Sterilization (microbiology)

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Sterilization_(microbiology) 

Sterilization refers to any process that removes, kills, or deactivates all forms of life (particularly microorganisms such as fungi, bacteria, spores, and unicellular eukaryotic organisms) and other biological agents such as prions present in or on a specific surface, object, or fluid. Sterilization can be achieved through various means, including heat, chemicals, irradiation, high pressure, and filtration. Sterilization is distinct from disinfection, sanitization, and pasteurization, in that those methods reduce rather than eliminate all forms of life and biological agents present. After sterilization, an object is referred to as being sterile or aseptic.

Organisms growing on an agar plate

Applications

Foods

One of the first steps toward modernized sterilization was made by Nicolas Appert, who discovered that application of heat over a suitable period slowed the decay of foods and various liquids, preserving them for safe consumption for a longer time than was typical. Canning of foods is an extension of the same principle and has helped to reduce food borne illness ("food poisoning"). Other methods of sterilizing foods include food irradiation and high pressure (pascalization).

Medicine and surgery

Joseph Lister, a pioneer of antiseptic surgery.

 

Apparatus to sterilize surgical instruments (1914–1918)

In general, surgical instruments and medications that enter an already aseptic part of the body (such as the bloodstream, or penetrating the skin) must be sterile. Examples of such instruments include scalpels, hypodermic needles, and artificial pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.

Preparation of injectable medications and intravenous solutions for fluid replacement therapy requires not only sterility but also well-designed containers to prevent entry of adventitious agents after initial product sterilization.

Most medical and surgical devices used in healthcare facilities are made of materials that are able to go under steam sterilization. However, since 1950, there has been an increase in medical devices and instruments made of materials (e.g., plastics) that require low-temperature sterilization. Ethylene oxide gas has been used since the 1950s for heat- and moisture-sensitive medical devices. Within the past 15 years, a number of new, low-temperature sterilization systems (e.g., vaporized hydrogen peroxide, peracetic acid immersion, ozone) have been developed and are being used to sterilize medical devices.

Spacecraft

There are strict international rules to protect the contamination of Solar System bodies from biological material from Earth. Standards vary depending on both the type of mission and its destination; the more likely a planet is considered to be habitable, the stricter the requirements are.

Many components of instruments used on spacecraft cannot withstand very high temperatures, so techniques not requiring excessive temperatures are used as tolerated, including heating to at least 120 °C (248 °F), chemical sterilization, oxidization, ultraviolet, and irradiation.

Quantification

The aim of sterilization is the reduction of initially present microorganisms or other potential pathogens. The degree of sterilization is commonly expressed by multiples of the decimal reduction time, or D-value, denoting the time needed to reduce the initial number ${\displaystyle N_{0}}$ to one tenth (${\displaystyle 10^{-1}}$) of its original value. Then the number of microorganisms ${\displaystyle N}$ after sterilization time ${\displaystyle t}$ is given by:

${\displaystyle {\frac {N}{N_{0}}}=10^{\left(-{\frac {t}{D}}\right)}}$.

The D-value is a function of sterilization conditions and varies with the type of microorganism, temperature, water activity, pH etc.. For steam sterilization (see below) typically the temperature, in degrees Celsius, is given as an index.

Theoretically, the likelihood of the survival of an individual microorganism is never zero. To compensate for this, the overkill method is often used. Using the overkill method, sterilization is performed by sterilizing for longer than is required to kill the bioburden present on or in the item being sterilized. This provides a sterility assurance level (SAL) equal to the probability of a non-sterile unit.

For high-risk applications, such as medical devices and injections, a sterility assurance level of at least 10⁻⁶ is required by the United States Food and Drug Administration (FDA).

Heat

Steam

See also: Moist heat sterilization

Steam sterilization, also known as moist heat sterilization, uses heated saturated steam under pressure to inactivate or kill microorganisms via denaturation of macromolecules, primarily proteins. This method is a faster process than dry heat sterilization. Steam sterilization is performed using an autoclave, sometimes called a converter or steam sterilizer. The article is placed in the autoclave chamber, which is then sealed and heated using pressurized steam to a temperature set point for a defined period of time. Steam sterilization cycles can be categorized as either pre-vacuum or gravity displacement. Gravity displacement cycles rely on the lower density of the injected steam to force cooler, denser air out of the chamber drain. In comparison, pre-vacuum cycles draw a vacuum in the chamber to remove cool dry air prior to injecting saturated steam, resulting in faster heating and shorter cycle times. Typical steam sterilization cycles are between 3 and 30 minutes at 121–134 °C (250–273 °F) at 100 kPa (15 psi), but adjustments may be made depending on the bioburden of the article being sterilized, its resistance (D-value) to steam sterilization, the article's heat tolerance, and the required sterility assurance level. Following the completion of a cycle, liquids in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is released. This may be achieved by gradually depressurizing the sterilization chamber and allowing liquids to evaporate under a negative pressure, while cooling the contents.

Proper autoclave treatment will inactivate all resistant bacterial spores in addition to fungi, bacteria, and viruses, but is not expected to eliminate all prions, which vary in their resistance. For prion elimination, various recommendations state 121–132 °C (250–270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The 263K scrapie prion is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, and strains of Creutzfeldt-Jakob disease (CKD) and bovine spongiform encephalopathy (BSE) are more resistant. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134–138 °C (273–280 °F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity.

Most autoclaves have meters and charts that record or display information, particularly temperature and pressure as a function of time. The information is checked to ensure that the conditions required for sterilization have been met. Indicator tape is often placed on the packages of products prior to autoclaving, and some packaging incorporates indicators. The indicator changes color when exposed to steam, providing a visual confirmation.

Biological indicators can also be used to independently confirm autoclave performance. Simple biological indicator devices are commercially available, based on microbial spores. Most contain spores of the heat-resistant microbe Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), which is extremely resistant to steam sterilization. Biological indicators may take the form of glass vials of spores and liquid media, or as spores on strips of paper inside glassine envelopes. These indicators are placed in locations where it is difficult for steam to reach to verify that steam is penetrating there.

For autoclaving, cleaning is critical. Extraneous biological matter or grime may shield organisms from steam penetration. Proper cleaning can be achieved through physical scrubbing, sonication, ultrasound, or pulsed air.

Pressure cooking and canning is analogous to autoclaving, and when performed correctly renders food sterile.

To sterilize waste materials that are chiefly composed of liquid, a purpose-built effluent decontamination system can be utilized. These devices can function using a variety of sterilants, although using heat via steam is most common.

Dry

See also: Dry heat sterilization

 

Dry heat sterilizer

Dry heat was the first method of sterilization and is a longer process than moist heat sterilization. The destruction of microorganisms through the use of dry heat is a gradual phenomenon. With longer exposure to lethal temperatures, the number of killed microorganisms increases. Forced ventilation of hot air can be used to increase the rate at which heat is transferred to an organism and reduce the temperature and amount of time needed to achieve sterility. At higher temperatures, shorter exposure times are required to kill organisms. This can reduce heat-induced damage to food products.

The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects. Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam (e.g. it does not cause rusting of steel objects).

Flaming

Flaming is done to inoculation loops and straight-wires in microbiology labs for streaking. Leaving the loop in the flame of a Bunsen burner or alcohol burner until it glows red ensures that any infectious agent is inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating, infectious material may be sprayed from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave carbon or other residues on the object if the object is not heated enough. A variation on flaming is to dip the object in a 70% or more concentrated solution of ethanol, then briefly touch the object to a Bunsen burner flame. The ethanol will ignite and burn off rapidly, leaving less residue than a gas flame

Incineration

Incineration is a waste treatment process that involves the combustion of organic substances contained in waste materials. This method also burns any organism to ash. It is used to sterilize medical and other biohazardous waste before it is discarded with non-hazardous waste. Bacteria incinerators are mini furnaces that incinerate and kill off any microorganisms that may be on an inoculating loop or wire.

Tyndallization

Main article: Tyndallization

Named after John Tyndall, Tyndallization is an obsolete and lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, and then repeating the process a total of three to four times. The incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth, and will not sterilize non-nutritive substrates like water. Tyndallization is also ineffective against prions.

Glass bead sterilizers

Glass bead sterilizers work by heating glass beads to 250 °C (482 °F). Instruments are then quickly doused in these glass beads, which heat the object while physically scraping contaminants off their surface. Glass bead sterilizers were once a common sterilization method employed in dental offices as well as biological laboratories, but are not approved by the U.S. Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) to be used as a sterilizers since 1997. They are still popular in European and Israeli dental practices, although there are no current evidence-based guidelines for using this sterilizer.

Chemical sterilization

Chemiclav

Chemicals are also used for sterilization. Heating provides a reliable way to rid objects of all transmissible agents, but it is not always appropriate if it will damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics. In these situations chemicals, either in a gaseous or liquid form, can be used as sterilants. While the use of gas and liquid chemical sterilants avoids the problem of heat damage, users must ensure that the article to be sterilized is chemically compatible with the sterilant being used and that the sterilant is able to reach all surfaces that must be sterilized (typically cannot penetrate packaging). In addition, the use of chemical sterilants poses new challenges for workplace safety, as the properties that make chemicals effective sterilants usually make them harmful to humans. The procedure for removing sterilant residue from the sterilized materials varies depending on the chemical and process that is used.

Ethylene oxide

Ethylene oxide (EO, EtO) gas treatment is one of the common methods used to sterilize, pasteurize, or disinfect items because of its wide range of material compatibility. It is also used to process items that are sensitive to processing with other methods, such as radiation (gamma, electron beam, X-ray), heat (moist or dry), or other chemicals. Ethylene oxide treatment is the most common chemical sterilization method, used for approximately 70% of total sterilizations, and for over 50% of all disposable medical devices.

Ethylene oxide treatment is generally carried out between 30 and 60 °C (86 and 140 °F) with relative humidity above 30% and a gas concentration between 200 and 800 mg/l. Typically, the process lasts for several hours. Ethylene oxide is highly effective, as it penetrates all porous materials, and it can penetrate through some plastic materials and films. Ethylene oxide kills all known microorganisms, such as bacteria (including spores), viruses, and fungi (including yeasts and moulds), and is compatible with almost all materials even when repeatedly applied. It is flammable, toxic, and carcinogenic; however, only with a reported potential for some adverse health effects when not used in compliance with published requirements. Ethylene oxide sterilizers and processes require biological validation after sterilizer installation, significant repairs or process changes.

The traditional process consists of a preconditioning phase (in a separate room or cell), a processing phase (more commonly in a vacuum vessel and sometimes in a pressure rated vessel), and an aeration phase (in a separate room or cell) to remove EO residues and lower by-products such as ethylene chlorohydrin (EC or ECH) and, of lesser importance, ethylene glycol (EG). An alternative process, known as all-in-one processing, also exists for some products whereby all three phases are performed in the vacuum or pressure rated vessel. This latter option can facilitate faster overall processing time and residue dissipation.

The most common EO processing method is the gas chamber method. To benefit from economies of scale, EO has traditionally been delivered by filling a large chamber with a combination of gaseous EO either as pure EO, or with other gases used as diluents; diluents include chlorofluorocarbons (CFCs), hydrochlorofluorocarbons (HCFCs), and carbon dioxide.

Ethylene oxide is still widely used by medical device manufacturers. Since EO is explosive at concentrations above 3%, EO was traditionally supplied with an inert carrier gas, such as a CFC or HCFC. The use of CFCs or HCFCs as the carrier gas was banned because of concerns of ozone depletion. These halogenated hydrocarbons are being replaced by systems using 100% EO, because of regulations and the high cost of the blends. In hospitals, most EO sterilizers use single-use cartridges because of the convenience and ease of use compared to the former plumbed gas cylinders of EO blends.

It is important to adhere to patient and healthcare personnel government specified limits of EO residues in and/or on processed products, operator exposure after processing, during storage and handling of EO gas cylinders, and environmental emissions produced when using EO.

The U.S. Occupational Safety and Health Administration (OSHA) has set the permissible exposure limit (PEL) at 1 ppm – calculated as an eight-hour time-weighted average (TWA) – and 5 ppm as a 15-minute excursion limit (EL). The National Institute for Occupational Safety and Health's (NIOSH) immediately dangerous to life and health limit (IDLH) for EO is 800 ppm. The odor threshold is around 500 ppm, so EO is imperceptible until concentrations are well above the OSHA PEL. Therefore, OSHA recommends that continuous gas monitoring systems be used to protect workers using EO for processing.

Nitrogen dioxide

Nitrogen dioxide (NO₂) gas is a rapid and effective sterilant for use against a wide range of microorganisms, including common bacteria, viruses, and spores. The unique physical properties of NO₂ gas allow for sterilant dispersion in an enclosed environment at room temperature and atmospheric pressure. The mechanism for lethality is the degradation of DNA in the spore core through nitration of the phosphate backbone, which kills the exposed organism as it absorbs NO₂. This degradations occurs at even very low concentrations of the gas. NO₂ has a boiling point of 21 °C (70 °F) at sea level, which results in a relatively highly saturated vapour pressure at ambient temperature. Because of this, liquid NO₂ may be used as a convenient source for the sterilant gas. Liquid NO₂ is often referred to by the name of its dimer, dinitrogen tetroxide (N₂O₄). Additionally, the low levels of concentration required, coupled with the high vapour pressure, assures that no condensation occurs on the devices being sterilized. This means that no aeration of the devices is required immediately following the sterilization cycle. NO₂ is also less corrosive than other sterilant gases, and is compatible with most medical materials and adhesives.

The most-resistant organism (MRO) to sterilization with NO₂ gas is the spore of Geobacillus stearothermophilus, which is the same MRO for both steam and hydrogen peroxide sterilization processes. The spore form of G. stearothermophilus has been well characterized over the years as a biological indicator in sterilization applications. Microbial inactivation of G. stearothermophilus with NO₂ gas proceeds rapidly in a log-linear fashion, as is typical of other sterilization processes. Noxilizer, Inc. has commercialized this technology to offer contract sterilization services for medical devices at its Baltimore, Maryland (U.S.) facility. This has been demonstrated in Noxilizer’s lab in multiple studies and is supported by published reports from other labs. These same properties also allow for quicker removal of the sterilant and residual gases through aeration of the enclosed environment. The combination of rapid lethality and easy removal of the gas allows for shorter overall cycle times during the sterilization (or decontamination) process and a lower level of sterilant residuals than are found with other sterilization methods. Eniware, LLC has developed a portable, power-free sterilizer that uses no electricity, heat or water. The 25 liter unit makes sterilization of surgical instruments possible for austere forward surgical teams, in health centers throughout the world with intermittent or no electricity and in disaster relief and humanitarian crisis situations. The four hour cycle uses a single use gas generation ampoule and a disposable scrubber to remove nitrogen dioxide gas.

Ozone

Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.

Ozone offers many advantages as a sterilant gas; ozone is a very efficient sterilant because of its strong oxidizing properties (E=2.076 vs SHE) capable of destroying a wide range of pathogens, including prions, without the need for handling hazardous chemicals since the ozone is generated within the sterilizer from medical-grade oxygen. The high reactivity of ozone means that waste ozone can be destroyed by passing over a simple catalyst that reverts it to oxygen and ensures that the cycle time is relatively short. The disadvantage of using ozone is that the gas is very reactive and very hazardous. The NIOSH's immediately dangerous to life and health limit (IDLH) for ozone is 5 ppm, 160 times smaller than the 800 ppm IDLH for ethylene oxide. NIOSH and OSHA has set the PEL for ozone at 0.1 ppm, calculated as an eight-hour time-weighted average. The sterilant gas manufacturers include many safety features in their products but prudent practice is to provide continuous monitoring of exposure to ozone, in order to provide a rapid warning in the event of a leak. Monitors for determining workplace exposure to ozone are commercially available.

Glutaraldehyde and formaldehyde

Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 22 hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may lengthen the required period or render the treatment ineffective. Sterilization of blocks of tissue can take much longer, due to the time required for the fixative to penetrate. Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation. Glutaraldehyde has a short shelf-life (<2 weeks), and is expensive. Formaldehyde is less expensive and has a much longer shelf-life if some methanol is added to inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.

Hydrogen peroxide

Hydrogen peroxide, in both liquid and as vaporized hydrogen peroxide (VHP), is another chemical sterilizing agent. Hydrogen peroxide is a strong oxidant, which allows it to destroy a wide range of pathogens. Hydrogen peroxide is used to sterilize heat- or temperature-sensitive articles, such as rigid endoscopes. In medical sterilization, hydrogen peroxide is used at higher concentrations, ranging from around 35% up to 90%. The biggest advantage of hydrogen peroxide as a sterilant is the short cycle time. Whereas the cycle time for ethylene oxide may be 10 to 15 hours, some modern hydrogen peroxide sterilizers have a cycle time as short as 28 minutes.

Drawbacks of hydrogen peroxide include material compatibility, a lower capability for penetration and operator health risks. Products containing cellulose, such as paper, cannot be sterilized using VHP and products containing nylon may become brittle. The penetrating ability of hydrogen peroxide is not as good as ethylene oxide and so there are limitations on the length and diameter of the lumen of objects that can be effectively sterilized. Hydrogen peroxide is a primary irritant and the contact of the liquid solution with skin will cause bleaching or ulceration depending on the concentration and contact time. It is relatively non-toxic when diluted to low concentrations, but is a dangerous oxidizer at high concentrations (> 10% w/w). The vapour is also hazardous, primarily affecting the eyes and respiratory system. Even short term exposures can be hazardous and NIOSH has set the IDLH at 75 ppm, less than one tenth the IDLH for ethylene oxide (800 ppm). Prolonged exposure to lower concentrations can cause permanent lung damage and consequently, OSHA has set the permissible exposure limit to 1.0 ppm, calculated as an eight-hour time-weighted average. Sterilizer manufacturers go to great lengths to make their products safe through careful design and incorporation of many safety features, though there are still workplace exposures of hydrogen peroxide from gas sterilizers documented in the FDA MAUDE database. When using any type of gas sterilizer, prudent work practices should include good ventilation, a continuous gas monitor for hydrogen peroxide and good work practices and training.

Vaporized hydrogen peroxide (VHP) is used to sterilize large enclosed and sealed areas, such as entire rooms and aircraft interiors.

Although toxic, VHP breaks down in a short time to water and oxygen.

Peracetic acid

Peracetic acid (0.2%) is a recognized sterilant by the FDA for use in sterilizing medical devices such as endoscopes. Peracetic acid, which is also known as peroxyacetic acid, is a chemical compound often used in disinfectants such as sanitizers. It is most commonly produced by the reaction of acetic acid and hydrogen peroxide with each other by using an acid catalyst. Peracetic acid is never sold in unstabilized solutions which is why it is considered to be environmentally friendly. Peracetic acid is a colorless liquid and the molecular formula of peracetic acid is C₂H₄O₃ or CH3COOOH. More recently, peracetic acid is being used throughout the world as more people are using fumigation to decontaminate surfaces to reduce the risk of Covid-19 and other diseases.

Potential for chemical sterilization of prions

Prions are highly resistant to chemical sterilization. Treatment with aldehydes, such as formaldehyde, have actually been shown to increase prion resistance. Hydrogen peroxide (3%) for one hour was shown to be ineffective, providing less than 3 logs (10⁻³) reduction in contamination. Iodine, formaldehyde, glutaraldehyde, and peracetic acid also fail this test (one hour treatment). Only chlorine, phenolic compounds, guanidinium thiocyanate, and sodium hydroxide reduce prion levels by more than 4 logs; chlorine (too corrosive to use on certain objects) and sodium hydroxide are the most consistent. Many studies have shown the effectiveness of sodium hydroxide.

Radiation sterilization

Sterilization can be achieved using electromagnetic radiation, such as ultraviolet light, X-rays and gamma rays, or irradiation by subatomic particles such as by electron beams. Electromagnetic or particulate radiation can be energetic enough to ionize atoms or molecules (ionizing radiation), or less energetic (non-ionizing radiation).

Non-ionizing radiation sterilization

Further information: Ultraviolet germicidal irradiation

Ultraviolet light irradiation (UV, from a germicidal lamp) is useful for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt (which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages some plastics, such as polystyrene foam if exposed for prolonged periods of time.

Ionizing radiation sterilization

Efficiency illustration of the different radiation technologies (electron beam, X-ray, gamma rays)

The safety of irradiation facilities is regulated by the International Atomic Energy Agency of the United Nations and monitored by the different national Nuclear Regulatory Commissions (NRC). The radiation exposure accidents that have occurred in the past are documented by the agency and thoroughly analyzed to determine the cause and improvement potential. Such improvements are then mandated to retrofit existing facilities and future design.

Gamma radiation is very penetrating, and is commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets, and food. It is emitted by a radioisotope, usually cobalt-60 (⁶⁰Co) or caesium-137 (¹³⁷Cs), which have photon energies of up to 1.3 and 0.66 MeV, respectively.

Use of a radioisotope requires shielding for the safety of the operators while in use and in storage. With most designs, the radioisotope is lowered into a water-filled source storage pool, which absorbs radiation and allows maintenance personnel to enter the radiation shield. One variant keeps the radioisotope under water at all times and lowers the product to be irradiated in the water in hermetically-sealed bells; no further shielding is required for such designs. Other uncommonly used designs use dry storage, providing movable shields that reduce radiation levels in areas of the irradiation chamber. An incident in Decatur, Georgia, US, where water-soluble caesium-137 leaked into the source storage pool, requiring NRC intervention has led to use of this radioisotope being almost entirely discontinued in favour of the more costly, non-water-soluble cobalt-60. Cobalt-60 gamma photons have about twice the energy, and hence greater penetrating range, of caesium-137-produced radiation.

Electron beam processing is also commonly used for sterilization. Electron beams use an on-off technology and provide a much higher dosing rate than gamma or X-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. Because electrons carry a charge, electron beams are less penetrating than both gamma and X-rays. Facilities rely on substantial concrete shields to protect workers and the environment from radiation exposure.

High-energy X-rays (produced by bremsstrahlung) allow irradiation of large packages and pallet loads of medical devices. They are sufficiently penetrating to treat multiple pallet loads of low-density packages with very good dose uniformity ratios. X-ray sterilization does not require chemical or radioactive material: high-energy X-rays are generated at high intensity by an X-ray generator that does not require shielding when not in use. X-rays are generated by bombarding a dense material (target) such as tantalum or tungsten with high-energy electrons, in a process known as bremsstrahlung conversion. These systems are energy-inefficient, requiring much more electrical energy than other systems for the same result.

Irradiation with X-rays, gamma rays, or electrons does not make materials radioactive, because the energy used is too low. Generally an energy of at least 10 MeV is needed to induce radioactivity in a material. Neutrons and very high-energy particles can make materials radioactive, but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.

Sterilization by irradiation with gamma rays may however affect material properties.

Irradiation is used by the United States Postal Service to sterilize mail in the Washington, D.C. area. Some foods (e.g. spices and ground meats) are sterilized by irradiation.

Subatomic particles may be more or less penetrating and may be generated by a radioisotope or a device, depending upon the type of particle.

Sterile filtration

Fluids that would be damaged by heat, irradiation or chemical sterilization, such as drug solution, can be sterilized by microfiltration using membrane filters. This method is commonly used for heat labile pharmaceuticals and protein solutions in medicinal drug processing. A microfilter with pore size of usually 0.22 µm will effectively remove microorganisms. Some staphylococcal species have, however, been shown to be flexible enough to pass through 0.22 µm filters. In the processing of biologics, viruses must be removed or inactivated, requiring the use of nanofilters with a smaller pore size (20–50 nm). Smaller pore sizes lower the flow rate, so in order to achieve higher total throughput or to avoid premature blockage, pre-filters might be used to protect small pore membrane filters. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems also reduce particulate accumulation and blockage.

Membrane filters used in production processes are commonly made from materials such as mixed cellulose ester or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre-sterilized disposable units in sealed packaging or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure proper functioning of the filter, the membrane filters are integrity tested post-use and sometimes before use. The nondestructive integrity test assures the filter is undamaged and is a regulatory requirement. Typically, terminal pharmaceutical sterile filtration is performed inside of a cleanroom to prevent contamination.

Preservation of sterility

A curette in sterile packaging.

Instruments that have undergone sterilization can be maintained in such condition by containment in sealed packaging until use.

Aseptic technique is the act of maintaining sterility during procedures.

Infrared Nanospectroscopy (AFM-IR)

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Infrared_Nanospectroscopy_(AFM-IR) 

 

An atomic-force microscope with its controlling computer

AFM-IR (atomic force microscope-infrared spectroscopy) or infrared nanospectroscopy is one of a family of techniques that are derived from a combination of two parent instrumental techniques. AFM-IR combines the chemical analysis power of infrared spectroscopy and the high-spatial resolution of scanning probe microscopy (SPM). The term was first used to denote a method that combined a tuneable free electron laser with an atomic force microscope (AFM, a type of SPM) equipped with a sharp probe that measured the local absorption of infrared light by a sample with nanoscale spatial resolution.

Originally the technique required the sample to be deposited on an infrared-transparent prism and be less than 1μm thick. This early setup improved the spatial resolution and sensitivity of photothermal AFM-based techniques from microns to circa 100 nm. Then, the use of modern pulsed optical parametric oscillators and quantum cascade lasers, in combination with top-illumination, have enabled to investigate samples on any substrate and with increase sensitivity and spatial resolution. As most recent advances, AFM-IR has been proved capable to acquire chemical maps and nanoscale resolved spectra at the single-molecule scale from macromolecular self-assemblies and biomolecules with circa 10 nm diameter, as well as to overcome limitations of IR spectroscopy and measure in aqueous liquid environments.

Recording the amount of infrared absorption as a function of wavelength or wavenumber, AFM-IR creates an infrared absorption spectra that can be used to chemically characterize and even identify unknown samples. Recording the infrared absorption as a function of position can be used to create chemical composition maps that show the spatial distribution of different chemical components. Novel extensions of the original AFM-IR technique and earlier techniques have enabled the development of bench-top devices capable of nanometer spatial resolution, that do not require a prism and can work with thicker samples, and thereby greatly improving ease of use and expanding the range of samples that can be analysed. AFM-IR has achieved lateral spatial resolutions of ca. 10 nm, with a sensitivity down to the scale of molecular monolayer and single protein molecules with molecular weight down to 400-600 kDa.

AFM-IR is related to techniques such as tip-enhanced Raman spectroscopy (TERS), scanning near-field optical microscopy (SNOM), nano-FTIR and other methods of vibrational analysis with scanning probe microscopy.

History

Early history

Atomic force microscope inside a FTIR spectrometer with the optical interface

 

Left: The original AFM-IR configuration with bottom side illumination and the sample mounted on an infrared-transparent prism. Right: Topside illumination, enabling sample measurements on arbitrary substrates

The earliest measurements combining AFM with infrared spectroscopy were performed in 1999 by Hammiche et al. at the University of Lancaster in the United Kingdom, in an EPSRC-funded project led by M Reading and H M Pollock. Separately, Anderson at the Jet Propulsion Laboratory in the United States made a related measurement in 2000. Both groups used a conventional Fourier transform infrared spectrometer (FTIR) equipped with a broadband thermal source, the radiation was focused near the tip of a probe that was in contact with a sample. The Lancaster group obtained spectra by detecting the absorption of infrared radiation using a temperature sensitive thermal probe. Anderson took the different approach of using a conventional AFM probe to detect the thermal expansion. He reported an interferogram but not a spectrum; the first infrared spectrum obtained in this way was reported by Hammiche et al. in 2004: this represented the first proof that spectral information about a sample could be obtained using this approach.

Both of these early experiments used a broadband source in conjunction with an interferometer; these techniques could, therefore, be referred to as AFM-FTIR although Hammiche et al. coined the more general term photothermal microspectroscopy or PTMS in their first paper. PTMS has various subgroups; including techniques that measure temperature measure thermal expansion use broadband sources. use lasers excite the sample using evanescent waves, illuminate the sample directly from above etc. and different combinations of these. Fundamentally, they all exploit the photothermal effect. Different combinations of sources, methods, methods of detection and methods of illumination have benefits for different applications. Care should be taken to ensure that it is clear which form of PTMS is being used in each case. Currently there is no universally accepted nomenclature. The original technique dubbed AFM-IR that induced resonant motion in the probe using a Free Electron Laser has developed by exploiting the foregoing permutations so that it has evolved into various forms.

The pioneering experiments of Hammiche et al and Anderson had limited spatial resolution due to thermal diffusion - the spreading of heat away from the region where the infrared light was absorbed. The thermal diffusion length (the distance the heat spreads) is inversely proportional to the root of the modulation frequency. Consequently, the spatial resolution achieved by the early AFM-IR approaches was around one micron or more, due to the low modulation frequencies of the incident radiation created by the movement of the mirror in the interferometer. Also, the first thermal probes were Wollaston wire devices that were developed originally for Microthermal analysis (in fact PTMS was originally considered to be one of a family of microthermal techniques). The comparatively large size of these probes also limited spatial resolution. Bozec et al. and Reading et al. used thermal probes with nanoscale dimensions and demonstrated higher spatial resolution. Ye et al described a MEM-type thermal probe giving sub-100 nm spatial resolution, which they used for nanothermal analysis. The process of exploring laser sources began in 2001 by Hammiche et al when they acquired the first spectrum using a tuneable laser (see Resolution improvement with pulsed laser source).

A significant development was the creation by Reading et al. in 2001 of a custom interface that allowed measurements to be made while illuminating the sample from above; this interface focused the infrared beam to a spot of circa 500μm diameter, close to the theoretical maximum. The use of top-down or top-side illumination has the important benefit that samples of arbitrary thickness can be studied on arbitrary substrates. In many cases this can be done without any sample preparation. All subsequent experiments by Hammiche, Pollock, Reading and their co-workers were made using this type of interface including the instrument constructed by Hill et al. for nanoscale imaging using a pulsed laser. The work of the University of Lancaster group in collaboration with workers from the University of East Anglia led to the formation of a company, Anasys Instruments, to exploit this and related technologies (see Commercialization).

Spatial resolution improvement with pulsed laser sources

An infrared optical parametric oscillator (OPO), 1997

 

Schematic of the AFM-IR instrument using an OPO light source constructed at the University of East Anglia by Hill et al in 2007

In the first paper on AFM-based infrared by Hammiche et al., the relevant well-established theoretical considerations were outlined that predict that high spatial resolution can be achieved using rapid modulation frequencies because of the consequent reduction in the thermal diffusion length. They estimated that spatial resolutions in the range of 20 nm-30 nm should be achievable. The most readily available sources that can achieve high modulation frequencies are pulsed lasers: even when the rapidity of the pulses is not high, the square wave form of a pulse contains very high modulation frequencies in Fourier space. In 2001, Hammiche et al. used a type of bench-top tuneable, pulsed infrared laser known as an optical parametric oscillator or OPO and obtained the first probe-based infrared spectrum with a pulsed laser, however, they did not report any images.

Nanoscale spatial resolution AFM-IR imaging using a pulsed laser was first demonstrated by Dazzi et al at the University of Paris-Sud, France. Dazzi and his colleagues used a wavelength-tuneable, free electron laser at the CLIO facility in Orsay, France to provide an infrared source with short pulses. Like earlier workers, they used a conventional AFM probe to measure thermal expansion but introduced a novel optical configuration: the sample was mounted on an IR-transparent prism so that it could be excited by an evanescent wave. Absorption of short infrared laser pulses by the sample caused rapid thermal expansion that created a force impulse at the tip of the AFM cantilever. The thermal expansion pulse induced transient resonant oscillations of the AFM cantilever probe. This has led to the technique being dubbed Photo-Thermal Induced Resonance (PTIR), by some workers in the field. Some prefer the terms PTIR or PTMS to AFM-IR as the technique is not necessarily restricted to infrared wavelengths. The amplitude of the cantilever oscillation is directly related to the amount of infrared radiation absorbed by the sample. By measuring the cantilever oscillation amplitude as a function of wavenumber, Dazzi's group was able to obtain absorption spectra from nanoscale regions of the sample. Compared to earlier work, this approach improved spatial resolution because the use of short laser pulses reduced the duration of the thermal expansion pulse to the point that the thermal diffusion lengths can be on the scale of nanometres rather than microns.

Fast Fourier Transform of cantilever vibrations after a laser pulse; the height of a characteristic peak measures the amount of infrared light absorbed by the sample

 

Spectrum obtained from the AFM measurement by changing the laser wavelength (below); it has good agreement with a conventional FTIR spectrum (above)

A key advantage of the use of a tuneable laser source, with a narrow wavelength range, is the ability to rapidly map the locations of specific chemical components on the sample surface. To achieve this, Dazzi's group tuned their free electron laser source to a wavelength corresponding to the molecular vibration of the chemical of interest, then mapped the cantilever oscillation amplitude as function of position across the sample. They demonstrated the ability to map chemical composition in E. coli bacteria. They could also visualize polyhydroxybutyrate (PHB) vesicles inside Rhodobacter capsulatus cells and monitor the efficiency of PHB production by the cells.

At the University of East Anglia in the UK, as part of an EPSRC-funded project led by M. Reading and S. Meech, Hill and his co-workers followed the earlier work of Reading et al. and Hammiche et al. and measured thermal expansion using an optical configuration that illuminated the sample from above in contrast to Dazzi et al. who excited the sample with an evanescent wave from below. Hill also made use of an optical parametric oscillator as the infrared source in the manner of Hammiche et al. This novel combination of topside illumination, OPO source and measuring thermal expansion proved capable of nanoscale spatial resolution for infrared imaging and spectroscopy (the figures show a schematic of the UEA apparatus and results obtained with it). The use by Hill and co-workers of illumination from above allowed a substantially wider range of samples to be studied than was possible using Dazzi's technique. By introducing the use of a bench top IR source and topdown illumination, the work of Hammiche, Hill and their coworkers made possible the first commercially viable SPM-based infrared instrument (see Commercialization).

Broadband pulsed laser sources

Reading et al. have explored the use of a broadband QCL combined with thermal expansion measurements. Above, the inability of thermal broadband sources to achieve high spatial resolution is discussed (see history). In this case the frequency of modulation is limited by the mirror speed of the interferometer which, in turn, limits the lateral spatial resolution that can be achieved. When using a broadband QCL the resolution is limited not by the mirror speed but by the modulation frequency of the laser pulses (or other waveforms). The benefit of using a broadband source is that an image can be acquired that comprises an entire spectrum or part of a spectrum for each pixel. This is much more powerful than acquiring images bases on a single wavelength. The preliminary results of Reading et al. show that directing a broadband QCL though an interferometer can give an easily detectable response from a conventional AFM probe measuring thermal expansion.

Commercialization

The free-electron laser FELIX at the FOM Institute for Plasma Physics Rijnhuizen Nieuwegein, The Netherlands (2010); a large and uncommon piece of equipment

The AFM-IR technique based on a pulsed infrared laser source was commercialized by Anasys Instruments, a company founded by Reading, Hammiche and Pollock in the United Kingdom in 2004; a sister, United States corporation was founded a year later. Anasys Instruments developed its product with support from the National Institute of Standards and Technology and the National Science Foundation. Since free electron lasers are rare and available only at select institutions, a key to enabling a commercial AFM-IR was to replace them with a more compact type of infrared source. Following the lead given by Hammiche et al in 2001 and Hill et al in 2008, Anasys Instruments introduced an AFM-IR product in early 2010, using a tabletop laser source based on a nanosecond optical parametric oscillator. The OPO source enabled nanoscale infrared spectroscopy over a tuning range of roughly 1000–4000 cm⁻¹ or 2.5-10 μm.

The initial product required samples to be mounted on infrared-transparent prisms, with the infrared light being directed from below in the manner of Dazzi et al. For best operation, this illumination scheme required thin samples, with optimal thickness of less than 1 μm, prepared on the surface of the prism. In 2013, Anasys released an AFM-IR instrument based on the work of Hill et al. that supported top-side illumination. "By eliminating the need to prepare samples on infrared-transparent prisms and relaxing the restriction on sample thickness, the range of samples that could be studied was greatly expanded. The CEO of Anasys Instruments recognised this achievement by calling it " an exciting major advance" in a letter written to the university and included in the final report of EPSRC project EP/C007751/1. The UEA technique went on to become Anasys Instruments' flagship product.

Comparison to related photothermal techniques

It is worth noting that the first infrared spectrum obtained by measuring thermal expansion using an AFM was obtained by Hammiche and co-workers without inducing resonant motions in the probe cantilever. In this early example the modulation frequency was too low to achieve high spatial resolution but there is nothing, in principle, preventing the measurement of thermal expansion at higher frequencies without analysing or inducing resonant behaviour. Possible options for measuring the displacement of the tip rather than the subsequent propagation of waves along the cantilever include; interferometry focused at the end of the cantilever where the tip is located, a torsional motion resulting from an offset probe (it would only be influenced by the motions of the cantilever as a second order effect) and exploiting the fact that the signal from a heated thermal probe is strongly influenced by the position of the tip relative to the surface thus this could provide a measurement of thermal expansion that wasn't strongly influenced by or dependent upon resonance. The advantages of a non-resonant method of detection is that any frequency of light modulation could be used thus depth information could be obtained in a controlled way (see below) whereas methods that rely on resonance are limited to harmonics. The thermal-probe based method of Hammiche et al. has found a significant number of applications.

A unique application made possible by the top-down illumination combined with a thermal probe is localized depth profiling, this is not possible using either using the Dazzi et al. configuration of AFM-IR or that of Hill et al. despite the fact the latter uses top-down illumination. Obtaining linescans and images with thermal probes has been shown to be possible, sub-diffraction limit spatial resolution can be achieved and the resolution for delineating boundaries can be enhanced using chemometric techniques.

In all of these examples a spectrum is acquired that spans the entire mid-IR range for each pixel, this is considerably more powerful than measuring the absorption of a single wavelength as is the case for AFM-IR when using either the method of Dazzi et al. or Hill et al. Reading and his group demonstrated how, because thermal probes can be heated, localized thermal analysis can be combined with photothermal infrared spectroscopy using a single probe. In this way local chemical information could be complemented with local physical properties such melting and glass transition temperatures. This in turn led to the concept of thermally assisted nanosampling, where the heated tip performs a local thermal analysis experiment then the probe is retracted taking with it down to femtograms of softened material that adhere to the tip. This material can then be manipulated and/or analysed by photothermal infrared spectroscopy or other techniques. This considerably increases the analytical power of this type of SPM-based infrared instrument beyond anything that can be achieved with conventional AFM probes such as those used in AFM-IR when using either the Dazzi et al. or the Hill et al. version.

Thermal probe techniques have still not achieved the nanoscale spatial resolution that thermal expansion methods have attained though this is theoretically possible. For this, a robust thermal probe and a high intensity source is needed. Recently, the first images using a QCL and a thermal probe have been obtained by Reading et al. A good signal to noise ratio enabled rapid imaging but sub-micron spatial resolution was not clearly demonstrated. Theory predicts improvements in spatial resolution could be achieved by confining data analysis to the early part of the thermal response to a step change increase in the intensity of the incident radiation. In this way pollution of the measurement from adjacent regions would be avoided, i.e. the measurement window could be confined to a suitable fraction of the time of flight of the thermal wave (using a Fourier analysis of the response could provide a similar outcome by using the high frequency components). This could be achieved by tapping the probe in synchrony with the laser. Similarly, lasers that provide very rapid modulations could further reduce thermal diffusion lengths.

Although most effort to date has been focused on thermal expansion measurements, this might change. Truly robust thermal probes have recently become available, as have affordable compact QCL's that are tuneable over a broad frequency range. Consequently, it may soon be the case that thermal probe techniques will become as widely used as those based on thermal expansion. Ultimately, instruments that can easily switch between modes and even combine them using a single probe will certainly become available, for example, a single probe will eventually be able to measure both temperature and thermal expansion.

Recent improvements and single-molecule sensitivity

The original commercial AFM-IR instruments required most samples to be thicker than 50 nm to achieve sufficient sensitivity. Sensitivity improvements were achieved using specialized cantilever probes with an internal resonator and by wavelet based signal processing techniques. Sensitivity was further improved by Lu et al. by using quantum cascade laser (QCL) sources. The high repetition rate of the QCL allows absorbed infrared light to continuously excite the AFM tip at a "contact resonance" of the AFM cantilever. This resonance-enhanced AFM-IR, in combination with electric field enhancement from metallic tips and substrates led to the demonstration of AFM-IR spectroscopy and compositional imaging of films as thin as single self-assembled monolayers. AFM-IR has also been integrated with other sources including a picosecond OPO offering a tuning range 1.55 μm to 16 μm (from 6450 cm⁻¹ to 625 cm⁻¹).

In its initial development, with samples deposited on transparent prisms and using OPO laser sources, the sensitivity of AFM-IR was limited to a minimal thickness of the sample of circa 50-100 nm as mentioned above. The advent of quantum cascade lasers (QCL) and the use of the electromagnetic field enhancement between metallic probes and substrates have improved the sensitivity and spatial resolution of AFM-IR down to the measurement of large (>0.3 μm) and flat (~2–10 nm) self-assembled monolayers, where still hundreds of molecules are present. Ruggeri et al. have recently developed off-resonance, low power and short pulse AFM-IR (ORS-nanoIR) to prove the acquisition of infrared absorption spectra and chemical maps at the single molecule level, in the case of macromolecular assemblies and large protein molecules with a spatial resolution of ca. 10 nm.

Nanoscale chemical imaging and mapping

Nanoscale resolved chemical maps and spectra

AFM-IR enables nanoscale infrared spectroscopy, i.e. the ability to obtain infrared absorption spectra from nanoscale regions of a sample.

Chemical compositional mapping AFM-IR can also be used to perform chemical imaging or compositional mapping with spatial resolution down to ~10-20 nm, limited only by the radius of the AFM tip. In this case, the tuneable infrared source emits a single wavelength, corresponding to a specific molecular resonance, i.e. a specific infrared absorption band. By mapping the AFM cantilever oscillation amplitude as a function of position, it is possible to map out the distribution of specific chemical components. Compositional maps can be made at different absorption bands to reveal the distribution of difference chemical species.

Examples of AFM-IR Nanospectroscopy

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  AFM-IR nanospectroscopy of a laser printer toner particle, showing spatially resolved chemical analysis. Toner particles are typically complex composites of various binders and transfer agents; these can be revealed by AFM-IR

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  AFM-IR compositional mapping of Streptomyces bacteria. Left: AFM topographic image of bacterial cells. Middle: AFM-IR absorption at 1650 cm⁻¹, corresponding to the amide I band associated with protein. Right: AFM-IR absorption at the carbonyl band 1740 cm⁻¹, indicating the distribution of triglyceride vesicles within bacterial cells.

Complementary morphological and mechanical mapping

Complementary elasticity mapping via simultaneous contact resonance measurements.

The AFM-IR technique can simultaneously provide complementary measurements of the mechanical stiffness and dissipation of a sample surface. When infrared light is absorbed by the sample the resulting rapid thermal expansion excites a "contact resonance" of the AFM cantilever, i.e. a coupled resonance resulting from the properties of both the cantilever and the stiffness and damping of the sample surface. Specifically, the resonance frequency shifts to higher frequencies for stiffer materials and to lower frequencies for softer material. Additionally, the resonance becomes broader for materials with larger dissipation. These contact resonances have been studied extensively by the AFM community (see, for example, atomic force acoustic microscopy). Traditional contact resonance AFM requires an external actuator to excite the cantilever contact resonances. In AFM-IR these contact resonances are automatically excited every time an infrared pulse is absorbed by the sample. So the AFM-IR technique can measure the infrared absorption by the amplitude of the cantilever oscillation response and the mechanical properties of the sample via the contact resonance frequency and quality factor.

Applications

Applications of AFM-IR have include the characterisation of protein, polymers composites, bacteria, cells, biominerals, pharmaceutical sciences, photonics/nanoantennas, fuel cells, fibers, skin, hair, metal organic frameworks, microdroplets, self-assembled monolayers, nanocrystals, and semiconductors.

Polymers

Polymers blends, composites, multilayer films and fibers AFM-IR has been used to identify and map polymer components in blends, characterize interfaces in composites, and even reverse engineer multilayer films Additionally AFM-IR has been used to study chemical composition in Poly(3][4-ethylenedioxythiophene) (PEDOT) conducting polymers. and vapor infiltration into polyethylene terephthalate PET fibers.

Protein science

The chemical and structural properties of protein determine their interactions, and thus their functions, in a wide variety of biochemical processes. Since Ruggeri et al. pioneering work on the aggregation pathways of the Josephin domain of ataxin-3, responsible for type-3 spinocerebellar ataxia, an inheritable protein-misfolding disease, AFM-IR was used to characterize molecular conformations in a wide spectrum of applications in protein and life sciences. This approach has delivered new mechanistic insights into the behaviour of disease-related proteins and peptides, such as Aβ42, huntingtin and FUS, which are involved in the onset of Alzheimer's, Huntington's and Amyotrophic lateral sclerosis (ALS). Similarly AFM-IR has been applied to study studying protein based functional biomaterials.

Life sciences

AFM-IR has been used to characterise spectroscopically in detail chromosomes, bacteria and cells with nanoscale resolution. For example in the case of infection of bacteria by viruses (Bacteriophages), and also the production of polyhydroxybutyrate (PHB) vesicles inside Rhodobacter capsulatus cells and triglycerides in Streptomyces bacteria (for biofuel applications). AFM-IR has also been used to evaluate and map mineral content, crystallinity, collagen maturity and acid phosphate content via ratiometric analysis of various absorption bands in bone. AFM-IR has also been used to perform spectroscopy and chemical mapping of structural lipids in human skin, cells and hair.

Fuel cells

AFM-IR has been used to study hydrated Nafion membranes used as separators in fuel cells. The measurements revealed the distribution of free and ionically bound water on the Nafion surface.

Photonic nanoantennas

AFM-IR has been used to study the surface plasmon resonance in heavily silicon-doped indium arsenide microparticles. Gold split ring resonators have been studied for use with Surface-Enhanced Infrared Absorption Spectroscopy. In this case AFM-IR was used to measure the local field enhancement of the plasmonics structures (~30X) at 100 nm spatial resolution.

Pharmaceutical sciences

AFM-IR has been used to study miscibility and phase separation in drug polymer blends, the chemical analysis of nanocrystalline drug particles as small 90 nm across, the interaction of chromosomes with chemotherapeutics drugs, and of amyloids with pharmacological approches to contrast neurodegeneration.