reproduce themselves, creating more diploid cells with pairs of chromosomes (23 in humans, for a total of 46 chromosomes)

Interphase - 90 % of cell cycle spent here

• phase of cell life cycle where cells are not dividing - doing other cellular functions
• chromosomes exist as chromatin, a diffuse granular material in nucleus - not visible as separate chromosomes
• cells prepare for division in this phase - increase cell size, replicate DNA and other cell components, including centrioles

• chromatin condenses to form chromosomes; each chromosome contains 2 identical strands called chromatids, joined together at the centromere
• centrioles move toward opposite sides of cell, project spindle fibers toward the middle of the cell, the equator
• nuclear envelope and nucleoli disappear, chromosomes become visible

• chromosomes attach by their centromeres to the equator of the spindle fibers

• centromeres separate, chromatids now called chromosomes again
• chromosomes are pulled by spindle fibers toward the opposite ends of cell; each ½ of cell now has 2 identical sets of chromosomes

• cytokinesis begins, producing a cleavage furrow
• cytokinesis continues until 2 separate cells are formed
• new nuclear membrane forms around chromosomes in each cell
• chromosomes convert to chromatin

Magnification - compound light microscope bends light waves using a dual lens system (ocular + objective lenses) to enlarge the image of a small object

Resolution - the ability to distinguish 2 objects as separate entities; the compound light microscope enhances resolution so we can see the fine detail of a viewed object (resolution of light microscope = 0.2 micrometers)

Contrast - use of dyes or stains to enhance our ability to tell things apart based on their relative lightness or darkness (cheek epithelial cells)

Total Magnification = Ocular X Objective

4. Letter "e" wet mount - what happens to the orientation of an object viewed under the microscope?

5. What structures do you think you would be able to see in the cheek epithelial cells without the use of dyes and stains?