Lecture 5 - microscopy

Question 1

What are the main components of a standard brightfield microscope?

Answer 1

light source, condensor lens, stage (holding specimen), objective and ocular (projection lenses, and detector (eye)

Question 2

Where does diffracted and undiffracted light go in a standard brightfield microscope?

Answer 2

light diffracted by specimen and undiffracted light focused by objective lens

Question 3

How is the image captured in a standard brightfield microscope?

Answer 3

by video camera
- more sensitive to low light intensities - living cells can be viewed with limited photo (light) damage
- record image as digital file - different light intensities converted into 2D array of numbers (quantified)

Question 4

What is the overall magnification of a brightfield microscope?

Answer 4

objective lens x ocular lens

Question 5

If you continue to magnify an image on a brightfield microscope, will it improve the quality?

Answer 5

no - empty magnification

Question 6

What is the most important aspect of today’s microscope?

Answer 6

resolution

Question 7

What is the goal for resolution in a microscope?

Answer 7

the minimum distance that can separate two points that still remain identifiable as separate points

Question 8

What are the two factors that the resolving power of a microscope depends on?

Answer 8

1. wavelength of illumination light
2. numerical aperture - light gathering qualities of objective lens and specimen mounting medium

Question 9

How is the resolution of a microscope maximized?

Answer 9

• use shorter wavelengths of illuminating light
• increase NA - alter mounting medium
• limit of resolution for most standard brightfield - 200 nm

Question 10

What do electric microscopes use for resolution?

Answer 10

electrons rather than photons

Question 11

What are two major limitations of brightfield microscopy?

Answer 11

• specimens poor contrast
• specimens usually fixed embedded then sectioned with microtome and stain with molecules specific dye - fixation results in cell death, embedding and sectioning can lead to structural artifacts

Question 12

What is fluorescence microscopy?

Answer 12

microscopy technique for visualizing fluorescent molecules in living or fixed specimens

Question 13

What does fluorescent microscopy rely on?

Answer 13

endogenous fluorescence, applied fluorescent dyes or dye conjugated antibodies and or autofluorescence proteins

Question 14

What is a pro and con of fluorescent microscopy?

Answer 14

pro - provides increased contrast and allows study of structure and when not fixed, it is a dynamic process in living cells and it is also 3D
con - out of focus fluorescence from thick specimen results in blurred image

Question 15

What are the principles of fluorescence?

Answer 15

• certain atoms in fluorescent molecules can absorb photon of certain wavelength
• atoms electron becomes excited and moves up to higher energy state
• excited electron is highly unstable - loses energy and returns to ground state by emitting photo with lower energy
• emitting electron has lower energy (longer wavelength) bc some energy lost as heat

Question 16

what type of microscopy is confocal laser scanning microscopy? (CLSM)

Answer 16

fluorescence microscopy

Question 17

What are some characteristics of confocal laser scanning microscopy CLSM?

Answer 17

• specimen viewed with CLSM usually living: allows for viewing dynamic processes live
• lasers can penetrate into thicker liver specimens

Question 18

What is the technical process of CLSM?

Answer 18

• specimen rapidly scanned with point laser light at specific excitation wavelength
• emitted fluorescent light from only a single layer (focal plane) within the specimen is focused through the pinhole and then collected/viewed
• all out-of-focus fluorescence from the specimen is excluded

Question 19

What does CLSM yield?

Answer 19

2D z-section optical slice of the specimen that is less blurry, than images obtained with standard fluorescence

Question 20

What can you do with z-sections obtained by CLSM?

Answer 20

individual z sections collected at different depths in sample and combined to form z-stack and generate 3D image

Question 21

What are the limitations to CLSM?

Answer 21

• rapid but cannot capture very dynamic cellular processes
• point lase light can photobleach fluorescent molecules and damage live cells by phototoxicity
• not efficient for imaging deep into thicker specimens/tissues
• limited spatial resolution

Question 22

What is super-resolution CLSM?

Answer 22

10x better res than CLSM
various techniques - use multiple lasers with different wavelengths, angles and beam widths

Question 23

What is the pros and cons of super-resolution CLSM?

Answer 23

pro - useful for visualizing small intra-cellular structures
con - longer specimen scanning time, not efficient for capturing very dynamic cellular processes and imaging deeper into thicker specimens