Lecture 2

Question 1

Refraction of Light

Answer 1

• light waves transmittedthrough a vacuum travel at a fixed velocity
  
  • velocity is slowed through air,water , or glass
  • EAch medium transmits light at acharacterisitc velocity
• Refractive index(R.I.)
  
  • RI=Velocity of light/(velocity of light inside transmitting medium)
  • Refractive index of air=1
  • Refraction refers to then bending of light waves ,travel at a fixed velocity
  • refracitve index is awlays greater then 1

Question 2

Does light bend when it travels through a medium?

Answer 2

• Yes light bends when it travels through a medium
• Degree of bending depends on
  
  • R.I.
  • Angle at which light strikes the surface
• Light waves follow the same path in either direction
• Note that light striking a perpendicular surface continues on through without bending

Question 3

What is refractive power

Answer 3

Refractive power is a meausre of how much a lens bends light waves. Measured in diopters

• 1 diopter=1 meter divided by the focal length of a lens

Question 4

Convex lens

Answer 4

Binding of all individual lights will converge at a single point at a certain distance past the lens

Question 5

Focal point

Answer 5

The point through which all parallel rays of light will pass after passing through each part of the lens

Question 6

Focal Length

Answer 6

• The distance from the center of the lens to the focal point

Question 7

Image formation and magnification by lenses

Answer 7

• Light waves traveling through a convex lens will converge at a focal point
• a real image is formed when the object is placed outside the focal point
  
  • Real image is inverted
  • Real image can be projected onto a screen
  • Real image differs in size from the object
    
    • greatest magnification will be obtained from lenses having a very short focal length with the object as close as possible to the focal point
  • how much it varies depends where it is in relationship to the focal point
• Convext lens will create real image if hte object is outside the focal point

Question 8

When is a virtual image formed

Answer 8

Virtual image is formed when the object is placed insdie the focal point

• Virtula image is not inverted
• virtual image cannot be projected onto a screen virtual image can be magnified
• no points exist at any plane in space at which rays radiating from the objects are brought to a focus
• rays radiate outward and do not form a real image when object is inside focal point
• when you look at a mirror its a virtual image

Question 9

Resolution

Answer 9

• ability of a microscope(or any optical instrument) to distinguish two small points as separate points
• To accomplish this, the diamter of diffraction lines around the points must be reduced
• Resolution is important to be able to see objects
• Resolving power is how close two objects be and still be seen as seperate objecs
• when light waves strike an object as they go off the edge of the object will raidate and wavelengths will mi and be seen as one blurry object if we don’t have high resolution to seperate wavelengths

Question 10

What is the resolution equation

Answer 10

• d=.61(lambda)/(nsina)
• lambda=wavelength of light(3800A)
• .61=constant relating to defined overlap allowed for two points to be recognized as separate by a human observer
• n=refractive index of medium surrounding object
• sin a= angle of cone of light entering objective lens(max 180)
• n sina=numerical aperture
  
  • Max=1.4
• D actual distance that two objects can be seen as separate things
  
  • Want D to be smaller becasue it means that the objects can be closer together and still be seen as sepearate objects
• Light microscope never gets a NA of above 1.25 usually

Question 11

Resolution in light and TEM microscope?

Answer 11

• light microscope the highest resolution is about .5um
• In TEM the highest is theoretically .01 angstorms, but the actual resolution achievable is 5-10 angstorms(=.5-1.0nm)
• Don’t see the .01 angstorms becasue electrons have a defraction pattern and create sometimes blurry images at lower and lower resolutions

Question 12

Compound optical(light) microscope

Answer 12

• Also referred to as birght field microscope
• Direct descendent of the microscopes used in 1800s
• Components
  
  • Light sources
  • Condenser
  • Stage
  • objective lens
  • Ocular lens
• Good microscope is made up of seperate lenses glued together to create a better resolution
• Objective lens has a rotating ability to chagne the magnification
• stage where all the action happens
• Condenser -to create optimal cone of light that will pass throuh the objective lens and very detailed instruction on how to set condenser,creating cone of light that is consistent with the objective lens

Question 13

Pros and cons of the Light Microscope

Answer 13

• Ability to magnify
• Ability to resolve strucutural detail
• Specimen must be thin
• Relatively little contrast in the unstained specimen
• Important thing is the resoltuion not the magnification
• Specimen must be thin is a con because it takes a lot of work to get a specimen thin enough , and there is a little contrast in a non stained specimen

Question 14

Phase contrast microscope

Answer 14

• Converts phase shifts(invisible to the eye) 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
• Different parts of a cell have small differences in the refractive index
• Light passing through these regions become deflected and out of phase with the main stream of light waves
• Out of phase wavelengths are matched with other induced out of phase wavelengths which cnacels their amplitude
• Creates light waves that can be seen in contrast to other light waves that have not been retarded
  
  • Phase contrast chagnes the phase into patterns of darkness and light
  • The condenser system is special and doesn’t allow light to pass directly through the specimen which allows for the light to hit specimen at different angles

Question 15

Fluorescence Microscope

Answer 15

• Detects molecules that emit light of wavelengths in the visible range when exposed to a UV light source
• Detects naturally occurring fluorescent(autofluorescent) molecuels such as Vitamin A
• Most widespread application is to detect induced fluorescence:
  
  • Detection of antigens or antibodies in immunochemical staining procedures
  • detection of fluorescent tracers injected into animals or cells

Question 16

Confocal scanning microscope

Answer 16

• Increases optical resolution and contrast by means of adding a pinhole placed at the confocal plane of the lens
• enables the reconstruction of three-dimensional images
• combines light microscope components fitted with fluorescence equipment and scanning system that employs a laser beam
• The illuminating laser light is strongly convergent produces a shallow scannign spot
• Light emerging from the spot is directed to a photomultiplier tube and analyzed
• A mirror system moves the alser beams across the specimen and data from each spot of the specimen is recorded and stored in a computer
• The information is then displayed on a monitor to create a visual image
• 6-10um thick which his thicker then a typical
• usually see 2 layers of cell , and sometimes you get a single layer cell(highly resolution)
• looking at the specimen with multiple layers at the same time

Question 17

Advantages of Confocal Scanning Microscope

Answer 17

• Very thin optical images of hte specimen are created(typically 1um thick)
• out of focus images are subtracted from the image by the computer program
• Computer can make 3-D reconstruction of the speciment b stacking individual images

Question 18

Transmission Electron Microscope

Answer 18

• TEM utilizes a beam of electrons rather than light
• Components
  
  • Cathode
  • Heated tungsten filament(electron source)
  • series of electromagnets:
    
    • Condenser lens, objective lens,projective lens
  • Specimen holder
  • Viewing screen and photographic film
• Potential difference between the anode and cathode impart an aceleartion voltage of between 20,000 and 200,000 volts
• Electrons will pass through glass beyond easy , cna’t focus electrons with glass
• Control electrons with a series of electromagnets to focus the elcctrons and get a clear image
• electrons need to be in a vacuum to reach velocity necessary