Microscopy Flashcards
Refraction of light
Light waves transmitted through a vacuum travel at a fixed velocity
RI = Velocity of Light/(Velocity of light inside transmitting medium)
What is the refractive index of air?
One. R.I. will always be more than one
When light travels from one medium to another, it bends. What does the degree of bending depend on?
- Refraction of light
2. Angle at which light strikes surface
What is the focal point?
The point through which all parallel rays of light will pass after passing through each part of the lens
What is the focal length?
The distance from the center of the lens to the focal point
What are diopters?
Diopters measure how much a lens bends light waves (refractive power).
1 meter / focal length of a lens = the diopter
How do you increase the # of diopters for a lens?
Have a stronger lens. The stronger it is, the closer the convergence(shorter focal length) which increases diopters.
Lenses: When is a real image formed?
A real image is formed when the object is placed outside the focal point
What are the characteristics of a real image?
- A real image is inverted
- A real image divers in size from the object
How can you achieve greatest magnification?
From lenses having a very short focal length, with the object as close as possible to the focal point.
Lenses: When is a virtual image formed?
When the object is placed inside the focal point
What are the characteristics of a virtual image?
- Not inverted, cannot be projected onto a screen, and no points exist at any place at which rays radiating from the object are brought to a focus
- Can be magnified
What is resolution?
The ability of a microscope to distinguish two small points as separate points.
How do you improve resolution?
- Use higher refractive index
- Use shorter wavelengths
What is the equation for resolution?
Resolution = d = (0.61λ) / n*sin(a)
Microscopes: Compound Optical (Light) Microscope
- also referred to as a bright-field microscope
What does a condenser of a microscope do?
It is a lens that concentrates the light
What does the objective less do?
The objective lens is usually made up of multiple lenses glued together in order to correct the focal points from a bad lens. Also fixes the halo rights that sometimes show up
What are the pros/cons of a compound light microscope?
Pros
- Ability to magnify
- Ability to resolve structural detail
Cons:
- Specimen must be thin (takes a lot of work)
- Relatively little contrast in the unstained specimen (hard to see if not stained)
Microscopes:
Phase Contrast Microscope
- Converts phase shifts (invisible to the ye) in light passing through a transparent specimen to brightness changes (visible to the eye) in the image
- Can be used to examine unstained cells and tissues
- Useful for examination of living cells
- No real clinical application. Mostly for lab
Microscopes:
Fluorescence Microscope
- Detects molecules that emit light of wavelengths in the visible range when exposed to a UV light source
- Detects naturally occurring fluorescent molecules such as Vitamin A
- Used to detect induced fluorescence - antigens or antibodies
- fluorescent tracers injected into animals or cells
Microscopes:
Confocal Scanning Microscope
- Very high resolution. Very sharp, focused points of light using a laser. Can be focused on a specimen but can only go through a small area and has to constantly be moved. But it will pick up images and sends it to a computer to focus at different levels and make a 3D image
What are the advantages of the Confocal Scanning Microscope?
- very thin optical images of the specimen
- out-of-focus images are subtracted from the computer
- computer can make 3D reconstructions by stacking images
Microscopes:
Transmission Electron Microscope
- Utililzes a beam of electrons rather than light
- Catode, anode,
- heated tungsten fliament (electron source)
- electromagnets - by moving magnets around, you can focus the light beams
