Lecture 1- Light Microscopy

Question 1

What are the types of light microscopy

Answer 1

1. Brightfield microscopy
2. Phase contrast microscopy
3. Differential interference contrast (DIC) microscopy
4. Widefield fluorescence microscopy

Question 2

Brightfield microscopy

Answer 2

cell will appear on a bright background

visible light passes through the specimen

stains are often needed to make cells visible

Question 3

Phase contrast microscopy

Answer 3

Visible light passes through specimen

Complex lenses make darks darker and lights lighter

puts a halo around cells

Question 4

Differential Interference Contrast (DIC) microscopy

Answer 4

Visible light passes through the specimen

Complex lenses makes darks darker and lights lighter

Lighter than phase contrast

puts a shadow on cells

Question 5

Similarity of phase contrast and DIC

Answer 5

They have the same function: How to make cells visible without the need for stains

Question 6

How to tell phase contrast and DIC apart?

Answer 6

Phase contrast: puts a halo around cells

DIC: puts a shadow around cells

Question 7

Widefield fluorescence microscopy

Answer 7

a short wavelength hits a specimen and it will emit a longer wavelength

ex: blue light excited fluorescein and it will emit green

Question 8

Composite images

Answer 8

made from multiple images

monochromatic images are captured, given artificial colors, and superimposed

Question 9

Confocal fluorescence microscopy

Answer 9

more expensive

shows a 2D optical plane within a 3D specimen

same as widefield fluorescence microscopy but instead of a regular light source, we are using a laser

Question 10

Confocal fluorescence vs. Widefield fluorescence

Answer 10

Confocal fluorescence show smore detail than widefield fluorescence

Question 11

Tomography

Answer 11

taking 2D images to build 3D images

several confocal fluorescence images can be assembled to make a 3D model

Question 12

Use of light microscope (STEPS)

Answer 12

1. Getting the specimen onto the slide
2. Making things visible

Question 13

Colored Stains

Answer 13

Synthetic molecules with two properties:
1. affinity for target
2. absorbs light

Question 14

Why does H&E staining makes nuclei blue and cytoplasm pink?

Answer 14

because there’s a lot of protein in the cytoplasm

Question 15

Fluorescent stains

Answer 15

Synthetic molecules with two properties:
1. affinity for target
2. fluoresces light
ex: DAPI - high affinity for DNA

Question 16

Fluorescent probes

Answer 16

two molecules joined together by covalent bond

Molecules that absorb light at a specific wavelength and emit light of a longer wavelength

Question 17

Antibodies

Answer 17

small proteins made by white blood cells (B-cells) in mice and other animals

have high affinity for target

will bind to target

Question 18

Alexa Fluor 488

Answer 18

fluoresces green

example of fluorescent dye- synthetic molecules

Question 19

Can you reveal a specific object in brightfield microscopy?

Answer 19

Yes - use colored stains

Question 20

Can you see the whole cell in brightfield microscopy?

Answer 20

Yes

Question 21

Can you reveal a specific object in phase contrast/ DIC?

Answer 21

No

Question 22

Can you see the whole cell in a phase contrast/ DIC?

Answer 22

Yes

Question 23

Can you reveal a specific object in widefield/ confocal fluorescence?

Answer 23

Yes - use fluorescent stains and fluorescent probes

Question 24

Can you see the whole cell in Widefield/ Confocal fluorescence?

Answer 24

No