Lecture #12

Question 1

the first microscopes were invented in the 16th century

Answer 1

• allowed science to move past vitalism
• thought living and non-living things had a fundamental difference in elements

Question 2

microscopes in 17th century

Answer 2

-Robert Hooke: writes the basis for cell theory (smallest building blocks) -also studied by Antonie Van Leeuwenhoek -used to observe small things, find common features

Question 3

five key properties of light

Answer 3

1. both particle and wave 2. usually involves visible light (400-700 nm) 3. the speed of light depends on the medium it’s traveling through (can be bent/separated by glass) 4. absolute limit of resolution exists- there is a smallest thing that can be seen 5. energy and wavelength are inversely related

Question 4

microscopes work by…

Answer 4

generation of contrast (6 diff ways) 1. absorption 2. refraction 3. scattering 4. fluorescence 5. fluorescence with sectioning 6. fluorescence super-resolution

Question 5

Huygens Principle

Answer 5

-how waves interact with objects -waves have set wavelength and create ripples –> target pattern

Question 6

the Airy disk

Answer 6

a dimensionless unit equal to the diameter of the central spot (contains about 84% of the photons from the source)

Question 7

the smallest possible object you can see will be determined by…

Answer 7

the wavelength of light that you use -can’t see a protein with a light microscope, not enough resolution

Question 8

bioluminescence

Answer 8

chemical reaction where something gives off light

Question 9

fluorescence

Answer 9

involves taking up light of a lower wavelength with higher energy and giving off light at a higher wavelength with lower energy

Question 10

things that fluoresce

Answer 10

-toothpaste -detergent -marine invertebrates -tonic water -chlorophyll -some minerals

Question 11

stokes shift

Answer 11

-difference between absorbed and emitted energy in fluorescence -only goes one way

Question 12

confocal microscope

Answer 12

-invented by Martin Minsky in 1955 -not used widespreadly bc light sources were too weak -put pinhole in front of the detector to block all out of focus light and get higher resolution -made feasible by invention of lasers

Question 13

several newer methods have increased resolution greatly

Answer 13

still not high enough to see a protein, but pretty close

Question 14

super-resolution microscopy

Answer 14

refers to light microscopy with resolution below the diffraction limit

Question 15

structured illumination microscopy

Answer 15

-involves projection of a moire pattern onto the object and deconvolving to the moire pattern -creates 3D model

Question 16

transporters are placed on the tips of the membranes on sea urchin embryos because…

Answer 16

it will release the chemical in a place where it will be carried away by the tide rather than reabsorbed

Question 17

selective plane resolution microscopy

Answer 17

not intended to impact resolution, instead changes amount of light used so it can be observed for longer

Question 18

kohler illumination

Answer 18

-for transmitted light microscopy -ensures that the filament image is in a different focal plane than the sample image and that the field is illuminated completely

Question 19

factors that govern image brightness

Answer 19

-refractive index and depth of the medium -the sample (thickness, opacity, etc…) -fluorescence characteristics of the label -higher magnification isn’t always better, consider numerical aperture too

Question 20

brightness equation for microscope image

Answer 20

=(NA/magnification)^2

Question 21

brightness equation for fluorescence

Answer 21

Question 22

choosing a fluorescent marker

Answer 22

fluorescent labels have big differences in size and brightness, need to choose an appropriate size for the size of the molecule

Question 23

numerical aperture influences…

Answer 23

both resolution and brightness