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Biol201 - Topic 1 - Cells and Microscopy
NA
22
Biology
Undergraduate 1
11/15/2009

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Cards

Term
What are two similarities between eukaryotes and prokaryotes?
Definition

both are membrane-bound

both have DNA and ribosomes

Term
What is the main difference between eukaryotes and prokaryotes?
Definition

Eukaryotes have membrane-bound organelles

Prokaryotes don't

Term
Tissues
Definition
similar cell types connected
Term
What are the principles of microscopy?
Definition
  • illumination passes through specimen and then through additional lenses
  • illumination source travels in waves of a specific wavelength
  • the way a specimen changes the wavelength of illumination is relevant to what is seen
Term
limit of resolution
Definition
the minimum distance objects can be apart and yet still be distinguishable as separate entities
Term

Resolving power of:

 

human eye

 

light microscope

 

electron microscope

Definition

human eye: mm range

 

light microscope: 250-300nm

 

electron microscope: 2nm

Term

What entities are on the order of micrometers/microns?

 

What entities are on the order of nanometers?

Definition

Animal cell: 20 uM

Mitochondria: 1 uM

 

Ribosome 25-30nm

DNA: 2nm

Term
What three factors affect microscop resolution?
Definition
  • illumination wavelength
  • angular aperture
  • refractive index (oil immersion)
Term
List at least four methods of sample preparation
Definition
  • fixation - add acids or aldhydes to corsslink proteins in place
  • permeabilization - put holes in membrane
  • sectioning - using microtone
  • dehydrate/freeze
  • staining - dyes increase contrast
Term
What are three types of microscopy?
Definition

brightfield

phase contrast

fluorescence

Term

Describe brightfield microscopy.

 

What is two limitations?

Definition

Visible light passes through specimen; reveals color and opacity.

 

Limits:

  • usually dead specimens - may show artifacts
  • requires color or some sort of property that affects light
Term

Define phase contrast microscopy

 

Advantage?

Definition

converts the density/thickness differences into differences in visible contrast

 

advantage: can be used to see living cells

Term

What is fluorescence?

 

How do fluorescent dyes work?

Definition

Fluorescence: absorption and emission of light

 

Fluorescent dyes absorb light at one wavelength and emit light at a longer/lower energy wavelength. Atoms are excited, and emit light as the electrons return to ground state

Term

Describe the steps of fluorescent microscopy.

 

What is a drawback to fluorescent microscopy?

Definition
  1. shine light of all wavelengths
  2. filters allow only a specific wavelength through
  3. light illuminates object
  4. fluorescent dye absorbs the light, then emits light
  5. emitted light is of longer wavelength, lower energy (e.g. blue in, green out)
  6. another filter removes the excitation wavelength

drawback: focus only on one plane of the sample, but light is emitted from all planes, which creates background illumination and causes blurriness.

Term
What are two ways to couple antibodies to fluorescent markers?
Definition

Method 1

  • create and use antibodies to detect and purify proteins
  • link antibodies to fluorescent dyes
  • stain the cell with fluorescent antibodies to detect the localization of specific proteins

Method 2

  • primary antibody binds to target
  • fluorescent secondary antibody binds to primary antibody
Term
How does GFP work?
Definition
  1. make a construct by adding genetic sequence for GFP to DNA of gene of interest
  2. transfect cells with construct
  3. cells express fusion protein of GFP-Protein of interest, which fluoresces
Term
Describe confocal microscopy
Definition

only a single plane of the sample is

illuminated at a time

make 3D reconstruction based on all the planes

→ microscope takes a series of images and combines into one

Term

What are radioisotopes?

 

Give some examples.

Definition

unstable atomic nuclei that give off electrons or gamma rays which can be detected

 

3H, 14C, 32P (in DNA), 35S (in proteins)

Term
What is autoradiography?
Definition

technique used to detect radioactive molecules by overlaying the sampel with X-ray film

 

silver grains indicate locations

Term
Describe sample preparation for electron microscopy
Definition

use electron-dense mateirals (e.g. gold, platinum) and coat sample.

 

fix, section, and probe with antibodies or radioisoptopes

Term

What three types of electron microscopy?

 

Briefly describe

Definition

TEM (transmission)

electrons travel through thin (~100nm) sample

 

HVEM (high voltage)

electrons travel through thicker (1um) sample

 

SEM (scanning)

sputter coating: coat whole sample with electron-dense sample. Electrons bounce off sample, yielding 3D image

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