what can we see with light vs electron microscopes
Light microscopes are used to observe objects that are too small to be seen with the unaided eye, such as most cells or larger organelles.
Electron microscopes are used to observe individual organelles, membranes, and large macromolecular assemblies such as ribosomes.
how do you clean the lens of a microscope
Any dirt on the surface of a lens will be magnified an interfere with your observations. Use a piece of folded lens tissue from the booklets provided to clean a lens. Never use Kimwipes, paper towels, or other material, also don’t use water or other solvents.
how do you care for a microscope
Always store a microscope with lower power objective in position
Never take a lens apart
Clean each lens with tissue after use
Always use immersion oil with the 100X objective, and only with the 100X objective. Remove oil from the 100X immediately after use
what are the light intensity controls of a microscope
light intensity control - located at the base of the microscope. The light intensity is adjusted with a knob which varies the output voltage to the light source and therefore its brightness.
what is the field iris diaphragm
Field Iris diaphragm - on the base of the microscope centred below the condenser. Turning its outer dial controls the diameter of the visible illuminated field
what are the coarse and fine focus adjustment knobs
Coarse and fine focus adjustment knobs - These focus the image by adjusting the distance between the objective lens and the specimen. Don’t use the coarse adjustment knob with the 40 or 100X objectives.
what is the condenser
Condenser - a series of lenses immediately below the specimen stage that focuses the light onto the specimen slide
what is the Condenser focus knob
Condenser focus knob - moves the condenser up and down, the light source is focused at the different focal lengths of different objective lenses. This ensures optimal illumination of the specimen.
what are the Condenser Centering Screws
Condenser Centering Screws - Center the condenser so that the source light is centered in the field of view. Two screws on the metal ring holding the free-floating condenser lens unit control the position of the condenser.
what is the Condenser iris diaphragm
- Condenser Iris Diaphragm - controls the diameter of the cone of light entering the objective lens tube. Reduces glare and enhances contrast by “sharpening” the light beam. The diaphragm is controlled by means of a short lever on the condenser unit. Opened wide for high power objective, and closed to its lower setting for lower power objectives.
what is the stage and slide holder
Stage and slide holder - supports the specimen slide and make carefully controlled movement of the object possible in both the x and y planes. Because of the lens system inverts and reverses the image of the specimen, the object appears to move in a direction opposite to the stage’s movement when the stage control knobs are turned.
what is the objective lens
Objective lenses - these provide the primary magnification power and produce an enlarged but inverted projection of the object. Objectives providing different magnification can be rotated in place (.35 or 5X, 10X, 40X, 100X). They are designed to be parfocal and parcentral, meaning that the objective remains in focus, and in the centre of the field, when a different objective is swung into place. Always commence veiwing specimens with th elowest power objective in position.
what are the ocular lenses
Eyepiece (ocular lenses) - They further magnify the object by 10X. The total magnification of a microscope is the product of the magnifications of the oculars and objective in use.
how can you focus the ocular lenses
Each ocular lens can be independently focuses to suit your own vision by pulling the oculars fully apart than, while looking through them, move them closer together into you see a single image. Now focus one of the oculars using the coarse and fine focus knobs. While looking through both oculars rotate the other ocular until it too is focused.
what is the depth of field
depth of field: High power lenses have a shorter depth of field than low power ones. This means that less of the depth of an object is in focus on any specific plane of focus when progressively higher powers are used. If you want to see the entire object in focus, use a lower power lens, but it will be seen at lower magnification.
what is the working distance
Working distance: High power lenses have a shorter working distance than lower power ones. The working distance is the distance between the specimen (or glass coverslip over it) and the objective lens when it is in focus.
how should you always adjust the fine focus knob
When using the high power objectives, always adjust the fine focus by rotating the know counterclockwise towards you so the lens moves away from the slide.
what is the field of view
Field of view: High power lenses have a smaller field diameter than low power lens. The actual diameter of the visible area of the slide as seen through the microscope is its field diameter.
what is Koehler illumination
Koehler Illumination is a standardized procedure used to obtain uniform illumination, thus ensuring the best resolution and image quality possible.
what is PFA
Paraformaldehye (PFA) was used in lab 2 to fix the cells and permeabilize the membrane, which will allow the dyes to enter the cell and nucleus.
what are fluorescent molecules
Fluorescent molecules absorb light at a particular wavelength and subsequently emit light of a longer wavelength. the emitted light can be visualized on a fluorescent microscope.
what is DAPI
DAPI is a fluorescent dye that binds to A-T rich regions of DNA. It is excited by UV light (358nm) and emits a blue light (461 nm)
what is Rhodamine
Rhodamine is a family of fluorescent dyes that can be bound to other chemicals for targeted labeling. For this exercise we used rhodamine conjugate to a toxin found in death cap mushrooms. This toxin called Phalloidin, binds to F-actin, part of the cytoskeleton. It is excited at 540nm and emits red light at 565 nm.
what fluorescent imager did we use
We are using the ZOE fluorescent cell imager, it allows us to image our DAPI and rhodamine-phalloidin stained cheek cells.
