HISTORY of the MICROSCOPE 1590: The first compound microscope was used 1655 – Robert Hooke used a compound microscope to observe pores in cork He called them “cells” They reminded him of prison cells

HISTORY of the MICROSCOPE 1674 – Anton van Leeuwenhoek built a simple microscope with only one lens to examine blood, yeast, insects and many other tiny objects.Anton van Leeuwenhoek

Compound Microscope standard microscope two lenses: ocular lens and objective lens two or three objective lenses (low, medium, and high magnification) Better than magnifying glass, but… Specimens must be thin so light can pass through Specimens may need to be stained to see structures Image viewed is a mirror image of the object

Parts of the Microscope Eyepiece Body Tube Revolving Nosepiece Arm Objective Lens Stage Stage Clips Coarse Focus Fine Focus Base Diaphragm Light

FUNCTIONS a. Magnifies image (10X) b. Used for rough focus (use with low power) c. Used for fine adjustments (use with high power) d. Enlarges image (scanning 4x, low power 10x, high power 40x) e. Used for carrying microscope f. Platform for holding the slide g. Can be a mirror or light bulb h. Used for carrying the microscope i. Adjusts the amount of light

10x x 4x = 40x Magnification magnification total of eyepiece lens of obj. lens magnification *The greater the total magnification, the smaller the field of view (FOV) or area that you see. The lower the total magnification, the larger the field of view (FOV). Light Compound Microscopes have 3 magnifications: Scanning: find the object; uses course focus mainly Low: make clearer High: only fine focus Each objective will have written the magnification and the ocular lens (eyepiece) has a magnification. The total magnification is the ocular x objective

Microscope Magnification  Calculate the total magnification: Eyepiece Lens Objective Lens Total Magnification a. 10x4x b. 10x10x c. 10x40x

HIGH POWER FIELD OF VIEW -How much can you see? In High Power we see 25% of the low power FOV (low power 100 x is 25% of high power 400x) We see less of the specimen but we see more details of the specimen under high power LOW POWER

Answer the following Questions 1. After switching from high power to low power the area of the field of view will appear a. larger and brighter b. Smaller and brighter c. larger and darker d. smaller and darker 2. What should a student adjust if the field of view seems too dark? ________________________________ 3. Is the field of vision smaller or larger under low power? _______________________________

4. To locate and observe a specimen under a slide, a student should begin by using what objective and what adjustment knob? 5. What adjustment knob should you use if you are using high power? 6. Why should a specimen be centered in the middle of the field of view when focusing under low power?

Microscope Vocabulary Magnification: increase of an object’s apparent size Resolution: power to show details clearly Both are needed to see a clear image

Stereoscopic Microscope (Dissecting Microscope) An ocular lens for each eye Gives a 3-D image Can view whole organisms Great for studying external or surface structure of a specimen Images are not reversed

Electron Microscope The limit of resolution restricts the usefulness of light microscopes for studying VERY small specimens such as viruses. Electron microscopes use a stream of electrons to view these specimens. Electron microscopes have a limit of resolution more than 1000 times finer than light microscopes.

Electron Microscope: TEM Best magnification (250,000 X) Can see things that are smaller than a cell (organelles and viruses) Objects must be thinly sliced and stained with metal for viewing Bamboo Fiber Cells

Electron Microscope: SEM Best detail / depth Less magnification than a TEM Able to view a whole organism and provide a 3-D image Foot of House Fly staple

FIELD OF VIEW How do we find the FOV of a microscope? Low Power (100x) 1. Find the diameter of the LP FOV 2. Use the clear metric ruler (mm side) 3. Be sure to line up the first mm mark with the left side of the field. LP FOV = 1.5 mm

The mm is too large to measure microscopic objects, so you need to use the micron (micrometer) µm 1 mm = 1,000 µm 1 µm = 1/1000 mm or 0.001mm LP FOV = 1500µm

So how do we determine how big something is in the microscope? Use the following formula: FOV # of cells (that can fit across diameter) Let’s see how it works: FOV = 2mm =.5mm # of 4 cells OR.5mm x 1000 =500 microns

CARRYING THE MICROSCOPE AB

FOCUSING 1. Always start with the SCANNING objective. Odds are, you will be able to see something on this setting. Use the Coarse Knob to focus then the fine adjustment knob until clear, image may be small **Do not use stage clips, try moving the slide around until you find something.

2. Once you've focused on Scanning, switch to Low Power. Use the Coarse Adjustment Knob to refocus Use the Fine Adjustment Knob to make the image crystal clear *If you haven't focused on this level, you will not be able to move to the next level. 3. Now switch to High Power. (If you have a thick slide, or a slide without a cover, do NOT use the high power objective). ONLY use the Fine Adjustment Knob to focus specimens.

1. Use pencil - you can erase and shade areas 2. All drawings should include clear and proper labels (and be large enough to view details). Drawings should be labeled with the specimen name and magnification. 3. Labels should be written on the outside of the circle. The circle indicates the viewing field as seen through the eyepiece, specimens should be drawn to scale - ie..if your specimen takes up the whole viewing field, make sure your drawing reflects that. DRAWING SPECIMENS

Occasionally you may have trouble with working your microscope. Here are some common problems and solutions. 1. Image is too dark! Adjust the diaphragm, make sure your light is on. 2. There's a spot in my viewing field, even when I move the slide the spot stays in the same place! Your lens is dirty. Use lens paper, and only lens paper to carefully clean the objective and ocular lens. The ocular lens can be removed to clean the inside. The spot is probably a spec of dust. 3. I can't see anything under high power! Remember the steps, if you can't focus under scanning and then low power, you won't be able to focus anything under high power. Start at scanning and walk through the steps again. 4. Only half of my viewing field is lit\ You probably don't have your objective fully clicked into place.. TROUBLESHOOTING

Occasionally you may have trouble with working your microscope. Here are some common problems and solutions. 1. Image is too dark! Adjust the diaphragm, make sure your light is on. 2. There's a spot in my viewing field, even when I move the slide the spot stays in the same place! Your lens is dirty. Use lens paper, and only lens paper to carefully clean the objective and ocular lens. The ocular lens can be removed to clean the inside. The spot is probably a spec of dust. 3. I can't see anything under high power! Remember the steps, if you can't focus under scanning and then low power, you won't be able to focus anything under high power. Start at scanning and walk through the steps again. 4. Only half of my viewing field is lit\ You probably don't have your objective fully clicked into place.. TROUBLESHOOTING

OTHER TOOLS Metric Ruler: Length (meters) Graduated Cylinder: Volume (g/mL) To read the volume, you must look at the bottom of the _____________ which is a curve surface. Triple Beam Balance: Mass (grams) M VD

OTHER TOOLS a magnifying glass One lens Low magnification and low resolution

OTHER TOOLS Centrifuge- technique used to separate substances based on density Spins material at very high rates of speed Materials suspended in a liquid are spun around very rapidly. Heaviest particles settle at bottom; lightest particles settle on top.

OTHER TOOLS Spectrophotometer Measures amount and kind of light absorbed by a material. Spectrophotometry: is the use of light to analyze samples.

OTHER TOOLS Chromotography- technique used to separate substances based on their chemical make up ( the different colors of leaves are separated this way) A family of techniques for the separation of mixtures. Separates different substances from each other on the basis of their chemical or physical properties Be familiar with paper chromatography

OTHER TOOLS Gel electrophoresis -technique used to separate substances based on their electrical charge. (DNA is separated this way) Separates substances based on size and electrical charge An electrical current is run through a gel that contains the substance being studied Different components of the substance move at different rates through the gel