Microscope Handbook PDF

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Microscope Handbook...

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Microscopy: Defined as the use of or investigation performed via the use of a microscope. Micro = small, scope = to see. Bright Field Microscopy is the focus of this week’s laboratory. In it you will: -

Work with light microscopes

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Distinguish cells and cellular structures from debris

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Compare various histological slides of cells and tissues

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Focus and compare what can be visualized at different magnification levels

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Measure specimens using an ocular micrometer

Bright Field Microscopy Bright field microscopy, as the name implies, is the use of light to visualize a darker sample within the otherwise bright field of light produced. In bright field microscopy we utilize visible (white) light to view objects and samples at a proximity and with detail that is impossible for the naked eye. Unfortunately, when looking at biological samples, without additional help, what is mostly visible is:

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The overall cell shape or general tissue structure (e.g., thick, thin, square, etc.,)

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The overall membrane (outline) of the cell or tissue

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The nucleus is normally visible

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Large organelles may be visible

Most of the rest of the internal structures and organelles are invisible as most biological samples do not absorb enough visible light to produce contrast that would allow other structures to become clearly visible. In order to increase contrast and view through bright field microscopy, specific dyes can be employed which allow us to better view these images. Unfortunately, the use of dyes is typically applied to these samples that are no longer alive, meaning we cannot observe these cells in their living state (as they move, respire, procreate, etc.,). The upside of using stains is that structures that were invisible become visible and more easily analyzed. Table 1 gives examples of stains and their uses in histological (tissue) preparations by pathologists

1

Adapted from Robert Bagnell, Jr. Ph.D. 2012, Chapter 8 Bright Field, med.unc.edu What Is Stained

The Stain Used

DNA

Fulgen, Methyl Green Pyronine

Collagen

Masson Trichrome / Sirius Red

Carbohydrate

Periodic Acid-Schiff (PAS), Alcian Blue

Calcium

VonKassa, Alizarin Red S

Bacteria

Brown-Brenn

Beta Cells

Scott

Amoeba

Wheatly, Crystal Violet

Amyloid

Congo Red

Alpha Cells and Pancreas

Grimelius

General Polychromatic Stain

Hematoxylin and Eosin (H&E)

Using Oil Immersion Bacteria and other cellular structures that are generally not large enough to be clearly seen under a microscope at lower magnifications (4x, 10x, 40x) and require the use of 100x or oil immersion microscopy. To further view these kinds of cells (bacterial), one can utilize a simple stain: 1. Smear the sample on a clean slide (from liquid or solid media) and let either air dry or allow to pass briefly through a Bunsen burner flame and allow to fully dry. 2. Apply stain to slide for 30 seconds (e.g., Crystal Violet, Safranin, etc.,). 3. Gently rinse with water. 4. Gently blot dry using bibulous paper. This will allow the microscopist to visualize the general morphology (shape) and size of the bacteria present, though it does not differentiate between the most common classification division: Gram (+) positive versus Gram (-) negative. In terms of eukaryotic cells, stained cells can be visualized with greater detail utilizing the 100x magnification. This allows visualization of some tissue microstructures or even small smaller structures within larger cells that are more difficult or impossible to visualize at 10x or 40x magnification.

Magnification & Measurement The light microscope enlarges objects so that they are visible to the human eye. Most light microscopes are equipped with 3 objective lenses with varying magnification power: 10x (low power), 40x (high power), 100x (oil immersion), whereas some may also include a 4x objective lens. Firstly, the objective lens (closest to the sample) magnifies the object being viewed, the image is then projected up the body tube into the ocular pieces which magnify the specimen by an additional 10x. The final image magnification is a multiplication of these two. FILL-IN the final magnification of a given sample given the use of the following lenses: OBJECTIVE LENS

OCULAR PIECE

4x

10x

10x

10x

40x

10x

100x

10x

FINAL MAGNIFICATION

Note: light microscopes have limitations as to the resolution (the smallest distinguishable size) of objects in the samples. This is called resolving power. Light microscopes can maximally resolve along the lines of 0.2 to 0.3 m (when everything is working correctly). There are two normal/typical rulers (micrometers) used to measure the size of what is present in a sample. One is etched on the slide used (stage micrometer) which is mounted onto the slide stage.

Stage Micrometer from mecanusa.com

The stage microcometer is used in conjunction with the second type: the ocular micrometer which is calibrated using the stage micrometer. Using this ruler which does not change when switching between objective lens magnifications, this provides an arbitrary tool by which we can measure (after calibration). The following table provides and example of some of the relative lengths of the full ocular micrometer rule that has been calibrated to a stage micrometer: 10 ocular dimensions (10 o.d.): Objective

Full Ocular Micrometer

Actual Size

Length 4x Objective

10 o.d.

2.5mm (2500m)

10x Objective

10 o.d.

1mm (1000m)

40x Objective

10 o.d.

0.25mm (250m)

100x Objective

10 o.d.

0.1mm (100m)

The Light Microscope:

Diagrams from the AccuScope Instructions Manual for the Microscopes You Will be Using

Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece usually contains a 10X or 15X power lens. Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to correct for any difference in vision between your two eyes. Body tube (Head): The body tube connects the eyepiece to the objective lenses. Arm: The arm connects the body tube to the base of the microscope. Coarse adjustment: Brings the specimen into general focus. Fine adjustment: Fine tunes the focus and increases the detail of the specimen. Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the nosepiece to select different objective lenses. Objective lenses: One of the most important parts of a compound microscope, as they are the lenses closest to the specimen. A standard microscope has three, four, or five objective lenses that range in power from 4X to 100X.

When focusing the microscope, be careful that the objective lens doesn’t touch the slide, as it could break the slide and destroy the specimen. Specimen or slide: The specimen is the object being examined. Most specimens are mounted on slides, flat rectangles of thin glass. The specimen is placed on the glass and a cover slip is placed over the specimen. This allows the slide to be easily inserted or removed from the microscope. It also allows the specimen to be labeled, transported, and stored without damage. Stage: The flat platform where the slide is placed. Stage clips: Metal clips that hold the slide in place. Stage height adjustment (Stage Control): These knobs move the stage left and right or up and down. Aperture: The hole in the middle of the stage that allows light from the illuminator to reach the specimen. On/off switch: This switch on the base of the microscope turns the illuminator off and on. Illumination: The light source for a microscope. Older microscopes used mirrors to reflect light from an external source up through the bottom of the stage; however, most microscopes now use a low- voltage bulb. Iris diaphragm: Adjusts the amount of light that reaches the specimen. Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed. Base: The base supports the microscope and it’s where illuminator is located.

HANDLING THE MICROSCOPE: 1. Use TWO hands when carrying the microscope, they are sensitive and COSTLY. Keep the instrument upright and ocular pieces visible as these can fall out and break or be otherwise damaged. 2. ONLY clean using lenses using LENS PAPER, any other type will SCRATCH and DAMAGE the lenses (e.g., paper towel, Kim Wipes, etc.,) 3. Do not remove ocular pieces or objective lenses (magnification pieces). 4. Turn the objective lenses by grasping the RING of the revolving nosepiece until desired lens CLICKS into place. 5. Always CLEAN the lenses using LENS PAPER when finished using. 6. When putting the microscope away:

a. Place the lowest power objective or none in the primary position. b. Make sure slides are removed and that the slide stage is clean. c. Wrap the cord loosely around the arm. USING THE MICROSCOPE: 1. After step 4 above, plug-in the microscope and identify the ON/OFF switch – and turn the microscope’s power source ON. 2. Place the lowest power objective in place using the ring of the revolving nosepiece. 3. Adjust the ocular pieces to best resolve your sample. NOTE: ONCE THE SAMPLE IS IN FOCUS – Switching Objective Lenses should require little adjustment to return the sample to focus. 4. Adjust the light source as needed: a. The main light source power –and/orb. The condenser 5. Only utilize the COARSE focus adjustment knob at 4x and 10x. At 40x (and 100x) ONLY USE FINE FOCUS knob. After you are done, turn the microscope OFF and unplug. Then follow steps 5 and 6 in the section above to finish.

This handbook is modified by Dr. Javier Rivera Guzman from the preparation by Dr. Karen Whitworth and Dr. Cynthia Wagner as developed for the Department of Biological Sciences at UMBC. Portions of this is adapted from: Cell Biology Laboratory. Robert D. Horner. Johns Hopkins University. Molecular th Biology of t he Cell. Alberts et al. (4 Ed.) Garland Science. Robert Bagnell, Jr. Ph.D. 2012, Chapter 8 Bright Field, med.unc.edu Microscopy I: Bright-Field. Yvonee Vaillancourt. University of Maryland Baltimore ....