Introduction to cells and microscopical techniques

Question 1

What is The nature of light

Answer 1

The ‘size of light’ in microscopy terms is the wavelength (λ) or colour used in a particular situation.
The wavelength of an electron is 100,000x smaller than that of a photon.

Question 2

What is the Light microscopy?
What is Electron microscopy?

Answer 2

1. Brightfield, fluorescence and confocal
   Lower magnification but can be used to image living cells
2. Transmission (TEM) and Scanning (SEM)
   Cells must be fixed (i.e. killed)

Question 3

Light vs electron wavelengths?

Answer 3

Light wavelengths are much longer than that of the electron, i.e. the basketball diameter compared to the tennis ball diameter.
Can you see why you get more information about the surface by bouncing a tennis ball off of a rough wall instead of a basketball?

Question 4

Who is Robert Hooke?

Answer 4

1665 - Robert Hooke (1635-1703) - in the book Micrographia, published in 1665, he coined the word ‘cell’ to describe the small ‘boxes’ that organisms seem to be composed of because they reminded him of the small cells that monks live in in a monastery:

Question 5

What are Modern microscopes?

Answer 5

Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens.

Question 6

What are Magnification and
resolution/Highest Typical Resolution?

Answer 6

• Magnification
  Image size / Object size
• Resolution
  The fineness of detail that can be distinguished in an image
• Highest Typical Resolution
  In other words, the smallest thing that can be seen
  Light microscopy ~ 200 nm Electron microscopy ~ 0.1 nm

Question 7

What is Specimen preparation for light microscopy?

Answer 7

a) Paraffin wax embedded sections
-Tumor biopsy embedded for sectioning
-A ‘microtome’ for cutting sections
(Slide 20)

Question 8

Specimen preparation for light microscopy?

Answer 8

(b) Frozen and embedded sections
Possibly better preservation of some cellular constituents than by wax embedding
-Section of a whole frozen pregnant mouse
-A ‘cryostat’ for cutting frozen sections
(Slide 21)

Question 9

How to see Cells that are colourless and transparent, and, therefore, pretty much invisible?

Answer 9

They usually need to be stained or labelled to improve contrast. This is done using:
1.Chemical stains / dyes
2.Enzyme labels (often used in immunocytochemistry)
3.Fluorescent labels (often used in immunocytochemistry
and also live-cell imaging)
4. Electron dense labels (used in electron microscopy)

Question 10

Light microscopy techniques?

Answer 10

Transmitted light contrast modes
Brightfield
Phase contrast
Differential interference contrast (DIC or Nomarski)
(Slide 23)

Question 11

Go to Goodnotes for slide 23-26

Question 12

What is Fluorescence microscopy?

Answer 12

Dark cellular background labelled with bright fluorescent stains
Often used in immunocytochemistry and also live cell imaging.
Multiple labelling (more than one colour) possible
Can be used with conventional (but specially designed) light microscopes and increasingly used in confocal microscopy

Question 13

What is Confocal microscopy?

Answer 13

Confocal microscopy is a more recent improvement on normal fluorescence microscopy

Question 14

Who invented Confocal microscopy?

Answer 14

Marvin Minsky invents the idea as a student at Harvard University in the 1950s.
His confocal worked on paper, i.e. in principle, but didn’t produce the sort of stunning images that we are used to now.

Question 15

What are The key
components of Confocal microscopy?

Answer 15

• Research microscope equiped as for fluorescence.
• Lasers of various output wavelengths.
• A scanning mechanism.
• Light detectors and amplifiers.
• Computer with substantial processing power.
• Suitable fluorochromes.

Question 16

Why couldn’t Minsky build a functioning confocal microscope in the 1950s?

Answer 16

The ‘pinhole’ limits the amount of light that gets to the detector to such an extent that it needs to be amplified to form an image, and light amplifiers (photo multiplier tubes - PMTs) weren’t really available back then.

Question 17

What are Fluorescent proteins?

Answer 17

-Continually produced within living cells and subject to cellular targeting, partitioning, and turnover processes as with all other proteins.

-Fluorescent proteins are not ‘stains’. They remain glowing
as permanent markers in living cells!

-These proteins are very bright and non-toxic which means that cell and tissue development can be monitored over the long term.

-Importantly, fluorescent protein expression and sub-cellular localisation can be controlled using molecular biological techniques.

Question 18

Fluorescent proteins - discovery and development?

Answer 18

-Osamu Shimomura first noticed green fluorescent protein in 1962.

-Douglas Prasher cloned the GFP gene in 1992 but didn’t get to test it.

-Martin Chalfie expressed the gene in bacteria in 1994. It worked!

Question 19

Fluorescent proteins - transforming living cells and organisms

Answer 19

Fluorecent proteins have been used to transform bacteria, fungi, Dictyostelium, C. elegans, plants, Drosophila, and mammals

Question 20

What is Transmission electron microscopy?

Answer 20

For making high magnification views of very thin sections of fixed biological tissues