1e can Cells, study uted nee cal sts. J-ese light and n ina-1ose his in 1e gh-ere-·too o-'er ,peci-cular enses 1pe of .e of tru-.re all le. FIGURE 2.1a LAB TOPIC 2 Microsco12_es and Cells ~5 b \{10(;\~ \l\{lilv'tj -OiJ'{r{\ a--~ 0 \OWV\f $v\) ,~ ._ \\v)V't\-~OV\Y-l'-f r~\A. ~~ \N½!'f( J t,Q)\>-V'S{ ~ (AA~ y \V"\-t' fuo-,t,S -The compound binocular light microscope. Locate the parts of your microscope described in Exercise 2.1 and label this photograph. Indicate in the margin of your lab manual any features unique to your microscope. FIGURE 2.1b Enlarged photo of compound light microscope as viewed from under the stage. This microscope is ~l\~~'(uq~ipped with phase-contrast optics. Locate the condenser, condenser adjustment knob, phase-contrast revolving turret, and iris diaphragm on your microscope (if present) and label them on the diagram.)Uf ;epiece time you letter ~ ,er n ,yslowly :he ion, the tr lab .cale he . S il the he both ---------- -------------~LAB TOPIC 2 Microscopes and Cells 39 k. I. For binocular microscopes, cover your left eye and use the fine adjust-ment knob to focus the fixed (right) ocular until the letter e is in maxi-mum focus. Now cover the right eye and, using the diopter ring on the left ocular, bring the Image into focus. The letter e should now be in focus for both of your eyes. Each new lab day, as you begin to study your first slide, repeat this procedure. You can increase or decrease the contrast by adjusting the iris dia-phragm opening. Note that the maximum amount of light provides little contrast. Adjust the aperture until the image is sharp. m. Move the slide slowly to the right. In what direction does the image in the ocular move? \t\ \ n. Is the image in the ocular inverted relative to the specimen on the stage? o. Center the specimen in the field of view; then rotate the 40X objective into position while watching from the side. If it appears that the objective will hit the slide, stop and ask for assistance. Most of the microscopes have parfocal lenses, which means that little refocusing is required when moving from one lens to another. If your scope is not parfocal, ask your instructor for assistance . p. After the 40X objective is in place, focus using the fine adjustment knob.40 LAB TOPIC 2 Microscopes and Cells [\ . tion if the ocular were 20 x and \he t I inagni c,1 b. Whal would be the to a ' . )? This magnification approach1cs 0 , 111mmers1on • objective were \0 x ,o . ·is in a specimen using a light micro-. • g fine det,11 ' the upper limit tor s_1ee1:'\ or\ '-.I scope. V V "-d f ieW Once you determine the size 4. Measure the diameter of the fiel O v f · ,lar and ob·1ective lenses you b' ation o oct • of the field of view for any com tn . 11. that field. can determine the size of any structure wit m . ·.tl 11 and remove the letter slide. a. Rotate the 4 x objective into poSI O ' d focus on its edge. b. Place a clear ruler on the stage, an . the ruler is 1 mm. What is the c. The distance between two \mes on diameter (mm) of the field of view? '\~ d. Convert this measurement to micrometers (µm), a more commonly used unit of measurement in microscopy (1 mm = 1,000 µm). e. Measure the diameters of the field of view for the lOX and 40X objectives, and enter all three in the spaces below to be used for future reference. 4X = ½~D~\IJ~ox = 1 ~a~ µ.wt1ox = L\ QO AA.~V\ f. What is the relationship between the size of the field of view and magnification? .~\\-\)( \\.t\c}.~ ~( ¼oitlf ~y'\.k~ s. Determine spatial relationships. The depth of field is the thickness of the specimen that may be seen in focus at one time. Because the depth of focus is very short in the compound microscope, focus up and down to clearly view all planes of a specimen. a. Rotate the 4X objective into position and remove the ruler. Take a slide of crossed threads, wipe it with a Kimwipe"', and place the slide on the stage. Center the slide so that the region where the two threads cross is in the center of the stage opening. b. Focus o n the region where the threads cross. Are both threads in focus at the same time? c. Rotate the lOX objective into position and focus on the cross. Are both threads in focus at the same ti~ Does the 4X or the lOX objective have a shorter depth of field? \t 'f.. N'-~ ~\\O\f ~-u ~~~ -hf\-~ d. Focus up· both lh l! acljustn11 under or e. Rotate l using ti have a • 6. At the enc store your a. Ralat! b. Remo c. Retur phas, d. Set ti e. Unp f. Repl g. Rett sho These steps EXERC The St Materii stereosco1 dissect in livingf.h lntrod The ster 7X to 31 Thebi1· scope i' p)The so obi< direct< viewlr object objeclLABTOPIC2 Microscopesand Cells 41 d. Focus upward (1110\'e the stage up) with the coarse ndjustmcnt until both threads are just out of focus. Slowly focus down using the fine adjustment. 'Which thread come~ into focus first? Is this thread lying undec °' ove, the ~1At~ceo~ { { ) e. Rotate the 40 x objective into position and slowly focus up nnd down, using the fine adjustment only. Does the lOX or the 40X objective haveashoctec depth offleld?\\ \)} ~ ~\j\O\\\( (M;~ 6. At the end of your microscope session, use the following procedures to store your microscope. a. Rotate the 4x objective into position. b. Remove the slide from the stage. c. Return the phase-contrast condenser to the O setting if you have used phase-contrast. d. Set the light intensity to its lowest setting and turn off the power. e. Unplug the cord and wrap it around the base of the microscope. f. Replace the dust cover. g. Return the microscope to the cabinet using two hands; one hand should hold the arm, and the other should support the base. These steps should be followed every time you store your microscope. EXERCISE 2.3 The Stereoscopic Microscope Materials stereoscopic microscope dissecting needles Jiving Elodea Introduction microscope slides droppers of water coverslips The stereoscopic (dissecting) microscope has relatively low magnification, ?x to 30X, and is used for viewing and manipulating relatively large objects. The binocular feature creates the stereoscopic effect. The stereoscopic micro-scope is similar to the compound microscope except in the following ways: (1) The depth of field is much greater than with the compound microscope, so objects are seen in three dimensions, and (2) the light source can be directed down onto as well as up through an object, which permits the viewing of objects too