ZOOL 409, Week 1 Objective 1: Lab Notebook. Objective 2: Meet your microscope Drawing is one of the most powerful study-aids for Basic elements (italics indicates user-adjustable learning visual information, such as the microscopic appearance of tissues. features) Each lab period should receive a notebook entry for each slide that you observe, including: Date, time, slide number and slide label for each slide that is observed. A list of expected observations. From your prior study (text, atlas, lecture), what are you looking for? Sketches of your actual observations. As an aid to sketching, use the the circular field of view in your microscope as a frame. Imagine a circle drawn halfway between the center of this field and the edge. Then imagine the circle as a clock-face, with four quarters and each quarter divided into three parts. Your sketches might usefully include: A map (overview) of the specimen, as a guide for locating (and relocating) specific features. o Anything visible by naked eye should be included in this map. Examples of features which you can identify, with appropriate labels. o Honestly, from your own observation, what features do you actually see? (Real specimens do not always display all typical features.) o Which of those can you confidently identify? Examples of features which you cannot identify (labelled as "unidentified"). A list of features that you expected to find (e.g., based on reference materials) but could not see (and/or could not reliably identify). If you have a question about some feature that you have found, the best way to ask is by way of a sketch that shows its location in the field of view and its relationship to other features. Thus, "What is this, that I have drawn here?" works much better than, "What's the red thing we see in the microscope?" Your instructor may refuse to address your question until you have made such a sketch. A well-kept notebook, with recognizable and accurately labelled drawings, may be submitted for grade consideration. Illuminator (light source) The illuminator may include adjustable field diaphragm. Condenser (light gathering lens) Proper condenser adjustment is important for optimal performance. The condenser includes an adjustable condenser diaphragm. The condenser may include focus adjustment. Stage (platform between condenser and objective) The stage supports the specimen (microscope slide). The stage allows specimen to move in x-y plane. The stage may move up and down for specimen focus. Specimen Note that the specimen mount (slide and coverslip) is part of the optical system. At high magnification, optimal optical adjustment requires that coverslip thickness be appropriate for the objective lens. Objective lens (principal image-forming lens) Functionally, the objective lens is the most critical (and most expensive) element of the microscope. Objectives are interchangeable (usually via a swivelling "nosepiece") to change magnification. Objectives may move up and down for specimen focus. Eyepiece (enlarging lens, paired in binocular m'scopes) If paired, interpupillary distance is adjustable; at least one eyepiece should be independently focussable. For more information on optimal adjustment, see http://www.siumed.edu/~dking2/intro/kohler.htm. Care of microscope (always follow these rules!) Three rules for carrying a microscope: 1. Your full attention is on the job, just as if you were carrying a baby. 2. Use both hands. 3. Always carry the microscope upright (otherwise the eyepieces can fall out). Three rules for cleaning a microscope: 1. Don't! It is too easy to cause damage Never the touch the objective lens surface, unless you have bought and paid for the microscope . Clean eyepieces and other optical surfaces only when absolutely necessary. It is better to ignore dirt than to cause permanent damage to the microscope. 2. Locate the dirt; use logic and experiment to determine where in the optical path the dirt is located. Dirt on a slide moves when you move the slide. Dirt on the condenser will go in and out of focus when you move the condenser up or down. Dirt on an eyepiece will move when you rotate the eyepiece. Dirt on an objective cannot be seen while viewing through the microscope, but manifests itself as a decrease in image quality. 3. If dirt cannot be ignored, use the least-intrusive procedure to make it go away. Some dirt can blown away without touching the surface. Use this procedure freely, but blow gently and be careful not to spit. Dirt on slides may be wiped off at will, using the same care you would use for eyeglasses. Dirt on an eyepiece is often accentuated by having the condenser aperture too small; such dirt may become inconspicuous when the diaphragm is opened to the correct setting. Dirt on the condenser may become inconspicuous if you lower the condenser (i.e., move the dirt out of focus). If dirt simply must be removed -o Begin by simply blowing dust away. o Use clean lens paper and moisture from breath. o Wipe gently in one direction. Do not use a circular motion; do not scrub. o Do not try to clean an objective lens; ask for help. Magnification o For the microscopes used in this course: Object. lens Field of view (diameter) Eyepiece pointer (diameter) Ocular micrometer (numbered units) 4x 10 x 40 x 4.5 mm 1.8 mm 450 µm 50 µm 20 µm 5 µm 260 µm 100 µm 25 µm o You may check these values with the calibration slide available from your instructor. o Use these values to estimate the size of the objects provided. Even with an uncalibrated microscope, sizes of tissue features can be estimated by reference to familiar objects of consistent size, such as red blood cells. (Mammalian RBCs are approx. 6-8 µm in diameter.) Resolution and numerical aperture "Resolution" refers to the "resolving power" of the microscope, i.e., the 'scope's ability to "resolve", or distinguish as separate, two nearby objects. Resolution is at least as important as magnification. An increase in magnification that is not accompanied by an increase in resolution is termed empty magnification, with the implication that nothing is really gained (i.e., you cannot see any additional detail by an empty increase in magnification. Magnification is determined in principle by multiplying the power of the Objective Lens by the power of the Eyepiece. Resolution is determined by optical quality and fundamentally limited by the wavelength of light and by the numerical aperture of the objective/condenser combination. Magnification is determined in practice by calibration with a specimen of known size. For more information on microscopes and microscopy, consult the following websites. o For research purposes, calibration is done with a reference standard, either a "calibration slide" (e.g.., a glass slide engraved with a very finely-calibrated ruler) or with standardized objects (e.g., microspheres). www.micrographia.com/tutoria/micbasic/micbpt01/micb0100.htm o Some microscopes include an "eyepiece micrometer", essentially a small, transparent ruler mounted within the eyepiece that is used to measure specimens. Such an eyepiece micrometer must initially be calibrated (at each magnification) using a specimen of known size (see preceding item). Objective 3: o Without an eyepiece micrometer, sizes can still be estimated in relation to the field of view or to the thickness of an eyepiece pointer (if there is one). Each of these reference standards must be initially calibrated at each magnification. On each of the following slides, find some cells. Estimate the size (diameter) for the whole cell and for its nucleus. www.microscopy.fsu.edu/primer/anatomy/anatomy.html ___________________________________ Meet some cells Recognizing that all animal tissues (plants, too) are composed of cells and cell products was a major accomplishment in the history of scientific observation. Slide number: 18, 35, 55, 91 11 July 2011