1.4.2. Good Clinical Practice (GCP)

According to the European regulations, all clinical trials must be performed in accordance with the rules of good clinical practice (GCP) that constitute a guarantee for patient protection and for test results.

That GCP guidelines of the International Conference on Harmonisation (ICH)^[1] have been followed since 1996 in Europe; they have also been adopted by the USA and Japan. They are a set of scientific and ethical quality requirements recognised internationally that must be fulfilled by the planning, execution, record and communication of clinical trials where human beings take part.
These rules must guarantee the rights, safety and well-being of trial subjects and the reliability of the results.

GCP arises from the need to reconciling the subject’s interests (who receives the treatment) and society’s interests in increasing its knowledge about drugs.

The main actors that participate in a clinical trial are the following:

▣      Promoter: legal entity or person interested in performing a clinical test and responsible for the same. It is usually a pharmaceutical multinational.

▣      Ethical committees: they belong to the centre where the research takes place.

▣      Regulatory agencies: they are usually QUANGOs with government oversight.
These two last participants must approve the protocol and supervise the experimental development. In addition, agencies evaluate the research results to authorise subsequent drug commercialisation.

▣      Researchers: carry out or manage the test research. They are normally physicians or health professionals that examine the compound response that is subject of study.

▣      Monitor: responsible for the direct follow-up of the clinical trial execution. Monitor is the intermediary between promoter and researcher and must guarantee the tracking of everything that occurs throughout the experiment.

▣      Patient: subjects who take part in the research and generally suffer from the disease of interest.

On occasion, some companies called CROs (contract research organisations) provide the trial management and monitoring.

After the II World War, the Nuremberg Code was promulgated to establish clinical research principles in human beings, which are as follows:

▣      Autonomy: respect the principle for subjects that they are treated as autonomous people to decide freely if they want to take part in the clinical test.
The informed consent^[2] signed by the patient is part of the fulfilment of this principle.

▣      Well-being: the physician is obligated to do good and to attend to the welfare of the patients, as well as their highest benefit.

▣      Non-malefience: to avoid harmful clinical tests for patients.

▣      Justice: obtained positive results must be shared with the people who suffer from the malady which is the focus of the trial (whether knowledge or new medication). This principle would not be fulfilled, for example, if a country from the third world, where the trial takes place, would not benefit from the treatment because of its high cost.

Phase IV clinical trials

Once a drug has been introduced to the market, studies continue to increase the knowledge about its safety and effectiveness, identify new risks and non-detected adverse response in previous research.
The group of controlled and randomised trials and studies that are performed at this given point is called phase IV of clinical trials.

The most common epidemiological (observational) studies after the drug authorisation are case-control and cohort studies.

Finally, the Yellow Card Scheme is the system for collecting suspected adverse reactions to drugs. This system is used especially when a medicine has just been marketed and it is mandatory for physicians (and current patients can also fill it out).

These cards consist of several sections among which we find the following:

▣      Patient details: first name or initials, age, sex, weight, height…

▣      Data about the suspected medicine causing adverse reactions.

▣      Description of those reactions.

▣      Reporter/clinician details: name, professional address, specialty…

---

[1] It matches the different regional requirements for the register of pharmaceutical products.
[2] The subject who participates in the clinical trial is informed by this document about the details, advantages and disadvantages of the test with an accessible language, being able to leave it freely at any time.

Sources: CEU Universidad San Pablo: Farmacología Básica, 2013.
              https://www.gov.uk/guidance/the-yellow-card-scheme-guidance-for-healthcare-professionals
              https://yellowcard.mhra.gov.uk/the-yellow-card-scheme/

              http://www.ich.org/home.html

Read more

4.1.2. The endomembrane system. The cytoskeleton

The endomembrane system is a group of organelles and membranes that work together to modify, pack and transport lipids and proteins. This system includes the nuclear envelope, lysosomes and vesicles that we have already mentioned in the previous post: “Prokaryotic and eukaryotic cells. Main components” , the Golgi apparatus and the endoplasmic reticulum.

The endoplasmic reticulum (ER)

The endoplasmic reticulum is made up of a set of membranous sacs and interconnected tubules that function colectively to modify proteins and synthesise lipids. These two functions are carried out in two different areas: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER).

Rough endoplasmic reticulum (RER)

The rough endoplasmic reticulum receives this name because of the large number of ribosomes that are stuck to its cytoplasmic surface.
The ribosomes transfer the proteins that they synthesise to the lumen of the RER where they undergo modifications and then they are incorporated into the plasma membrane or they are secreted outside the cell through vesicles that bud from the RER membrane.
Enzymes, hormones and even some phospholipids generated by the RER undergo these processes to become part of the cell membranes.
The rough endoplasmic reticulum is mainly found in cells that secrete a large amount of proteins, like hepatic cells.

Smooth endoplasmic reticulum (SER)

The smooth endoplasmic reticulum is next to the RER but, unlike this one, it has few or no ribosomes stuck to its surface.
Among its functions we can find the synthesis of lipids, proteins, steroid hormones, detoxification of poisons and drugs and the storage of calcium ions.
In the muscle cells, the smooth endoplasmic reticulum is called the sarcoplasmic reticulum (SR) and the SR is in charge of accumulating the necessary calcium ions for muscle contraction.

The Golgi apparatus

Before reaching their destination, the proteins and lipids that are carried by the vesicles of the ER must be tagged, classified, packed and distributed. The Golgi apparatus, which consists of a series of flattened membranes, performs these functions.
The receiving side of the Golgi apparatus is called the cis face, whereas the opposite side is called the trans face.

As lipids and proteins travel through the Golgi apparatus they undergo a set of modifications that enable classification; the most common modification is the addition of short chains of sugar molecules.

Afterwards, they are tagged with phosphate groups or other small molecules so that they can be delivered to their appropiate destinations.
Finally, they are packed into secretory vesicles that emerge from the trans face of the Golgi apparatus. Some of these lipids and proteins are placed in other parts of the cell, whereas others are fuse with the plasma membrane and they release their content outside the cell.

Immune system cells that secrete antibodies are characterised by having a large amount of Golgi.

Lysosomes

In addition to their digestive role and recycling of organelles, lysosomes are an important component of the endomembrane system too.
Lysosomes use their hydrolytic enzymes to destroy those pathogens that penetrate into the cell.
Lysosomes are used by a special type of white cell called macrophage. These cells via phagocytosis or endocytosis invaginate (fold) their plasma membranes to surround and enclose the pathogen. Later, these pathogens are destroyed under the action of the hydrolitic enzymes of lysosomes.

THE CYTOSKELETON

The cytoskeleton is the group of protein fibres that maintain the cell shape, attach the organelles in their appropiate positions, enable the vesicles to move in the cell and allow the cells of multicellular organisms to move.

Microfilaments

Of the three kinds of protein fibres of the cytoskeleton, microfilaments are the narrowest with a diameter around 7 nm.
They are two intertwined fibres of the actin protein, consequently they are also known as actin filaments.
They participate in processes that require movement such as the cell division in animal cells. They provide rigidity and shape to the cell.

Intermediate filaments

They purely have a structural function; they bear strain and fix the nucleus and other organelles in the necessary locations.
Their diameter is between 8 and 10 nm and they are made of several twisted protein fibres.
Within this category, keratin filaments are the best known; they strengthen nails, skin epidermis and hair.

Microtubules

Among the most important roles performed by microtubules, we find the movement of the replicated chromosomes to opposite ends of the cell during the cell division. They also provide a path for vesicles to move inside the cell and help the cell to maintain compression.
They are the widest components of the cytoskeleton with a diameter of around 25 nm.

Flagella and cilia

Flagella are moving appendages that extend from the cell membrane making possible the movement of those cells that have them (e.g., sperm).

On the contrary, cilia are short and are found widespread along the surface of the plasma membrane.
Like flagella, they enable the movement of cells (paramecia) or substances over the cell surface, like for example the cilia of the Fallopian tubes that move the ovule towards the uterus.

CELL CONNECTIONS

As you can guess, if cells have to work together they must communicate with each other. Let’s see what methods they use to achieve it.

Extracellular matrix of animal cells

The major role of the extracellular matrix is to hold cells together to form a tissue and to allow cell communication within that tissue.
The extracellular matrix is primarily made up of a sort of protein called collagen, intertwined with other types of proteins that contain carbohydrates (proteoglycans).

Overall, the cell communication is performed as follows:
Cells have quite a few receptors on the surface of their cell membranes.
When a molecule within the matrix joins the receptor, it modifies the molecular structure of the receptor. The receptor, in turn, changes the arrangement of the microfilaments located within the membrane. These changes produce chemical signals that reach the nucleus and they activate and deactivate the transcription of specific sections of DNA, which influences the creation of the related proteins.

Cells can also communicate through direct contact via intercellular joints, which are formed by diverse kinds of proteins. In animal cells, there are three categories of these joints:

▣      Tight junctions: they seal the plasma membranes of adjacent cells creating a waterproof barrier between them. Their main components are occludin and claudin proteins. These tight junctions are found, for instance, linking the epithelial cells of the urinary bladder.

▣      Desmosomes: they work like spot welds between the epithelial cells of organs and tissues that undergo contraction such as the skin, the heart and muscle cells. Desmosomes are composed of short proteins named cadherins.

▣      Gap junctions: they act like pores and channels that enable the transport of ions and nutrients. They play a significant role in the cardiac muscle, where they allow the movement of the electrical signal that contracts this muscle.
They are made up of a group of six proteins called connexins, which are arranged in the cell membrane in a donut-like configuration known as connexon.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
              http://www.oncoursesystems.com/images/user/9341/10845583/rough%20er.bmp
              http://apocketmerlin.tumblr.com/post/14923100822/a-summary-of-the-functions-of-major-eukaryotic
              http://iesicaria.xtec.cat/~SBG/BiologiaCurtis/Seccion%201/1%20-%20Capitulo%205.htm
              https://ohhaitrish.wordpress.com/2012/02/12/unit-one-compilation/

Read more

4.1.1. Prokaryotic and eukaryotic cells. Main components

We have already studied the different biological macromolecules, which comprise the main components of cell structure. So, we are now in position to deal with a new topic within this section: cell biology.

In the same way that bricks are the basic building blocks of a wall, cells are the building blocks of living organisms.

There is a huge range of cells, each one specialised in a particular function: growth, development and daily maintenance of the organism, but all of them share some fundamental features.
Among the diverse types of cells we find, for example, immune system cells that protect us against bacterial infections, blood cells that carry oxygen and nutrients throughout our body or epithelial cells that protect the body surface and cover organs and body cavities.

Cells are considered the smallest unit of a living being. When several cells of the same class interconnect with each other sharing a common function they become a tissue. Several tissues form an organ and several organs make up a system (nervous, circulatory, digestive systems…). Lastly, the union of several systems working together and in concert comprises an organism like the human being.

Cells are classified into two major groups: prokaryots and eukaryots. But all of them have four basic components: the cell membrane that protects it from outside, the cytoplasm that is composed of the cytosol with a jelly consistency and floating on it other cell components, the DNA that contains the genetic information of the cell and the ribosomes that synthesise proteins..

In prokaryotic cells, the organism (most of the time unicellular) lacks a nucleus with what its DNA located in the cell centre, or ‘nucleoid’. Bacteria and archaea^[1] belong to this category.

On the contrary, protist^[2], fungi, plants and animals cells are eukaryotic cells, which differ from the prokaryotic cells by having a nucleus that encloses its genetic material (DNA) surrounded by a membrane, a large variety of organelles^[3] limited by membranes and several rod-shaped chromosomes.

Due to the purpose of this blog, I will only focus on the components and functions of eukaryotic cells and particularly on animal cells.

The plasma membrane

We start the study of the diverse cell components with the plasma membrane or cell membrane, which separates the interior content of the cell from the external environment.

It consists of a lipid bilayer with many embedded proteins in it, which controls the movement of water, ions, oxygen, organic molecules and waste disposal (carbon dioxide and ammonia) between the inside and outside of a cell.

Microvilli located on the wall of the small intestine are an example of plasma membranes specialised in the absorption task. They are in charge of absorbing the nutrients from the digested food.
In the case of celiacs, the consumption of gluten (a cereal protein) causes an autoimmune respond that attacks these cells causing malnutrition, abdominal pain and diarrhoea.

The cytoplasm

The cytoplasm is the region between the plasma membrane and the nuclear membrane.
It has a jelly-like and semisolid consistency called cytosol, on which the organelles are suspended.

Also the cytoskeleton and a number of biochemical substances, such as amino acids, nucleic and fatty acids, polysaccharides, simple sugars and sodium, potassium and calcium ions form part of the cytoplasm.
In the cytoplasm a large range of metabolic reactions, like the synthesis of proteins, take place.

The nucleus

The nucleus is the most significant organelle in the cell. It contains the DNA and leads the synthesis of proteins and ribosomes.

The nuclear envelope

The nuclear envelope is a double-membrane structure, in which both the inner and the outer membranes are formed by phospholipid bilayers (to revisit what a phospholipid bilayer see the post: “Lipids”).
This structure has a set of pores on its surface to facilitate the movement of ions, molecules and RNA between the nucleoplasm and the cytoplasm.
The nucleoplasm is a semisolid fluid ubicated inside the nuclear membrane where the chromatin and the nucleolus are.

Chromosomes and chromatin

In eukaryotic cells, chromosomes are structures inside the nucleus made up of DNA and therefore, the hereditary genetic material.
Each eukaryotic species has a particular number of chromosomes in its cell nuclei, which in the case of human beings is 46 (23 pairs).
Chromosomes are only distinguishable when the cell is about to initiate the cell division process. When the cell is in the other phases of the cell cycle, the chromosomes join certain proteins (histones) forming a complex known as chromatin, which looks like a tangled skein.
Chromatin characterises the substance that forms the chromosomes both in decondensed and condensed states.

The nucleolus

The nucleolus is the part of the nucleus that aggregates the ribosomal RNA (rRNA) with specific proteins to assemble the ribosomal subunits. These subunits are then transported to the cytoplasm where they are put together. This is the way in which the nucleus performs one of its principal functions, the synthesis of ribosomes.

Ribosomes

Ribosomes are the organelles responsible for protein synthesis. They are made up of two subunits: large and small subunits.
They can be found in groups, or individually next to the cytoplasmic side of the plasma membrane, the endoplasmic reticulum or stuck to the outer membrane of the nuclear membrane.

Ribosomes receive instructions from the nucleus to produce proteins by means of DNA, this DNA is transcripted to mRNA and this one gets to the ribosome. Once there, the code provided by the nitrogenous bases of the mRNA is translated in a set of amino acids with a specific order, giving rise to the required protein.

Ribosomes are abundant in those cells that synthesise large amounts of protein, like pancreatic cells, which produce multiple digestive enzymes.

Mitochondria

Mitochondria are usually known as the energetic centre of cells, they take charge the production of the most crucial energetic molecule for cells: ATP (adenosine triphosphate). This molecule is generated using glucose and other nutrients during cellular respiration. During this process, mitochondria use oxygen and produce carbon dioxide as waste, which is expelled from the organism when we exhale.

Mitochondria are oval-shaped with a double membrane (phospholipid bilayer with embedded proteins) and have their own ribosomes and DNA.
Their inner layer has a set of folds called mitochondrial cristae, surrounded by the mitochondrial matrix. Both develop different roles in the cellular respiration.

Peroxisomes

Peroxisomes are small, spherical organelles surrounded by a monolayer membrane.
They carry out oxidation reactions that break down amino acids and fatty acids, as well detoxifying poisons that enter our body.
For instance, the peroxisomes of hepatic cells must detoxify the alcohol we consume.

Vesicles and vacuoles

These organelles are sac-shaped and their functions are transport and storage. The main difference between them is their size (vacuoles are larger) and the fact that vesicle membranes can fuse with the plasma membrane or with membranes of other components inside the cell.

The centrosome

The centrosome is a microtubule-organising centre (MTOC) located near animal cell nuclei. Centrosomes are made up of two structures that are perpendicular to each other named centrioles. These centrioles are cylinders of nine triplets of microtubules each.

The centrosome replicates before starting cell division and the centrioles pull the duplicated chromosomes to the opposite poles of the cell during its division stage.

Lysosomes

Lysosomes are dumps of cells. Their pH is more acidic than cytoplasm´s, which favours the fact that their enzymes are more active and assist the degradation of nucleic acids, lipids, polysaccharides and even the recycling of organelles that are no longer necessary for the cell by enclosing and digesting them.

---

[1] Unicellular microorganisms similar to bacteria.
[2] Unicellular organisms that form colonies at times.
[3] Small organs specialised in cell functions, in a similar way to our body organs.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
              http://michelleburden.weebly.com/cell-types-prokaryotes-v-eukaryotes-plant-v-animal.html
              http://cosbiology.pbworks.com/w/page/11556247/Lesson%204-02%20Prokaryotes%20and%20Eukaryotes
              http://ernsstev.com/bilayer-pattern-in-cell-membrane/
              http://www.biologyexams4u.com/2012/11/difference-between-chromatin-and.html#.Vu1BANI7uSp
              http://jptregularbio.pbworks.com/w/page/79985510/Cells
              https://oggisioggino.wordpress.com/2013/11/01/las-celulas-procariotas-y-eucariotas/
              http://www.studyrankers.com/2015/06/cell-the-unit-of-life-class-11th-ncert-solutions.html

Read more

1.4.1. Clinical research: Overview

Those compounds that have passed the preclinical phase are tested in human beings (clinical research).
This kind of research has significant associated issues of a scientific, ethical, social and economic nature.

In all developed societies it is mandatory to prove the efficacy and safety of drugs before commercialising them, a task that is fulfilled by clinical trials.

Clinical trials are defined as research studies in human beings to determine or confirm clinical, pharmacological, pharmacodynamic effects and/or detect adverse reactions and/or study the absorption, distribution, metabolism and excretion of a drug in order to establish its safety and efficacy.

Clinical tests are based on documentation, both national and international regulation and a series of ethical codes, declarations and international conventions.

Over the last fifty years hundreds of thousands of compounds have been produced, but only 10% of them have passed clinical evaluation, and even within this group some of them were withdrawn after being put on the market due to safety and efficacy reevaluations.

The methodology of clinical research can be carried out via one of the following procedures:

▣      Experimental: In experimental tests, researchers design the study, select the patients, establish the treatments and variables to study.

▣      Observational: Researchers are mere observers, without altering or intervening the habitual clinical practice.

Within the experimental tests, we find:

▣      Randomised trials. In these trials, patients are allocated to one treatment or another (often a placebo) in a random way. This is the procedure demanded by regulatory agencies to avoid bias in data interpretation before giving the authorisation to market the drug.

▣      Non-randomised trials. Here, patients are not allocated randomly.

Among the observational studies to develop the complete efficacy and safety profile of a drug after its commercialisation, we have:

▣      Case-control studies. Participants are chosen according to they have or not a certain disease and it is investigated if they were exposed or not to a factor of interest. The exposed group and the control group are compared.

▣      Cohort studies. The frequency of a sickness is compared between two populations, one exposed to a risk factor and the other population are not.

▣      Cross-sectional studies. They examine the relationship between an illness and a set of variables in a certain population at a given point in time.

Clinical trials chronologically go through three phases until achieving marketing authorisation for the drug. Once the drug is commercialised, it begins its phase IV, which lasts the whole lifespan of the drug. In Phase IV the drug can be removed from the market if undesired effects, not detected in the three previous stages, are identified.
These new side effects can be discovered when the drug is distributed among thousands of patients.

The measured efficacy in these real conditions is called effectiveness to distinguish it from the efficacy measured prior to drug distribution on the market.

Sources: CEU Universidad San Pablo: Farmacología Básica, 2013.
              http://cmidd.northwestern.edu/about/drug-discovery-and-development-process/
              http://indacea.org/desarrollo-de-medicamentos-1/

Read more

3.5. Nucleic acids

DNA and RNA are the most important biological macromolecules for the continuity of life.
DNA molecules contain the genetic material of all living organisms, from bacteria to multicellular mammals.
In prokaryotic cells DNA is not enclosed inside a membranous envelope, but is in eukaryotic cells where it is located inside the nucleus and organelles.

In eukaryotic cells, DNA along with some proteins, called histones, form chromatin (the material from which eukaryotic chromosomes are made), which can contain tens of thousands of genes.
DNA is in charge of controlling the activity of cells by activating and deactivating genes.

The other kind of nucleic acid, RNA, primarily conducts protein synthesis and its regulation through the action of three different sorts of RNA (rRNA or ribosomal RNA, tRNA or transfer RNA and miRNA or microRNA). The fourth type of RNA (mRNA or messenger RNA) acts as communication intermediary between DNA and the rest of the cell.

Nucleotides are nucleic acid monomers, which are combined to generate their polynucleotides (DNA and RNA). Each nucleotide is composed of three components: one nitrogenous base attached to a pentose sugar molecule, which, in turn, is attached to a phosphate group.

Nitrogenous bases are organic molecules made up of carbon (C) and nitrogen (N) that are divided into five kinds: adenine (A) and guanine (G) are known as purines, they have two fused rings of C-N. Cytosine (C), thymine (T) and uracil (U) are known as pyrimidines, they only have a fused ring of C-N.
Both purines and pyrimidines have different functional groups attached to these rings.

The DNA molecule comprises A, T, G and C, while the RNA molecule comprises A, U, G and C, therefore, tymine is substituted by uracil.
Another difference between DNA and RNA is that DNA sugar molecule is a deoxyribose (they have a hydrogen atom attached to the second carbon atom of the molecule) whereas RNA sugar molecule is a ribose (they have a hydroxyl group attached to that second carbon atom instead).

Carbon atoms of the sugar molecule are numbered as 1´, 2´, 3´, 4´ and 5´. The phosphate residue joins the hydroxyl group of the 5´carbon and the 3´ carbon of the sugar molecule of the next nucleotide forming a phosphodiester linkage between them.

DNA double-helix structure

In the DNA double-helix structure, the sugar molecule and phosphate group are located outside this structure, whereas the nitrogenous bases lie in the interior like the rungs of a ladder linked by hydrogen bonds, in such a way that A can only pair with T and G can only pair with C. This rule is known as the rule of complementary base pairing.
Each of these pairs is separated from the next one by 0.34 nm; both strands of the double-helix run in opposite directions in such a way that the 5' carbon of one strand faces the 3' carbon of the complementary strand.

During DNA replication, each of these strands are separated and they are used to synthesise a new complementary strand, in such a way that each daughter DNA double helix contains a new synthesised strand and one parental DNA strand.

RNA

Normally, RNA comprises a single ribonucleotide strand held together by phosphodiester bonds. These ribonucleotides contain, in turn, a phosphate group, a ribose molecule (pentose sugar) and one of the four nitrogenous bases (A, U, C and G).

The DNA that controls the activity of the cell uses the mRNA (messenger RNA) to carry its message
Thus, if a cell requires a certain protein, the gene for this product is activated and the nucleus synthesises the mRNA. This mRNA has a complementary sequence to the coding sequence of the DNA has been copied from.
The mRNA crosses the nuclear membrane, gets to the cytoplasm and there it interacts with the ribosomes and other cellular machinery.
To produce the required protein, the mRNA is read in groups of three bases called codons. Each codon codifies a single amino acid.

The second kind of RNA, rRNA, is responsible for the suitable alignment between the mRNA and ribosomes. It also has an enzymatic activity and catalyses the formation of peptide bonds between two aligned amino acids.

The tRNA is in charge of transporting the correct amino acid to the site where the protein synthesis takes place. The base pairing between the tRNA and the mRNA allows for the appropriate amino acid to be inserted in the polypeptide chain.

Lastly, the major function of the microRNA (the smallest of all types of RNA) is the regulation of gene expression by interfering with the expression of certain messages of the mRNA.

Hence, in an organism the information goes from DNA to RNA and from this one to proteins. During the first stage (transcription), the DNA dictates the structure of mRNA and during the second (translation), RNA rules the structure of a specific protein.
This process is known as the central dogma of molecular biology, which is valid for all living organisms except for some viral infections.

Sources: OpenStax College, Biology. OpenStax College. 30 May 2013.
              https://www.windowssearch-exp.com/images/search?q=Antiparallel+DNA+Replication&view=detailv2&&id=CE5B36CC30
              3DB6E9F7C902454DF516328A0882B8&selectedIndex=1&ccid=uEIdkd5u&simid=608029733518446573&thid=OIP.Mb84
              21d91de6e5ee9db88b338c5b77e9aH0&ajaxhist=0
              http://www.scienceprofonline.com/genetics/ribonucleic-acid-rna-structure-and-function.html
              http://guia.bio.br/tag/purinas/
              http://www.discoveryandinnovation.com/BIOL202/notes/lecture12.html
              http://www.ib.bioninja.com.au/standard-level/topic-3-chemicals-of-life/35-transcription-and-transl.html
              http://thelessonlocker.com/kvhs/biology/biology.html

Read more

2.2. Atomic force microscope (AFM)

In the post titled “Top-down and bottom-up approach” I mentioned that, in order to examine and handle objects at the nanoscale it is necessary to develop devices that enable observation of samples at the atomic scale. These instruments are called scanning probe microscopes (SPMs), from which 3D images are obtained at a very high resolution without damaging the analysed sample (an improvement on electron microscopes (TEMs)).

The scanning probe microscopes are divided, basically, into two types: scanning tunneling microscope (STMs), which we studied in the previous entry within this section, and the atomic force microscopes (AFMs), which we address below.

Atomic force microscopes are based on the following operating principle:
The sample topography is scanned by a microcantilever or strip around a few hundreds of microns (micrometres) long, built in silicon or silicon nitride, whose end has an extremely sharp tip and a curvature radius on the order of nanometers.

When this tip approximates the sample surface, the forces between them produce the cantilever deflection according to Hooke’s law^[1].
Depending on the type of tip-sample interaction (whether there is contact or not), the forces measured by AFMs can be: electrostatic, magnetic, capillary forces or Van der Waals interactions...
The deflection that the cantilever experiences is measured by a laser beam focused on the upper surface of this cantilever and reflected in an array of photodiodes that register the laser spot shift.

In contrast with STMs, AFMs can be used in the study of non-conductive samples, and they can perform two types of measurement depending on the application: image (which, in turn, it is divided into contact mode and dynamic mode) and force.

Contact mode

In this operating mode, the bending of the cantilever is kept constant during the sample scan, hence this cantilever is correspondingly moved up and down to keep invariable the pressure force of the tip on the sample. This up and down movement is registered and a specimen image is obtained.
A relatively fast scan speed and the opportunity to scan rough surfaces are its main advantages. Its principal drawback is that it may damage biological samples (soft and delicate), therefore these samples must be strongly adhered to the surface.

Dynamic mode

In the dynamic mode, the microcantilever tip does not come into contact with the sample surface. Instead the strip oscillates at its resonant frequency or just above by a piezoelectric actuator^[2] that determines the tip height over the sample.

The tip-sample interaction modifies the resonant frequency and amplitude. Depending on which of these two properties keeps constant during the surface scan, we have:

▣      Non-contact mode where the resonant frequency is stable. This mode is specially indicated for the study of soft biological samples and thin organic films. Because there is no contact, the samples can be subjected to countless analyses without being damaged.
Among its disadvantages, we find its low image resolution and samples must be examined in ultra-high vacuum environments, to avoid the presence of pollutants on the specimen surface that could interfere with imaging.

▣      Tapping mode where the resonant amplitude does not vary. This method provides high-resolution images of those samples that are susceptible to be easily damaged or are weakly adhered to the analysis surface. The major disadvantage is that the scanning has to be done at a slower speed.

Force mode

In the force operating mode, the tip oscillates vertically while the cantilever deflection is registered.
This method has as its main applications the examination of specific interactions between molecules (antigen-antibody, the complementary strands of DNA), structural interactions (protein folding) or the study of polymer elasticity.

---

[1] This law states that the lenghtening of materials is directly proportional to the applied force on the same.
[2] Mechanical devices whose function is provide force to move another mechanical device.

Sources: http://www.dme-spm.com/funktion.html
              https://es.wikipedia.org/wiki/Microscopio_de_fuerza_at%C3%B3mica
              http://www.phy.mtu.edu/nue/images/atomicforce/Nanoscope2.jpg
              http://slideplayer.com/slide/6856881/  
              http://www.biozentrum.unibas.ch/research/groups-platforms/overview/unit/lim/

Read more