Fields of Application for Fluorescent Nanoscopy

1. Primary Fields of Application

The primary fields of application for Fluorescent Nanoscopy are fundamental studies in life sciences and medicine, applied research in medicine, and research in different areas of chemistry and physics. Some of these fields of application relate to pure science and would open new markets only after years of research, but many of these fields could lead to the creation of new markets or market segments within the next five years.

The Fluorescent Nanoscope will help scientists conduct nanoscope research in such areas as real-time visualization of genome activity and DNA sequencing (see also paragraph 2.1 below), fluoresence in situ hybridization analysis, cell nuclei structure and function, stem cells, developing drugs that act on virus particles and not affected cells, transmembrane substance transportation, and nanobacteria as a disease agent (see also paragraph 2.3 below) and the nanoscopy of living cells.

We anticipate that the Fluorescent Nanoscope will be in great demand by researchers who work on fundamental medical issues in oncology, immunology, development of new drugs and vaccines, high-specificity diagnostics of virus agents, epidemiology, and forensics.

Fluorescent Nanoscopy also can be used in other applications that require high-precision 3D imaging of interior structures either for development of new materials or for quality assurance, such as metallurgy, development of nanomaterials and nanodevices, and the development of new materials (including highly porous, dendritic and spongy materials).

The Fluorescent Nanoscope will permit the study of fixed and living cells in their native liquid environment, visualization of the distribution of proteins, lipids, carbohydrates and DNA in different colors, reconstruction of 3D images of the interior structures of an object, and so forth. These are the undoubted advantages of Fluorescent Nanoscopy over electron and probe microscopes.

2. Promising Fields of Application

2.1. DNA Sequencing

“Sequencing” refers to the process of revealing the DNA sequence.

Sequencing is used in the research of the evolutionary processes and genetic nature of many diseases. Modern methods enable sequencing of the human genome for as little as $50,000, although expenses of $1 million are necessary to obtain the highest-quality and most reliable results. Even so, this cost is lower than the $20 million that was required for sequencing several years ago.

The development of cheaper sequencing methods (down to $1000 per human genome) will enable the development of new methods of diagnostics, predicting the likelihood that a patient will develop a particular disease, and predicting a patient’s receptiveness and contraindication to different drugs (tailored medicine).

Most of modern sequencing methods include these stages:
• Cutting DNA into a large number of short parts.
• Cloning these parts on a glass (also referred to as “growth of DNA colonies”).
• Determining the sequences of these DNA parts using fluorescent probes. This procedure includes several dozens of cycles of chemical reactions (one cycle per one nucleotide).

Each cycle produces an image that holds a number of spots of different colors (each spot – one colony) on a black background. The size of these colonies is approximately 1-2 µm. Developers of sequencing tools are forced to place colonies at a distance ~10 µm from each other so the fluorescence spots of these colonies do not overlap. This is the reason why the area for these chemical reactions is quite large, and costs for reagents become quite high ($50,000–200,000 for sequencers based on such technologies). A decrease of this area by a factor of 100 will allow developers to cut costs by 30-60 times, and fluorescent nanoscopy will enable them to achieve this result.

2.2. Chromosome Aberrations

“Chromosome aberrations” refers to mutations that cause changes in the chromosome structure. Such disorders in embryo genetics often lead to congenital diseases (for example, an additional chromosome number 21 causes Down’s syndrome) and various types of cancer (such as sarcomas, melanomas and lymphomas). Disorders can be of several types:
• Monosomy, or deletion of a chromosome (a cell has an incomplete set of chromosomes).
• Polysomy (extra chromosomes)
• Disruption of a chromosome
• Interchange of parts of chromosomes
• Other, more complicated disorders associated with non-linear geometrical figures

Several methods are used to reveal such disorders on the chromosome level in prenatal and ontology diagnostics. Oncology diagnostics require tissue to be punctured to test a large number of affected cells. Classifications of tumors and choice of specific medicines are made possible by conducting an analysis of 20-60 cells from the sample (FISH analysis – fluorescent in situ hybridization). The method can be applied only for several of the most studied diseases that correlate with dramatic changes in chromosome structure, such as the presence of extra chromosomes in cells, deletion of chromosomes, and distortions of chromosomes. Most of types of oncology cannot be diagnosed with FISH analysis or other methods (including sequencing) due to the high costs associated with these procedures, or the low specificity and reliability of results that can be obtained.

Obviously, large disorders such as bisections, deletions or additions of new chromosomes lead to serious diseases. But smaller disorders – disorders on the gene level – can also lead to serious health problems.

Fluorescence Nanoscopy can be used to improve the quality of FISH analysis. It is possible to improve the quality of diagnostics for diseases that are already diagnosed by FISH analysis, as well as to develop a new methodology to diagnose currently undiagnosed diseases. Another feature of modern FISH analysis is also important enough to be mentioned: the sample preparation procedure of tissue requires fixing the tissue in paraffin and thereafter slicing the tissue. Obviously, the tissue cells can be damaged during this procedure, and the chromosomes of these cells also can be damaged, which leads to quite misleading results. The Fluorescent Nanoscopy method will be better suited to localize membranes and proteins of these cells together with localization of chromosomes, enabling the device user to dismiss these cells from the list of cells that are reviewed during the test.

2.3. Ultra-Small Microorganisms

Viral particles range in size from 20 to 400 nm. Most bacteria have sizes from 500 to 5,000 nm (5µm), but some bacteria are as small as 200 to 400 nm. Even the largest bacteria (5µm) are seen through an optical microscope with only a low level of detail. Smaller bacteria are visible just as spots.

One type of small bacteria, mycoplasma (size ~500 nm) is known to cause mycoplasma pneumonia (which affects the respiratory system and leads to pneumonia and other diseases), mycoplasma genitalium (a poorly understood sexually transmitted disease), and mycoplasma bovis (which causes cow diseases that cause hundreds of millions of dollars of economic loss each year). Research and diagnostics of such diseases is very complicated because pathogenic organisms such as mycoplasma are very small and are able to mimic, and thus defend themselves from, the immune system.

The existence of another class of ultra-small bacteria is also assumed. Strictly speaking, their existence is not proven yet, although they have been researched for 80 years. These entities are known as nanobacteria, ultramicrobacteria, and filtered bacteria (this last term was coined in the beginning of the 20th century because these bacteria are so small that they can pass through filter paper). Modern methods allow researchers to distinguish nanobacteria of 140-300 nm in size in calcificates such as placenta calcificate, thrombus, and renal calculus. Their nature is not well studied yet, but it is assumed that these bacteria are the main reason of calcification that leads to, among other problems, the development of blood clots, and as such are a leading cause of death.

Small bacteria also have an important impact on agriculture. Ten percent of all crops are lost due to bacterial infections, and one of these – acidovorax – is a bacterium with size of 1µm.

Solutions for End Users

In summary, our product will be used at the research and development stage, and will provide solutions for various types of end users.

Currently research centers make their conclusions after analyzing either dead/modified cells, live cells at a poor quality (several hundred nanometers), or the surfaces of cells (no 3D research into the cell interiors). The Fluorescent Nanoscope will give research centers the opportunity to see the object as it is: live, at one of highest possible spatial resolutions (2-20 nm on the X and Y axes), in 3D (at 50 nm spatial resolution on Z axis), and in color. This spatial resolution is 10 to 100 times better than that of any common lens microscope, including different types of confocal microscopes, and it brings lens microscopy to the level of electron microscopy while also permitting the study of objects in liquid (e.g., in their native state), to obtain “color” images and to obtain 3D images. “Color” imaging here means separate visualization of different structures of the object, such as proteins and lipids. Furthermore, each object test (i.e., each image) will be cheaper to obtain and the time needed to prepare an object is shorter, providing researchers with a competitive edge.

Clinical research centers may well get wrong conclusions both in disease diagnostics and in drug development because the pictures they now obtain are generally vague – many viruses and cells under question are smaller than current spatial resolution can distinguish. This also prevents direct visualization of how damaged cells react to a specific medical treatment. Accordingly, most research conclusions are effectively hypotheses, and current specifications of diagnostic tools permit diagnosis of various diseases only in the development stage, which could be too late for efficient treatment. These problems can be solved if the Fluorescent Nanoscope is used and higher-quality data are obtained. The device will also help medical research centers minimize the cost of one test and the time needed to perform it.

The same problems are often met by medical treatment centers: if the objects of interest are smaller than the wavelength of light the image that can be obtained by a conventional microscope is vague. Diagnostic methods based on Fluorescent Nanoscopy will increase the accuracy of diagnostic processes and lower their costs, and lower the costs incurred from wrong diagnoses.

In new materials design, often there is a need to see the 3D structure of a material. If it is porous, dendritic or made of fibers and permits the injection of dye, it is possible to obtain a nanoscale 3D image of the material. As an example, this is needed for development of new types of concrete. The concrete is made of a dendritic polycrystal structure: each polycrystal is bound to other polycrystals by the crystals that protrude from it. The durability of the concrete is mainly dependent on this complex 3D structure. It is possible to visualize this structure with the Fluorescent Nanoscope and to obtain the information needed for development of a technology that would make a more durable concrete at a lesser cost. Similar problems can be solved in the development of new types of plastics, nanofibers and nanotube materials.

Metallurgy and nanotechnology can benefit from Fluorescent Nanoscopy because the methods used in these industries are sensitive to charge distribution and react on the change of charge distribution more rapidly than other methods. Our technology will enable industry researchers to obtain new information about their existing and potential products' characteristics and decrease operating expenses, which will increase competitive abilities of companies and their products. It can also give researchers the ability to conduct informative research into the interaction between nanostructures and live organisms.