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Biotechnology

Light inside the living cell

Laser tweezers and scalpel manipulate various biological particles

The manipulation of various types of biological particles through a system using laser beams is becoming a powerful tool in the field of biotechnology. When a laser beam is used, it is called an optical pair of tweezers and it able to grasp and move pieces of DNA, spermatozoids, bacteria and other components from the inside of a cell. With a dual beam, the system becomes an optical scalpel, able to pierce and cut parts of a cell.

Announced in 1986 by the physicists Arthur Ashkin, of Bell laboratories belonging to the AT&T company, nowadays known as Lucent, of the United States, they were brought to Brazil by professor Carlos Lenz Cesar, of the Photonic Laboratory of the Physics Institute of the Campinas State University  (Unicamp). Besides enabling the manipulation of microorganisms and particles in living cells without causing damage, the tweezers are also able to measure mechanical properties and very small forces in biological systems, such as the viscosity of fluids and the elasticity of cellular membranes.

Unicamp’s Photonic Laboratory is part of the Optics and Photonics Research Center, one of the ten Research, Innovation and Diffusion Centers (Cepid) recently created by FAPESP. With greater scope and a guarantee of continuity of the studies, professor Lenz expects to extend the technique of using optical tweezers and scalpel and to undertake research in collaboration with other researchers in the country.

At present, he is running two pieces of research in quite different fields: one in physics and the other in medicine. The distance between the two, however, is short. The researchers Adriana Fontes, of the Physics Institute, and Marcelo Mendes Brandão, of the Medical Science School work side by side. For the last four years, she has been researching the use of lasers in manipulating live cells. He is studying the deformability of hemocytes – the red blood cells – using optical tweezers, under the guidance of doctor Sara Teresinha Olalla Saad, of Unicamp’s Hematological Center. The new technique introduced by Lenz made possible a series of discoveries regarding hereditary diseases of the blood cells. With comparative data between healthy and diseased hemocytes and the effect of the pharmaceutical products used to treat cell disturbances.

Assembling equipment
Since 1990, Lenz’s challenge has been to show the countless applications of the optical tweezers. The greatest difficulty has been to put together all the sophisticated equipment necessary to make them work. This only happened through the research-aid project financed by FAPESP, Tweezers and Scalpel.  Optics for the Biological Manipulation and Study of Elastic Properties of Membranes. Two Nd:YAG (Neodymium, Yttrium Aluminum Garnet) lasers were bought – one continuous for the tweezers, and the other pulsing, for the scalpel – various special lenses and mirrors that guide the laser beams to a common optical microscope  coupled to a digital video camera.

The images captured by the camera are viewed on the computer and common TV screen. The images of the inside of a live cell are sharp. One can see  particles being trapped by the laser beam and moved with great accuracy, with no damage to the cells.

This is only  possible because the material being manipulated is transparent and operates in the infrared wavelength. As light is not absorbed, no heat is produced, thus avoiding any thermal damage. But, although weak, the power of the laser is sufficient to shift the particles within the cell.

Optical Strength
In crossing the lens, the laser beam forms a cone of light that works as a trap able to capture the corpuscles in its range of focus. This happens because the light behaves like a particle, transferring impulse whenever it is deflected or absorbed. When a particle pierces the cone of laser light and deflects its rays, it moves and remains captured in the cone’s focus. The optical force thus generated is tiny. Nonetheless, in this context, the acceleration is great because the mass of the microscopic particles is also very small. Normal optical strength is unable to move particles measuring millimeters, but it can measure in microns certain mechanical properties such as the elasticity and viscosity of the membranes, besides very small forces measured in picoNewtons (1/1trillion N), which cannot be gauged in any other way.

Optical tweezers have been used as a measuring instrument, for example, to measure the elasticity of a molecule of DNA or the motility of spermatozoids. Various studies have been published in this line of work, according to Lenz. To test the motility of spermatozoids, the tweezers are used to capture them and, when they attempt to escape from the optical trap, the strength of the laser is reduced until the point where they are able to release themselves. This strength is then recorded and can be compared to the standard motility considered normal.

More studies
The use of these optical tools has been growing as time goes by. From 1986 to 1998, optical tweezers have been the main subject of around 400 works published around the world, in the most varied of fields of research. In 1990, when Lenz began working with optical tweezers, fewer that ten works had been published. Nowadays, an average of 60 a year are published.

Many of the studies published by the Unicamp group were carried out with the Hemocenter in analyzing the hemocytes. These cells need to be very flexible to be able to perform the function of carrying oxygen and nutrients well. They have an average life of 120 days, measure from 7 to 9 microns (1 micron = 1 thousandth of a millimeter) in diameter and cover in this period around 250 km inside the blood vessels and capillaries of the human body. Inside the spleen these capillaries can measure 3 to 4 microns, less than half the diameter of the hemocytes, which, in order to pass through them, have to elongate to up to 230% of their original length. But over time, they become increasingly rigid to the point where they are retained in the spleen and withdrawn from circulation.

Some diseases, however, can damage this property of the hemocytes, making them prematurely stiff, thus reducing their useful life. As a result, there may be a sharp fall in the concentration of red blood cells, leading to anemia. In hereditary spherocytosis, for example, which affects 1 in 5 million individuals, a large number of hemocytes loses the ability to change shape and takes on a spherical shape (spherocytes).

In hereditary elliptocytosis, the red blood cells become ellipses because of mutations of the proteins making up the cellular frame, reducing the survival of the hemocytes. In diseases such as falciform anemia, the hemoglobin takes on the shape of a sickle, with little flexibility and a short life span. An increase in the blood viscosity also takes place making the flow through the capillaries more difficult, and leading to an oxygen deficiency in the tissues.

The importance of the elasticity of the hemocytes led researchers to develop a method of obtaining more accurate information about the flexibility of these cells using optical tweezers. Normal cells and those carrying various types of anemia, including cases where the patients had benefits submitted to treatment with hydroxyurea, a medication used in the treatment of falciform anemia. One by one, the hemocytes were captured by the tweezers and put into movement at 18 different speeds (ranging from 110 to 289 microns a second). Thus, it was possible to measure the elongation of the hemocytes using image-processing software. With this information, the elasticity of around 2,000 hemocytes from different patients was measured.

Blood storage
The characterization of the elasticity of the membrane of normal hemocytes and carriers of all these types of anemia resulting from the study is very useful in helping pathologists to understand better the mechanisms of these dysfunctions, the effect of certain pharmaceutical products. It has been discovered, for example, that the use of hydroxyurea improved the flexibility of hemocytes in patients showing some degree of stiffening of these cells. Another important conclusion has to do with the storage of pouches of blood. The study showed that the hemocytes thus stored for more than 15 days began to exhibit signs of loss of elasticity, and the normal period of storage is up to 35 days. This observation, however, does not impede blood transfusions, according to professor Sara.

Another field where optical tweezers have considerable application is that of experiments in genetic engineering. Coupled to another micro-tool, the optical scalpel, it can be used to transport particles from one cell to another. In contrast to the optical tweezers, the scalpel used a much more powerful pulse laser, the green Nd:YAG, in the visible range, able to pierce cellular membranes without damaging their functioning. Thus, the optical tweezers and scalpel enable a spermatozoid to be inserted into an egg with great accuracy and safety.

Tweezering in the Genome
In the Department of Quantum Electronics of Unicamp’s Physics Institute, the biologist Paulo Arruda, coordinator of the Sugar Cane Genome project, has closely followed experiments with the optical tweezers and scalpel. In his opinion, this new technology opens up fresh perspectives in all fields of biology. Within the project, one of the advantages of using micro-tools is the ability to transport large molecules of DNA, with hundreds of thousands of nucleotides, from one cell to another. “Using the scalpel, we can pierce the thick walls of vegetable cells and manipulate entire chromosomes”, he claims.

To judge by the reaction the subject has caused abroad, Lenz believes that in no more than five years, the technique will be widely used in Brazil. “Interest in the subject is growing every day”, says Lenz. With the experience acquired, Unicamp’s Photonics laboratory will undoubtedly contribute to spreading this technique in Brazil, involving other research centers interested in using the optical tweezers and scalpel.

Profile:
Carlos Lenz Cesar graduated in Physics from the Federal University – Ceará (UFC). He did his master’s and doctorate at Unicamp and completed his post-doctorate at the AT&T Bell Laboratories, in the United States.
Project: Optical Tweezers and Scalpel for Biological Manipulation and the Study of the Elastic Properties of Membranes
Investment: R$ 78,507.08 and US$ 108,081.68

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