Single-phase single-molecule fluorescence correlation spectroscopy
Related Terms
Antibody, antigen, autoimmune, bacteria, Brownian motion, conventional two-color cross-correlation FCS, cross-correlation spectroscopy, femtoliter, immune system, immunity, lupus, pathogen, photon, quantum, rheumatoid arthritis, solution-phase single-molecule fluorescence, SPSM-FCS, virus.
Background
Chemistry is the study of how atoms and molecules interact with one another. Simply put, the elements in a chemical reaction either combine or separate according to physical laws. For centuries scientists have studied chemical reactions in increasing detail, but only with large quantities of atoms and molecules in test tubes or glass bottles. Chemistry often takes place one molecule at a time, however, and cannot be studied in detail using standard laboratory methods.
For example, the immune system is a vastly complex mixture of single molecules reacting with single molecules. To study it definitively at the level of single molecule interactions has, up until now, surpassed the capabilities of modern science. Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) is the first technology that is able to study single molecules freely reacting in chemical solutions.
FCS is a solution-phase, optical methodology that uses a very thin laser beam to detect the random Brownian motion of a fluorescent-labeled molecule in a tiny volume of solution (one femtoliter, or one-quadrillionth of a liter) no bigger than the laser beam aimed through it. In this way the molecule is always in the laser beam.
Brownian motion is the constant random motion of all molecules when in a liquid solution; it is due to their intrinsic energy from the spinning of their electrons. Brownian motion can be seen readily under a microscope as tiny movements of particles, such as those in the cytoplasm of a cell. The warmer the solution, the greater the motion because heat gives the molecules greater energy.
The word laser is the acronym of "light amplification by stimulated emission of radiation," which is a method of producing pure, monochromatic light in a finely focused beam. "Monochromatic" refers to a single wavelength, whereas most light is a mixture of wavelengths that can be separated into a rainbow of colors using a prism. Certain molecules can be made to give off light (glow) when excited by ultraviolet light. The light they give off is in different colors depending upon the molecule.
Methods
Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) requires several steps. First, a small quantity of liquid is placed on a glass microscope slide. This tiny drop is calculated by dilution to contain a single molecule or two or three single but different molecules. The molecules are tagged with a special chemical component that glows when placed under a highly complex microscope that uses laser light. The volume of the liquid is so small that the entire drop is no bigger than the beam of laser light passing through it. Therefore, the molecules never leave the detection volume or field of view.
The molecule emits photons of fluorescent light. A photon is a tiny bit of energy that is seen as light. It is the smallest quantity of light possible, often called a quantum. The Brownian motion of the molecules, which is the constant, random motion of all molecules caused by the spinning of their electrons, causes subtle changes in the emitted photons that are detected by fluorescence correlation spectroscopy (FCS) This allows the study of the freely moving molecules as they interact with each other.
The process of seeing two different molecules emitting two different colors is called "conventional two-color cross-correlation FCS." It simultaneously measures fluorescence intensity fluctuations in two colors. The two colors represent the fluorescence emission spectra of the two different fluorescent tags attached to the two different molecules. They can be any color of the rainbow as long as they are a single color.
Research
The study of single molecule interactions is most promising in the field of immunity because immunity begins with one molecule, an antibody, which "recognizes" and attaches to another molecule, called an "antigen." An antigen is a molecule that usually comes from outside the body and that the immune system perceives as a threat. Chemicals produced by germs (e.g., bacteria or viruses) are examples of foreign antigens that are normally attacked by a healthy immune system. An antibody is a chemical, a protein, made by the immune system that renders an antigen harmless.
The immune system is highly complex and strikingly different at a genetic level from other chemical systems in the body. While other systems are designed to be constant and to have extensive mechanisms for avoiding changes in molecular structure, the immune system freely allows alterations in the molecules (antibodies) it produces. This generates millions of slightly different antibody molecules, which greatly expands the range of antigens that can be "recognized." Once recognition takes place, the immune system makes more of the successful antibody and continues to modify it for a better fit to the invading antigen. Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) is able to study these single molecule interactions, noting how and when the antigen and the antibody combine, whether it is a single-step or multiple-step process, how long it takes, and whether subsequent iterations with better fitting antibodies react more quickly.
Immunity is central to dealing with infectious diseases and many chronic diseases that represent dysfunction of the immune system (e.g., AIDS, allergies, and autoimmune conditions such as lupus and rheumatoid arthritis). A better understanding of immunity will ultimately produce better ways of dealing with this category of illnesses. The detailed interaction of the immune system with tissues and germs it attacks is as yet completely unknown. Nor does medical science have any idea why the immune system attacks targets it should recognize as harmless, such as allergens and its body's own tissues.
Implications
At present, single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) is so new that research is concentrated on refining the procedure itself. Specific applications have yet to appear. Certainly any chemical reaction that takes place on a scale of single molecules is a suitable object for this methodology. Immunity will be the first area to be investigated because of its central importance in allergy, autoimmune disease, infections, HIV, and cancer. But single molecule studies will have many more uses as the technology becomes more generally available.
Limitations
Single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) is currently limited primarily by being a brand new technology. It must be refined to produce reliable results, modified so that correct data can be extracted and properly interpreted, and standardized so that results are verifiable.
Future research
Fluorescence correlation spectroscopy (FCS) is a solution-phase, optical methodology that uses a very thin laser beam to detect the random Brownian motion of a fluorescent-labeled molecule in a tiny volume of solution that is no bigger than the laser beam aimed through it. FCS can contribute to research as one of many developing methodologies in the rapidly expanding universe of molecular biology.
The analysis of the behavior of single complex molecules is a new capability in biologic science devoted to detailed understanding of the molecular mechanisms of biological and immune processes. Wherever biologic activity takes place between very small quantities of molecules, single-phase single-molecule fluorescence correlation spectroscopy (SPSM-FCS) will be used to investigate the mechanics of these actions. Immune reactions are well known to begin with single molecules and are thus likely to be among the first to be studied by this new technology. Other possibilities include the first stages of generating a cancer and the development of antibiotic resistance on a molecular level.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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