Fluorescence Microscopy (Lecture 6)

Question 1

True or False: Green-fluorescent dye is specifically bound to lysosomes.

Answer 1

False, mitochondria

Question 2

True or False: Red-fluorescing dye that is specifically incorporated into lysosomes

Answer 2

True

Question 3

True or False: Dyes may be sensitive to pH or other ion concentrations, determining differential binding to organelles

Answer 3

True

Question 4

True or False: Immunofluorescence microscopy is direct.

Answer 4

False, immunofluorescence microscopy is indirect because the fluorochrome is on the secondary antibody

Question 5

What are the advantages of indirect immunofluorescence?

Answer 5

Amplified signal(several secondary antibodies bind)

Secondary antibodies are cheaper and may be used for other experiments

Question 6

True or False: Fluorochrome have to be attached to antibodies.

Answer 6

False, they do not have to be attached to antibodies.

Question 7

True or False: Images for different fluorescence colours (wavelengths) are taken separately.

Answer 7

True, they are electronically merged later.

Question 8

How else can antibodies be targeted to a protein of interest?

Answer 8

Make a recombinant protein that contains an epitope tag

Question 9

What are the two types of epitope tags?

Answer 9

—FLAG = AspTyrLysAspAspAspAspLys —  
myc = GluGlnLysIleSerGluGluAspLeu

Question 10

What is GFP

Answer 10

beta sheet barrel

Question 11

What are the two main drawbacks to fluorescence microscopy?

Answer 11

—Blurring of images caused by fluorescence outside of
the plane of focus
—
Can’t visualize thick specimens
— Collect serial images at various depths, then reconstruct

Question 12

What is deconvolution fluorescence microscopy?

Answer 12

Takes raw images and you can view them after an algorithm is applied

Question 13

What is confocal microscopy?

Answer 13

Illuminate and collect emitted fluorescent light from just one small area of a focal plane at a time

Takes a bunch of images and then stack them upon each other

Question 14

What are the two way to reveal molecular mobility and interaction dynamics.

Answer 14

—Fluorescence recovery after photobleaching (FRAP) —

Förster resonance energy transfer (FRET)

Question 15

True or False: During FRAP, after photobleaching if a protein recovers quick, it is slower moving and less dynamic.

Answer 15

False, it means its faster moving and more dynamic

Question 16

______ measures the distance between fluorochromes.

Answer 16

FRET

Question 17

How is fluorescence detected in FRET?

Answer 17

To be excited, have to look at the wavelength of the second partner

Question 18

_____ is the hort wavelength of EM high-energy electrons yields high resolution images

Answer 18

Electron microscopy

Question 19

True or False: Electron microscopy is better then a light microscope.

Answer 19

True

Question 20

What are the two main types of electron microscopy?

Answer 20

Transmission electron microscopy (TEM) —

Scanning electron microscopy (SEM)

Question 21

During ______ EM electrons pass through the sample and during ______ EM electrons are scattered off sample.

Answer 21

Transmission

Scanning

Question 22

Scanning EM is better for observing what?

Answer 22

Textures of specimens

Question 23

Transmission EM ia better for observing

Answer 23

Static image, specimen must be dead