A microbeam is a narrow beam of radiation

Radiation

In physics, radiation is a process in which energetic particles or energetic waves travel through a medium or space. There are two distinct types of radiation; ionizing and non-ionizing...

, of micrometer

Micrometre

A micrometer , is by definition 1×10-6 of a meter .In plain English, it means one-millionth of a meter . Its unit symbol in the International System of Units is μm...

 or sub-micrometer dimensions. Together with integrated imaging techniques, microbeams allow precisely defined quantities of damage to be introduced at precisely defined locations. Thus, the microbeam is a tool for investigators to study intra- and inter-cellular mechanisms of damage signal transduction

Signal transduction

Signal transduction occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response...

.

A schematic of microbeam operation is shown on the right. Essentially, an automated imaging system locates user-specified targets, and these targets are sequentially irradiated, one by one, with a highly-focused radiation beam. Targets can be single cells

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

, sub-cellular locations

Organelle

In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....

, or precise locations in 3D tissues. Key features of a microbeam are throughput, precision, and accuracy

Accuracy and precision

In the fields of science, engineering, industry and statistics, the accuracy of a measurement system is the degree of closeness of measurements of a quantity to that quantity's actual value. The precision of a measurement system, also called reproducibility or repeatability, is the degree to which...

. While irradiating targeted regions, the system must guarantee that adjacent locations receive no energy deposition.

History

The first microbeam facilities were developed in the mid-90s. These facilities were a response to challenges in studying radiobiological processes using broadbeam exposures. Microbeams were originally designed to address two main issues:

1. The belief that the radiation-sensitivity of the nucleus
   Cell nucleus
   In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
   
    was not uniform, and
2. The need to be able to hit an individual cell with an exact number (particularly one) of particles for low dose risk assessment.

Additionally, microbeams were seen as ideal vehicles to investigate the mechanisms of radiation response.

Radiation-sensitivity of the cell

At the time it was believed that radiation damage to cells was entirely the result of damage to DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

. Charged particle microbeams could probe the radiation sensitivity of the nucleus, which at the time appeared not to be uniformly sensitive. Experiments performed at microbeam facilities have since shown the existence of a bystander effect

Bystander effect

The bystander effect or Genovese syndrome is a social psychological phenomenon that refers to cases where individuals do not offer any means of help in an emergency situation to the victim when other people are present...

. A bystander effect is any biological response to radiation in cells or tissues that did not experience a radiation traversal. These "bystander" cells are neighbors of cells that have experienced a traversal. The mechanism for the bystander effect is believed to be due to cell-to-cell communication. The exact nature of this communication is an area of active research for many groups.

Irradiation with an exact number of particles

At the low doses of relevance to environmental radiation exposure, individual cells only rarely experience traversals by an ionizing particle and almost never experience more than one traversal. For example, in the case of domestic radon

Radon

Radon is a chemical element with symbol Rn and atomic number 86. It is a radioactive, colorless, odorless, tasteless noble gas, occurring naturally as the decay product of uranium or thorium. Its most stable isotope, 222Rn, has a half-life of 3.8 days...

 exposure, cancer

Cancer

Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...

 risk estimation involves epidemiological studies of uranium miners. These miners inhale radon gas, which then undergoes radioactive decay

Radioactive decay

Radioactive decay is the process by which an atomic nucleus of an unstable atom loses energy by emitting ionizing particles . The emission is spontaneous, in that the atom decays without any physical interaction with another particle from outside the atom...

, emitting an alpha particle

Alpha particle

Alpha particles consist of two protons and two neutrons bound together into a particle identical to a helium nucleus, which is classically produced in the process of alpha decay, but may be produced also in other ways and given the same name...

 This alpha particle traverses the cells of the bronchial epithelium, potentially causing cancer. The average lifetime radon exposure

Radiation poisoning

Acute radiation syndrome also known as radiation poisoning, radiation sickness or radiation toxicity, is a constellation of health effects which occur within several months of exposure to high amounts of ionizing radiation...

 of these miners is high enough that cancer risk estimates are driven by data on individuals whose target bronchial cells are subjected to multiple alpha particle traversals. On the other hand, for an average house occupant, about 1 in 2,500 target bronchial cells will be exposed per year to a single alpha particle, but less than 1 in 10⁷ of these cells will experience traversals by more than one particle. Therefore, in order to extrapolate from miner to environmental exposures, it is necessary to be able to extrapolate from the effects of multiple traversals to the effects of single traversals of a particle.

Due to the random distribution

Poisson distribution

In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time and/or space if these events occur with a known average rate and independently of the time since...

 of particle tracks, the biological effects of an exact number (particularly one) of particles cannot practically be simulated in the laboratory using conventional broadbeam exposures. Microbeam techniques can overcome this limitation by delivering an exact number (one or more) of particles per cell nucleus. True single-particle irradiations should allow measurement of the effects of exactly one alpha particle traversal, relative to multiple traversals. The application of such systems to low frequency processes such as oncogenic transformation depends very much on the technology involved. With an irradiation rate of at least 5,000 cells per hour, experiments with yields of the order of 10^-4 can practically be accomplished. Hence, high throughput is a desired quality for microbeam systems.

Charged particle microbeam

The first microbeam facilities delivered charged particles. A charged particle microbeam facility must meet the following basic requirements:

1. The beam spot size should be on the order of a few micrometres or smaller, corresponding to cellular or sub-cellular dimensions.
2. Irradiations of living cells should take place at atmospheric pressure.
3. Beam current must be reduced to levels such that targets may be irradiated with an exact number of particles with high reproducibility
   Reproducibility
   Reproducibility is the ability of an experiment or study to be accurately reproduced, or replicated, by someone else working independently...
   
   .
4. An imaging system is required to visualize and register cellular targets.
5. Cell positioning must have high spatial resolution and reproducibility
   Reproducibility
   Reproducibility is the ability of an experiment or study to be accurately reproduced, or replicated, by someone else working independently...
   
    in order that the ion beam hit the target with a high degree of accuracy and precision
   Accuracy and precision
   In the fields of science, engineering, industry and statistics, the accuracy of a measurement system is the degree of closeness of measurements of a quantity to that quantity's actual value. The precision of a measurement system, also called reproducibility or repeatability, is the degree to which...
   
   .
6. A particle detector
   Particle detector
   In experimental and applied particle physics, nuclear physics, and nuclear engineering, a particle detector, also known as a radiation detector, is a device used to detect, track, and/or identify high-energy particles, such as those produced by nuclear decay, cosmic radiation, or reactions in a...
   
    with high efficiency must count the number of particles per target and switch off the beam after the desired number of particles have been delivered.
7. Environmental conditions (humidity, for example) for cells must be maintained such that cells are under little or no stress.

Beam spot size

Beam spots with diameter down to about two micrometres can be obtained by collimating

Collimator

A collimator is a device that narrows a beam of particles or waves. To "narrow" can mean either to cause the directions of motion to become more aligned in a specific direction or to cause the spatial cross section of the beam to become smaller.- Optical collimators :In optics, a collimator may...

 the beam with pinhole apertures or with a drawn capillary. Sub-micrometre beam spot sizes have been achieved by focusing the beam using various combinations of electrostatic or magnetic lenses. Both methods are used at present.

Vacuum window

A vacuum window is necessary in order to perform microbeam experiments on living cells. Generally, this is accomplished with the use of a vacuum-tight window of a polymer

Polymer

A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...

 a few micrometres thick or 100-500 nm thick Silicon nitride

Silicon nitride

Silicon nitride is a chemical compound of silicon and nitrogen. If powdered silicon is heated between 1300° and 1400°C in an atmosphere of nitrogen, trisilicon tetranitride, Si3N4, is formed. The silicon sample weight increases progressively due to the chemical combination of silicon and nitrogen...

.

Cell registration and positioning

Cells must be identified and targeted with a high degree of accuracy. This can be accomplished using cell staining and fluorescence microscopy or without staining through the use of techniques such as quantitative phase microscopy or phase contrast microscopy. Ultimately, the objective is to recognize cells, target them, and move them into position for irradiation as fast as possible. Throughputs of up to 15,000 cells per hour have been achieved.

Particle counters

Particles must be counted with a high degree of detection efficiency in order to guarantee that a specific number of ions are delivered to a single cell. Generally, detectors can be placed before or after the target to be irradiated. If the detector is placed after the target, the beam must have sufficient energy to traverse the target and reach the detector. If the detector is placed before the target, the detector must have a minimal effect on the beam. When the desired number of particles are detected, the beam is either deflected or shut off.

Other considerations

Living cells must be maintained under conditions that do not stress the cell, causing an unwanted biological response. Additionally, cells must be attached to a substrate

Substrate (biology)

In biology a substrate is the surface a plant or animal lives upon and grows on. A substrate can include biotic or abiotic materials and animals. For example, encrusting algae that lives on a rock can be substrate for another animal that lives on top of the algae. See also substrate .-External...

 so that their position can be determined by the imaging system.

X-ray microbeam

Some facilities have developed or are developing soft x-ray microbeams. In these systems, zone plate

Zone plate

A zone plate is a device used to focus light or other things exhibiting wave character. Unlike lenses or curved mirrors however, zone plates use diffraction instead of refraction or reflection. Based on analysis by Augustin-Jean Fresnel, they are sometimes called Fresnel zone plates in his honor...

s are used to focus characteristic x rays

Characteristic x-ray

A high energy electron interacts with a bound electron in an atom and ejects it. The incident electron is scattered and the target electron gets displaced from its shell. The incident electron energy must exceed the binding energy of the electron to eject it...

 generated from a target hit by a charged particle beam. When using synchrotron x-rays as a source, x-ray microbeam can be obtained by cutting the beam with a precise slit system due to high directionality of synchrotron radiation

Synchrotron radiation

The electromagnetic radiation emitted when charged particles are accelerated radially is called synchrotron radiation. It is produced in synchrotrons using bending magnets, undulators and/or wigglers...

.

Biological endpoint

Many biological endpoints have been studied including oncogenic transformation, apoptosis

Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

, mutations, and chromosomal aberrations.

Microbeam systems worldwide

Microbeam facilities worldwide and their characteristics

Microbeam Facilities Worldwide	Radiation Type/LET	Beam Spot Size on Cell	Running Biology?
Radiological Research Accelerator Facility (RARAF), Columbia University	any cation, x rays low to very high	0.6 μm	yes
JAERI, Takasaki, Japan	high		yes
Special Microbeam Utilization Research Facility (SMURF), Texas A&M	low		no
Superconducting Nanoscope for Applied nuclear (Kern-)physics Experiments (SNAKE), University of Munich	From p to HI 2-10000 keV/μm	0.5 μm	yes
INFN-LNL, Legnaro, Italy	p, 3He+,++,4He+,++ 7-150 keV/μm	10 μm
CENBG, Bordeaux, France	p, α Up to 3.5 MeV	10 μm
GSI, Darmstadt, Germany	From α to U-ions Up to 11.4 MeV/n	0.5 μm	yes
IFJ, Cracow, Poland	p - Up to 2.5 MeV x ray - 4.5 keV	12 μm 5 μm	yes
LIPSION, Leipzig, Germany	p, 4He+,++ Up to 3 MeV	0.5 μm	yes
Lund NMP, Lund, Sweden	p Up to 3 MeV	5 μm
CEA-LPS, Saclay, France	p 4He+,++ Up to 3.75 MeV	10 μm	yes
Queen's University, Belfast, Northern Ireland UK	x ray 0.3-4.5 keV	< 1 μm	yes
University of Surrey, Guilford, UK	p, α, HI	0.01 μm (in vacuum)	yes
PTB, Braunschweig, Germany	p, α 3-200 keV/μm	< 1 μm	yes
SPICE, NIRS, Japan	p 3.4 MeV	5 μm	yes
W-MAST, Tsuruga, Japan	p, He	10 μm	no
McMaster University, Ontario, Canada			no
Nagasaki University, Nagasaki, Japan	x-rays 0.3-4.5 keV	< 1 μm	yes
Photon Factory, KEK, Japan	x-rays 4-20 keV	5 μm	yes
CAS-LIBB, Institute of Plasma Physics, CAS, Hefei, China	p 2-3 MeV	5 μm	yes
Centro Atómico Constituyentes, CNEA, Buenos Aires, Argentina	to H from U 15 MeV	5 μm	yes
FUDAN University, Shanghai, China	p,He 3 MeV	2 μm (in building)	yes
Institute of Modern Physics CAS, Lanzhou, China

Microbeam Workshops

There have been nine international workshops, held approximately once every two years, on Microbeam Probes of Cellular Radiation Response. These workshops serve as an opportunity for microbeam personnel to come together and share ideas. The proceedings of the workshops serve as an excellent reference on the state of microbeam-related science.

List of eight microbeam workshops held 1993-2010, plus the tenth workshop to be held in 2012.

International Workshops on Microbeam Probes of Cellular Radiation Response	Year	Number of Microbeams
Gray Laboratory, London	1993	3
Pacific Northwest Labs, Washington	1995	3
Columbia University, New York	1997	4
Dublin, Ireland	1999	7
Stresa, Italy	2001	12
Oxford, England	2003	17
Columbia University, New York	2006	28
NIRS, Chiba, Japan	2008	31
GSI, Darmstadt, Germany	2010
Columbia University, New York	2012