Microscopy

Question 1

Light vs. Electron Microscope: resolution and magnification

Answer 1

Light: 200nm, 1000X Electron: 2nm, 20 000X (SEM) and 100 000 (TEM)

Question 2

Light vs. Electron Microscope: Illumination, Lenses and Detection

Answer 2

Light: visible to glass OR UV light to quartz, to eye, film, CCD Electron: beam of electrons to electromagnets to monitor, film, CCD

Question 3

Types of Light Microscopy (5)

Answer 3

1. Bright field 2. Phase Contrast 3. DIC, differential interference contrast 4. Wide Field Fluorescence 5. Confocal Fluorescence Microscopy

Question 4

Bright Field Microscopy: how and preparation

Answer 4

visible light passes through specimen, but stains are often needed to make cells visible

Question 5

Phase Contrast Microscopy: how and preparation

Answer 5

visible light passes through specimen, and complex lenses exaggerate contrast: make darks darker and lights lighter, no staining needed

Question 6

DIC, differential interference contrast microscopy: how and preparation

Answer 6

visible light passes through specimen, complex lens make darks darker and lights lighter

Question 7

DIC vs phase contrast: image produced

Answer 7

phase contrast: halos around cells DIC: has a lighter edge and a darker edge around cell

Question 8

Widefield Fluorescence Microscopy: how and prep?

Answer 8

short wavelength light excites a fluorescent stain within the specimen, longer wavelength light is emitted and detected: has to be stained with fluorescent molecules

Question 9

Fluorescent Stain: excitation and emission (3)

Answer 9

DAPI: UV to blue Fluorescein: blue to green Rhodamine: green to red

Question 10

Confocal Fluorescence Microscopy: how and prep?

Answer 10

fluorescence microscope that reveals 2D optical plane within a 3D specimen, uses lasers to deliver excitation to a single point in the specimen, specimen has to have fluorescent molecules

Question 11

Widefield vs Confocal Fluorescence microscopy?

Answer 11

widefield: broad source of light confocal: laser, so image is more focused

Question 12

Preparing: whole cells (ex. yeast)

Answer 12

put cell culture (3μl) on slide, put coverslip on top - no staining needed (DIC microscope)

Question 13

Preparing: sections of cells or small organisms

Answer 13

cells/organisms are immobilized in wax or plastic, and then sectioned with a microtome into ribbons of thin sections, then those are placed onto glass slide, stained, and mounted under coverslip

Question 14

Regular stains, how to prepare and example

Answer 14

stain specimen, then wash off excess ex. orcein: small organic molecule, natural affinity for DNA

Question 15

Staining: DNA probes

Answer 15

collection of pieces of single stranded DNA that: have high affinity for target DNA (are antiparallel) and are attached to a fluorescent molecule

Question 16

Staining: Antibodies

Answer 16

small proteins made by white blood cells, high affinity for specific target molecules

Question 17

Immunofluorescence

Answer 17

Question 18

Indirect Imunofluorescence

Answer 18

Question 19

How antibodies are made?

Answer 19

purified molecule (ex YFP protein) put into animal (ex. rabbit) that doesn’t normally have this protein, then animal will produce antibodies against this molecule

Question 20

Direct vs indirect Immunfluorescence: which is more common and why?

Answer 20

Indirect is more common: more fluorescent dye goes to target molecules so microscopy becomes easier, if you are making primary antibodies yourself it’s easier and less risky to use secondary antibodies than attaching fluorescent molecules yourself

Question 21

Two types of electron microscopy

Answer 21

1. TEM: scanning
2. SEM: transmission

Question 22

TEM: how and image?

Answer 22

beam of electrons pass through specimen, reveals a 2D image - bright area = electron passes through, dark = electron couldn’t pass through

Question 23

SEM: how and image?

Answer 23

beam of electrons bounces