Basic techniques to study cells (Dr. Boucrot)

Question 1

What is a centrifuge ?

Answer 1

A centrifuge is an apparatus that rotates at high speed and by centrifugal force (an outward force on a body rotating about an axis) separates substances of different densities.

Question 2

What are the two main types of centrifugation techniques ?

Answer 2

The two main types of centrifugation techniques are differential and density gradient centrifugation.

Question 3

What is density gradient centrifugation ?

Answer 3

Density radiant centrifugation is a procedure for separating particles such as viruses, ribosomes or molecules like DNA in which the sample is placed on a preformed gradient such as sucrose or cesium chloride. Upon centrifugation, either by rate zonal or equilibrium procedures, the macromolecules are ‘banded’ in the gradient and can be collected as a pure fraction

Question 4

What is differential centrifugation ?

Answer 4

Differential centrifugation consists of successive centrifugation steps with increasing centrifugation forces and durations, generally aimed at isolating smaller from larger objects. Larger particles, assigned to be removed in the first centrifugation steps, sediment faster and leave most of the smaller particles in the supernatant. The supernatant is be centrifuged in subsequent steps.

Question 5

What is the advantage of density gradient centrifugation ?

Answer 5

The advantage of density gradient centrifugation is that faster sedimenting particles cannot be contaminated by the slow particles as occurs in differential centrifugation.

Question 6

What is the weakness of density gradient centrifugation ?

Answer 6

The weakness of density gradient centrifugation is that it limits the sample size (typically by 10%).

Question 7

What are the most dense organelles of the cell ?

Answer 7

The most dense organelle of the cell is the nucleus, followed by the peroxisome (1.23g/mL), the lysosome (1.18g/mL) and the mitochondrion (1.12g/mL).

Question 8

Why were Sir John Carew Eccles, Alan Lloyd Hodgkin and Andrew Fielding Huxley jointly awarded The Nobel Prize in Physiology or Medicine in 1963 ?

Answer 8

“[… ] for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane”. Differences in membrane potential during the action potential could be measured using the voltage clamp technique.

Question 9

What is voltage clamp ?

Answer 9

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level.

Question 10

Why were Erwin Neher and Bert Sakmann jointly awarded The Nobel Prize in Physiology or Medicine in 1991 ?

Answer 10

“[…] for their discoveries concerning the function of single ion channels in cells.” This was largely done by using the patch-clamp technique.

Question 11

What is patch clamp ?

Answer 11

Patch clamp is a technique in electrophysiology allowing the measurement of current flowing through single ion channels. The procedure involves pressing a glass micropipette against a cell in order to isolate a small “patch” of membrane that contains one or more ion channels.

Question 12

Where are there more K+ ions ?

Answer 12

Inside the cell (about 30 times more).

Question 13

Where are there more Na+ ions ?

Answer 13

Outside the cell (about 15 times more).

Question 14

How is charged distributed across the plasma membrane of nerve cells ?

Answer 14

The inside of the neuron is usually negative (-65mV) with respect to the outside.

Question 15

What is the smallest distance that the naked human eye can see ?

Answer 15

About 0.5mm.

Question 16

How big is an erythrocyte ?

Answer 16

About 10E-5m.

Question 17

How big is a C-C bond.

Answer 17

About 0.1nm.

Question 18

What is flow cytometry ?

Answer 18

Flow cytometry is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.

Question 19

What is cytometry used for ?

Answer 19

Cytometry is used to describe the characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology (shape and structure), cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm.

Question 20

What is Fluorescence-Activated Cell Sorting (FACS) ?

Answer 20

Fluorescence-Activated Cell Sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

Question 21

Why were Osamu Shimomura, Martin Chalfie and Roger Y. Tsien jointly awarded The Nobel Prize in Chemistry in 2008 ?

Answer 21

“[…] for the discovery and development of the green fluorescent protein, GFP”. The GFP was isolated and can now be injected in organisms to label different cells. By subtle mutations, the GFP proteins can reflect light in different colors .

Question 22

What is a fluorophore ?

Answer 22

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.

Question 23

Why were Ernst Ruska, Gerd Binnig and Heinrich Rohrer jointly awarded the nobel prize in physics in 1986 ?

Answer 23

The Nobel Prize in Physics 1986 was divided, one half awarded to Ernst Ruska “for his fundamental work in electron optics, and for the design of the first electron microscope”, the other half jointly to Gerd Binnig and Heinrich Rohrer “for their design of the scanning tunneling microscope”.

Question 24

What is Transmission Electoral Microscopy (TEM) ?

Answer 24

TEM is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through it.

Question 25

What is Scanning Electron Microscopy (SEM) ?

Answer 25

SEM is a technique that uses a focused beam of high-energy electrons to generate a variety of signals at the surface of solid specimens. The signals that derive from electron-sample interactions reveal information about the sample including external morphology (texture), chemical composition, and crystalline structure and orientation of materials making up the sample.

Question 26

What is the advantage of SEM ?

Answer 26

It is there-dimensional, and thus easier to interpret (contours more visible, etc.).

Question 27

What is the advantage of TEM ?

Answer 27

It reveals more details, and is thus more informative (reveals organelles, etc.).

Question 28

What are the pre-requisites to uses electron microscopy ?

Answer 28

The specimen must be electron dense. If this is not the case, we must make it so. To do this, we place the specimen on a mica surface, cover it with a metal replica and a fine layer of carbon film, and dissolve the bio-material with acid.

Question 29

What are the weaknesses of electron microscopy ?

Answer 29

By making the sample electron dense, we are effectively killing the cell and are thus unable to study any biological dynamics. Electron microscopy is also much more costly that conventional light microscopy. Moreover, sample observed by TEM need to be prepared appropriately and this process is lengthy and requires skill.

Question 30

What is the resolutions of light microscopy, TEM and SEM ?

Answer 30

200nm, 1nm and 10nm.

Question 31

Why were Eric Betzig, Stefan W. Hell and William E. Moerner jointly awarded the Nobel Prize in chemistry in 2014?

Answer 31

“[…] for the development of super-resolved fluorescence microscopy”.

Question 32

What is super-resolved microscopy ?

Answer 32

Super-resolved microscopy is an optic microscopic technique allowing images to be taken with a higher resolution than the diffraction limit (limit=200nm).