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Microscope maps channels on living cells

Device shows the way in and out of substances in organisms and paves the way for more efficient medications

In a small laboratory room in the Biomedical Science Institute of the University of São Paulo (USP), into which a just a few devices and a PC fit, a high-resolution instrument, the only one in Brazil, is operated: a Bioprobe model atomic force microscope applied to biological area. A technique created by the researcher Ricardo de Sousa Pereira enables the device, installed in the laboratory of the Department of Parasitology, to map the surface of living cells and accurately identify the points where, for example, phenomena such as the entry and exit of calcium ions take place. In this case, says Pereira, “this technique is based on the immobilization of calcium blocking medications at the apparatus’s probing point”, which should enable the development of more efficient calcium blockers to lower blood pressure.

The project, Use of Biosensors, Undertaken with the Probe of the Atomic Energy Microscope Applied to the Biological Area, to Map Ion Channels on the Surface of Cells, conducted by Pereira and financed by FAPESP, began in December 1998 and is expected to be completed in December 2001.

The researcher emphasizes that the great advantages of the device is the ability to view living material: “A scanning electron microscope has viewing power comparable to that of atomic force, but the fixer used in the preparation of the sample kills the cells”.

According to Pereira, the information obtained through the Bioprobe will enable more efficient medications to be developed, since they will have very localized action: the calcium blockers must act only on the cells’ calcium channels – where the mineral crosses the cellular membrane – which implies a high degree of efficiency in blocking any excess entry of the mineral into the cells. Another advantage is the near absence of side effects, since the medications will not interfere with any of the organism’s other chemical processes, not even in the reactions taking place in other parts of the cell.

Discovery by chance
The researcher relates that he and his colleague Nivaldo Antônio Parizzotto, of the Federal University of São Carlos, discovered the ability of the equipment to view living biological material – cells, tissue, organs, etc. – quite by chance. “In 1991, I was working on the atomic force microscope at the Photonic Laboratory of the Electrical Engineering Faculty of the State University of Campinas (Unicamp), headed by professor Vítor Baranauskas. This was the first equipment of the sort in Latin America. One day, we forgot a sample of biological yeast in it, prepared on a slide with a culture. The following day, on attempting to remove the slide, we realized that the microscope’s probe – called a cantilever – was stuck to the sample, because it had grown in size during the night. At this point, we suspected that the cells were alive. They had multiplied and grown onto the equipment’s probe”.

The possibilities of the discovery are many. Last July, for example, the researcher had an article published in the magazine Febs Letters, of the European Biochemical Companies Federation, in which he demonstrated how a biosensor made from the probe of the microscope could detect the absorption of glucose molecules by living Saccharomyces cerevisae cells – the yeast known as biological brewer’s yeast . According to Pereira, “this method will be interesting for scientists that study diabetes”.

Pereira relates that the publication of the article led the director of the Yale University Medical School, in the United States, to invite him to write a revision to be published in the magazine Biochemical Pharmacology: “In it, other interesting results with biosensors were included, notably the detection of ethyl alcohol with a cantilever covered with the alcohol enzyme dehydrogenase type II – which turns ethyl alcohol into glutaraldehyde – and the detection of reactive types of oxygen using the enzymes mutase superoxide, that detects superoxidic ions, and catalase that detects hydrogen peroxide, in other words, oxygenated water”.

The researcher also says that the scientists at IBM made a sort of DNA (deoxyribonucleic acid) chip out of the apparatus’s probe to identify gene mutations – the so-called SNPs (simple nucleotide polymorphisms).

Anti virus – Another application commented on in Pereira’s revision is that of immobilizing a particle of virus on the end of the equipment’s probe – the cantilever – to measure the strength of adhesion required for the virus to infect the cell: with this information, scientists can envisage medications that weaken this adhesive strength, thus avoiding infection by the virus.

The researcher calls attention to the fact that conventional atomic force microscopes, which he used in 1991, began being sold in 1989. Used in particular by physicists and chemists, they serve, for example, to compare the images of the synthetic diamonds used in industrial drills with those of natural diamonds.

It was only in 1995, tells Pereira, that the first model for use in biology emerged – the Bioscope, manufactured by Digital Instruments, of California, coupled to an inverted optical microscope and showing its great advantage: while with electronic  microscopes the fixer used in preparing the samples kills the cells, that of the atomic force microscope can examine living material without using a fixer – osmium tetroxide or glutaraldehyde or evaporated gold. This advantage appears both in relation to the transmission electron microscope, which looks into the inside of a cell, and the scanning electron microscope, which examines the surface of the material.

Pereira sees the equipment he uses as the most modern of its type. Launched at the end of 1998 by the American company Thermomicroscopes, of California, the Bioprobe was purchased in April 2000 after nine months of negotiation: its initial price of US$ 135,000 came down to US$ 100,000, since – as Pereira argued – besides being the first and only one sold in Brazil, the manufacturer would reap the benefit of the indirect advertising every time the researcher gave a lecture or had his work referred to.

Because of the many resources the equipment provides, Pereira considers it more appropriate for analyzing proteins three-dimensionally than the conventional atomic force microscope used by the Ministry of Science and Technology’s Synchrotron Light Laboratory in Campinas: “The microscope at the Synchrotron is appropriate for physics and chemistry, but it does not let you locate the protein crystal accurately, because the search is done by moving the probe blindly. The microscope I use is the best there is for any examination in the biological field – which does not mean that my laboratory is in any way better than the Synchrotron, which is a point of reference in research”.

Operating the microscope demands a workstation connected to a computer made up of the microscope, a television, and a high-definition video monitor.

The apparatus uses only low power laser radiation beams that keep the sample alive. This beam falls on the microstructure called a cantilever. Similar to a drawing square, the cantilever has one side measuring 100 micrometers. As each micrometer is equivalent to a thousandth of a millimeter, this side has the thickness of a hair.

The cantilever is made of two sheets – one of silicon  below, and the other of mirrored gold above. The laser falls on the mirrored part and is captured by a photocell or photodiode, an electronic component that turns light energy into electric power. The electric pulses are taken to the computer, which turns then into digital images.

On the lower part of the cantilever is an edge or probe, which scans the surface and, as it runs over the surface, it alters the direction of the laser beam to reach different areas of the photocell. This shifting of the beam produces more or fewer electric pulses. The number of pulses sent to the computer defines the lighter or darker areas, according to how the software was programmed: in this case, it was agreed that the higher topography of the surface the lighter the image would be. What can be seen on a high-definition computer screen, are three-dimensional images similar to mountains, with a more or less hilly relief, according to the smooth or abrupt variations of the surface of the sample. Above the place for the sample being examined, the Bioprobe has a blue plastic box – the heart of the apparatus – in which a small part containing the cantilever is inserted. Inverted below the sample, is an optical microscope. It is under the sample in order to capture both the image of the material being examined and the position of the cantilever. Alongside the sample, a camera sends the microscopic images of the moving cantilever to the television set.

Search for calcium
It remains to learn how Pereira builds the biosensors with the microscope’s probe, to map the ion channels of the surface of the cells. Let us take as an example the search for calcium ion channels, important in the development of medications for heart patients. He places a calcium ion blocker on the end of the cantilever: it is a heart medication, which may be nifedipine or one of its derivatives –  nitrendipine, nicardipine or amylodipine. Then, he passes the cantilever over the Saccharomyces cerevisae cells – baker’s yeast –, which serve as an experimental model because they have calcium channels similar to human cells.

To explain what happens then, Pereira reminds us of the so-called Van der Waals forces: these are very weak forces, related to temporary connections established whenever two living or inert materials get in touch . For example, when we walk, Van der Waals forces arise between our shoes and the ground. Naturally, they are very weak, otherwise we would not be able to move. The same thing happens when we put our hand on a table or elsewhere.

So, when the calcium ion blocker coupled to the end of the cantilever finds a calcium channel, the affinity between the two increases Van der Waals force slightly at that place, pulling the cantilever down and altering the direction of the laser beam that will strike the photocell. Such a small change does not prevent the cantilever from freeing itself and continuing its exploration of the sample. When it leaves the calcium channel, the Van der Waals force weakens again and, consequently, turns the direction of the laser beam. The result: the microscope produces a chart of the variation in these forces of interaction between the medication and the cell receptors – the calcium channels sought.

A graduate of the Pharmaceutical School of the Federal University of Ouro Preto (Ufop) in the State of Minas Gerais, Pereira did his master’s degree in Biochemistry in the field of drug metabolism at Unicamp – The São Paulo State University – Campinas  where he also did his doctorate in Organic Chemistry in the field of the synthesis of medications. He did a postdoctorate in atomic force microscopy at Yale University School of Medicine (United States).

The researcher places the studies he has been pursuing very closely  to those of Hermann E. Gaub, of the Physics Department of the Munich Technical University, Germany and of Julio Fernandez, of the Mayo Clinic, in Rochester, United States. Both measure the forces of interaction between biological molecules.

Because of his method of research into living cells, Pereira was the guest speaker at the 4th Pharmatech (Congress of the Brazilian Society of Pharmaceutical Technology – SBTF), in 1999, and he had the results of one of his molecular models published on the cover of the book summarizing the congress. The results obtained with the atomic force microscope earned a reference in the textbook Yeast Physiology and Biotechnology, by Graeme Walker, published in England, and were on the cover of the American magazine Applied Biochemistry and Biotechnology, says Pereira.

In October last year, he was in the United States to learn more about the potential of the apparatus. In April, he will go to Toronto, Canada, to learn about a new type of microscope, the Somatoscope: “This fabulous equipment can view the inside of a cell with a resolution of up to 150 angstroms” (1 angstrom = 10-8 cm – and is the unit of the length of light waves). In other words, “in the same way as the Bioprobe, it views biological material without using a fixer or colorant”.

The Project
Use of Biosensors, Made with the Atomic Force Microscope Probe Applied to the Field of Biology, to Map Ion Channels on the Surface of Cells (nº 98/07526-6); Type Young Researcher Program; Coordinator
Ricardo de Souza Pereira – USP’s Biomedical Sciences Institute; Investment R$ 78,240.42 and US$ 110,449