both are membrane-bound

both have DNA and ribosomes

Eukaryotes have membrane-bound organelles

Prokaryotes don't

• 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

Resolving power of:

human eye

light microscope

electron microscope

human eye: mm range

light microscope: 250-300nm

electron microscope: 2nm

What entities are on the order of micrometers/microns?

What entities are on the order of nanometers?

Animal cell: 20 uM

Mitochondria: 1 uM

Ribosome 25-30nm

DNA: 2nm

• illumination wavelength
• angular aperture
• refractive index (oil immersion)

• fixation - add acids or aldhydes to corsslink proteins in place
• permeabilization - put holes in membrane
• sectioning - using microtone
• dehydrate/freeze
• staining - dyes increase contrast

brightfield

phase contrast

fluorescence

Describe brightfield microscopy.

What is two limitations?

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

Define phase contrast microscopy

Advantage?

converts the density/thickness differences into differences in visible contrast

advantage: can be used to see living cells

What is fluorescence?

How do fluorescent dyes work?

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

Describe the steps of fluorescent microscopy.

What is a drawback to fluorescent microscopy?

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.

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

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

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

What are radioisotopes?

Give some examples.

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

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

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

silver grains indicate locations

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

fix, section, and probe with antibodies or radioisoptopes

What three types of electron microscopy?

Briefly describe

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