Microscopes

Care for your microscope Always carry your scope with 2 hands (One around the handle/Arm; One supporting the base) The Handle/Arm and oculars should be facing you when using the microscope. Only use lens paper to clean the objectives. (provided in each Student Drawer) Store the dust cover in your drawer/cabinet so it is out of the way Always use the plastic mat provided under your microscope

Types of Microscopes Light microscope – uses visible light to see stuff Simple microscope – one set of lenses (dissecting microscope) Compound microscope (two sets of lenses (ocular, objective) Imaging techniques Darkfield and phase contrast – see unstained samples based on denser structures Staining – used to color specific structures and identify specific subject Fluorescent – use invisible light to generate color (better contrast) Confocal microscopy – constrain field of view and move the stage around to see the whole image clearly Electron microscope – uses electrons to see stuff Scanning electron microscope – what bounces off of 3-dimensional subject Transmission electron microscope – what passes through a sliced sample

REVIEW! - Microscope focusing Initial Focusing Use Slide #3: Bacteria and Blood Place slide between stage clips, label facing up (coverslip OVER slide) Turn on the light until you have a WHITE background. (CONDENSER should be all the way up) View the slide through the OCULARS with both eyes. (adjust the for the distance between your eyes until you are able to see one field of vision) Use the STAGE ADJUSTMENT KNOBS until the specimen is in the middle of the light. Use the COURSE FOCUS to bring the specimen into focus. Start with the stage all the way DOWN (this is how the stage needs to be stored) and turn the coarse adjustment knob AWAY from you.

How to see your sample (better!) Improve the contrast Seeing difference between desired object and its neighbors (air, irrelevant cell etc.) Which image to the right has better contrast? Improved with fluorescence, staining, darkfield and phase contrast microscopy Improve the resolution Use of higher-power magnifications Use of bluer lightsources short wavelength/high energy Shorter wavelength allows one to see finer details Spread your sample out better Improve the intensity Increase amount of light entering the sample useful for high-power when light diffused Oil immersion – oil concentrates light, allowing more light to enter the objective lens Diaphragm ; increases light, but tightens focus

Moving to the next higher magnification (10x) Parfocal: the specimen will be in partial focus when moving to the next higher magnification; only need to adjust with FINE FOCUS with each magnification increase or decrease Move the revolving nosepiece clockwise to the next objective (Low Power-10X) Once specimen is into focus, you can move to the next higher objective (40x) by repeating the above steps Use only the FINE FOCUS for High-Power (40x) and higher magnifications to bring the specimen into focus Using the course focus at this time will damage the objective as it crashes into your microscope slide)

Calculating total magnification Compound microscopes use both objective lenses and ocular lenses Total magnification – ocular mag. × objective mag. Example: 40x objective and 10x ocular 40x × 10x = 400x total magnification Which Objective was used if a 25x ocular gave a total magnification of 2500x?

Care of the Microscope The High-Dry/HighPower objective (40X) is to NEVER touch oil. Same with the 4X and 10X objective lenses The oil will damage the lens and you will not be able to see anything clearly

Oil Immersion Once your specimen is brought to focus under the High-Dry, you can use Immersion oil for the oil objective ONLY. Move the revolving nosepiece to the scanning objective (40x→10x→4x). This gets the lenses out of the way DO NOT lower the stage. The stage and lighting is set for the best view of the specimen already. You worked hard to get to this stage! Add a drop of immersion oil onto the slide where light is passing through Rotate the revolving nosepiece counterclockwise (4x→100x) and adjust the focus using ONLY the FINE FOCUS. You may need to adjust the diaphragm to allow more light to pass

Storing your Microscope Scanning Objective (4X) over the Stage Oil Immersion Objective COMPLETELY wiped clean of oil (with lens paper!) Center the stage Stage in the full down position Scope switched off Phase Adapter in the OUT position Cord Wrapped around cord holder Dust Cover on Microscopes are stored with the handle facing out Clean up mat and put it away