Lecture Set 1

Question 1

How do cells differ?

Answer 1

Morphology and physiology

Question 2

Why do cells become differentiated?

Answer 2

Through development, even though cells all have same copies of DNA

Question 3

What are the three tenets of the cell theory?

Answer 3

All organisms consist of one or more cells
Cell is the basic unit of structure for all organisms
All cells arise from preexisting cells

Question 4

Discuss the difference between resolving power and limits of resolution

Answer 4

Resolving power = ability to distinguish objects as separate entities
Resolution = minimum distance between two objects such that they can still be recognized as separate

Question 5

What determines resolution, how is it calculated?

Answer 5

Abbe equation
r= 0.61λ/nsin(α)
r = resolution, want it to be as small as possible
lambda = wavelength, want as small as possible
n = refractive index (change in velocity of light as medium changes) (want as large as possible)
alpha = angular aperture (half angle of the cone of light entering objective lens (want to be as close to 90 as possible)

Question 6

How do you visualize cells with microscopy

Answer 6

1) fixate with preservative (formaldehyde) to complex with proteins/nucleic acids to maintain integrity of sample
2) Immobilize in wax
3) section –> cut thin slices
Can have light and fluorescent microscopy at same time b/c light deals with transmission, fluorescence relies on reflection

Question 7

What are simple stains, what are they used for?

Answer 7

Used to produce contrast
basic –> positively charged, adds to negative charged molecules (eg crystal violet, methylene blue)
acidic –> negatively charged, adds to positive charges
(eg eosin, acid fuschsin)

Question 8

What is Gram stain?

Answer 8

used to differentiate between gram + and gram - bacteria

uses certain dyes, gram + ends up blue, gram - ends up pink

Question 9

What is fluorescence microscopy, what is it used for?

example of a fluorescent dye

Answer 9

used to identify specific molecules within cell (ex proteins)
use fluorochromes (ex DAPI --> binds to nucleus) that absorb and emit light

Question 10

What is immunohistochemistry, what are the different types?

Answer 10

Attach fluorochrome to antibody that binds to specific proteins
Direct –> fluorochrome tag attachs to primary antibody bindign to protein
Indirect –> fluorochrome attaches to secondary antibody that binds to primary antibody binding to protein (signal heavily amplified)

Question 11

What is confocal microscopy

Answer 11

Much more detailed/clear images
uses lasers of specific wavelengths
uses pinholes to eliminate other light
scanning one pixel at a time
uses photomultiplier tubes instead of cameras
can create 3D construction

Question 12

What is deconvolution?

Answer 12

use of computer software to remove out of focus light from conventional microscopy to get to close to confocal clarity

Question 13

What is an example of a native fluorochrome?

Answer 13

GFP –> occurs in jelly fish

Question 14

What are the two types of electron microscopy?

Answer 14

TEM and SEM –> TEM = transmission, SEM = scanning, can only see surfaces in great detail