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Biophotonics

Spectrums of life

Researchers bring together optical tweezers and spectroscopy to facilitate the study of living cells

On the screen of a television set in the middle of a laboratory chock-full of lasers, microscopes and computers, it is possible to see a red blood cell being stretched, and even a living parasite, the Leishmania amazonensis protozoon, which causes the disease of leishmaniasis, thrashing about to escape from an invisible trap that prevents it from continuing to move about on in a culture plate of microorganisms. What makes the blood cell stretch and imprisons the unicellular microorganism are invisible laser beams that work like optical tweezers. A piece of equipment set up at the Gleb Wataghin Physics Institute (IFGW) of the State University of Campinas (Unicamp) uses these tweezers in work that has been carried out since the beginning of the 1990’s. The most recent innovation of the institute’s Biomedical Laser Applications Laboratory was to join up the optical tweezers with a spectroscopy system, for analyzing proteins, lipids, amino acids, calcium and other chemical substances existing in cells and in microorganisms. All this as if it were a film and with the analyses being carried out in real time on the living organisms caught and moving about.

The difference with the current spectroscopy systems is comparable with a photograph that freezes at a given moment, while the film shows the process throughout a given time. “Our intention was to join up optical tweezers, lasers and spectroscopy, for several kinds of analyses to be carried out simultaneously, without destroying the material under analysis”, says Carlos Lenz Cesar, the coordinator of the group that is developing the optical tweezers. He discovered the optical tweezers through their creator, physicist Arthur Ashkin, when he was doing postdoctoral studies at the Bell Laboratories of the AT&T telecommunications company, in the period from 1988 to 1990, in the United States. The work with optical traps began at the beginning of the 1970’s. To start with, Ashkin used the laser to move and to study solid particles, first with latex microspheres, and then with atoms. The first studies with biological material at cell level were also done by Ashkin, with the Escherichia coli bacterium and red blood cells, and they were published in the Nature magazine.

The spectroscopy system comes as a complement, with its capacity for microanalysis, the mechanical properties the optical tweezers in manipulating microorganisms and living cells. Accordingly, the sticking power can be observed of a parasite to the surface of a cell, at the exact moment of infection, from both the mechanical and biochemical points of view. Other examples of mechanical measures with optical tweezers are the analysis of the forces of impulsion of the microorganisms, the viscosity of fluids and the elasticity of cell membranes.

The work of joining spectroscopy with optical tweezers was part of the doctoral thesis of physicist Adriana Fontes and was accepted for publication in the Physical Review E magazine. The work also earned an award for the best poster presented at the Photonics West Congress, in the United States, which gathered together, in January this year, 15 thousand participants from the areas of photonics and biophotonics. Adriana, who is today doing postdoctoral studies at IFGW, has been working with lasers for eight years, since doing scientific initiation at the same laboratory, and is connected, like all of Professor Lenz’s team, to the Optics and Photonics Research Center, one of the ten Research, Innovation and Diffusion Centers (Cepids) financed by FAPESP.

In practice, the researchers joined a conventional optical microscope, which has a video camera coupled to it and is used for the observation of microorganisms, with a spectrometer installed alongside this classic laboratory instrument. The tweezers consist of a laser beam focused by the objective on a point of the image. By the screen of the monitor, it is possible to observe particles being trapped in the focus of the laser and moved with great precision, without any cell damage. The laser beam is invisible, operating in the infrared, precisely to avoid the absorption of light and the production of heat, which would cause thermal damage. The laser used as tweezers at IFGW is based on neodymium, one of the elements known as rare earths, whose light is emitted in the 1,064 nanometer (nm) wavelength. Absorption is necessary, on the other hand, when one wants to destroy corpuscles or to perforate cell walls using an optical scalpel. In this case, the researchers use another neodymium-based laser, with light emitted at half the infrared wavelength, 532 nm, which damages the cell only in the region desired.

By behaving like a particle, the light transfers an impulse, whenever the laser beam is diverted or absorbed, allowing a cone of rays of light to catch another particle. This behavior of light was discovered by Albert Einstein in 1905, in his study on the photoelectrical effect. He called these luminous particles photons (from the Greek photos, light), and he showed that they transport energy, as well as momentum. It was with this work that Einstein won the Nobel Prize in 1921, and not for the famous theory of relativity.

The forces generated by this optical trap are very small. An excellent pair of optical tweezers is capable of generating forces with maximum values around 200 picoNewtons (pN), equivalent to 1 billionth of 1 weight of 1 kilo. In these dimensions, the optical tweezers are capable of catching particles of sizes of from 40 to 50 nanometers (1 nanometer is 1 millimeter divided 1 million times) to 20 or 30 micrometers (1 micrometer is equal to 1 millimeter divided by a thousand). To catch a living microorganism, with its own motive power, trying to escape from the trap, the tweezers have to be capable of supplying forces of 50 pN at the least. An excellent test of the quality of optical tweezers is to show that they are capable of catching a live spermatozoid. Although these optical tweezers are very small, they are of the same order of magnitude as the forces that act in the cells and microorganisms. For this reason, optical tweezers are an ideal tool for measuring intensities of forces, besides other mechanical properties, in the microscopic universe.

In the ambit of spectroscopy, the work was carried out with several techniques, but always with the same objective of discovering the ‘signatures’ or ‘fingerprints’ that each substance or molecule emits when it interacts with light. One of these signatures results from molecular vibrations, the frequency of which depends on the masses and on the forces between the atoms of a molecule. The result is a spectrum in which the intensity of the electromagnetic waves emitted in each frequency is observed. “We discover the presence of a given molecule through the peak intensity of its frequency of vibration”, says Lenz.

Visible vibrations
As biological materials have many molecules that, in turn, show many peaks, the identification of the substances is done by means of a comparison with a spectrum library. “These molecular vibrations also appear as a modulation in a scattered beam of visible light and can be detected by means of the so-called Raman spectroscopy.” This is a process of scattering with an incident photon and a scattered photon, but it is also possible for processes to occur with two incident photons and one scattered photon, called hyper-Rayleigh scattering or spectroscopy. Multiphotonic processes like these only happen if all the photons involved collide with the same particle at the same time. For this reason, these processes need pulsed lasers, in which all the photons are emitted at the same time, instead of the constant emission of photons of the continuous lasers.

The light scattered by the processes of spectroscopy is captured in the same objective of the optical tweezers and sent to the spectrometer, where it is decomposed and analyzed to discover the molecular vibrations. “This is how we know which molecules are in that cell or living being”, Adriana says. “It’s chemical information.” With this system, it is possible to collect the spectrums of a parasite, such as the Leishmania protozoon, for example, while the optical tweezers keeps it captured in one and the same position, but alive and moving. It is also possible to accompany biochemical modifications that occur when another pair of tweezers takes it close to the cell that it likes to infect.

For one photon spectroscopies, like Raman, the researchers use a continuous titanium sapphire laser, the emission of which can be selected in the region of infrared between 780 and 1,000 nm; whereas for the multiphoton spectroscopies, they use a titanium sapphire laser with pulses lasting as little as 100 femtoseconds (fs), time for the light to cover a distance of only one third the diameter of a strand of hair. One femtosecond is equal to 1 second divided 1 quadrillion times.

Another much used molecular signature is fluorescence, a process in which certain molecules emit a typical light when illuminated by photons with greater energy than the photons emitted. However, as the substances with an efficient fluorescence are few, it is common to introduce colorants as markers. “The problem is that these colorants tend to be toxic, or cytotoxic, and to emit light for a short time, due to photodegradation”, Adriana says. One solution for these problems is the so-called quantum dot, which are semiconductor nanocrystals, called sulfide, selenide and cadmium telluride, indicated for biological applications. The greatest advantage of the quantum dot over the colorants is its great photostability, which makes possible the acquisition of images for hours on end with intense illumination. Furthermore, it shows a very low cytotoxicity. Another great advantage is that the size of the quantum dot controls the color of the fluorescence that it emits. It is possible to get fluorescence from cadmium telluride quantum dots, for example, in blue, green, yellow and red, with their diameter varying from 1 to 5 nanometers.

The group from Unicamp has been producing quantum dots since 1989, but in glasses, with a view to developing ultrarapid devices for optical communications. The work with quantum dots in solutions began at Unicamp in 1999, initially for a comparison with quantum dots produced in glass, and, simultaneously, at the Chemistry Department of the Federal University of Pernambuco (UFPE).

Wide study
The scientific and academic works have currently led to a more ample collaboration with UFPE, involving, at IFGW, Professors Lenz and Luiz Carlos Barbosa, besides Selma Giorgio, of Biology, and Sara Saad and Fernando Costa, from the Hemocenter, all from Unicamp. Taking part at UFPE are Ricardo Ferreira and Gilberto Sá, from the Fundamental Chemistry Department, Beate Santos, of Pharmacy, and Patrícia Farias, of Biophysics. Also collaborating are Professors Vivaldo Moura Neto and Jane Amaral, from the Anatomy Department of the Federal University of Rio de Janeiro (UFRJ). The collaboration with UFRJ involves studies with neurons and neuroglias, which are cells of the brain, while studies with the Leishmania amazonensis protozoon are done jointly with the Biology Institute at Unicamp. The applications of the optical tweezers at Unicamp started in the collaboration with the Hematology Center, with the team of physician Sara Saad, to characterize the mechanical properties of the red blood cells, relating them to diseases like sickle cell anemia and storage time in blood banks (please see Pesquisa Fapesp No.58).

The integration of optical tweezers with spectroscopies of one or more photons and with the use of quantum dots as markers unifies almost all the most modern techniques of biophotonics in just one system and opens up various new fields of research. “It’s a sea of possibilities”, says Lenz. “They are biological processes that can be observed with manipulation at cell level. For example, an American researcher won funding of almost US$ 1 million to assist a dairy product company to determine, with optical tweezers, the forces with which bacteria existing in milk stick to the walls of long-life packaging and how long they remain there. With the integrated system, it is possible to observe, besides the forces, which substances are released into the milk.” All this without killing the bacteria or destroying the substances that one wants to study.

The Project
Optical tweezers and spectroscopy; Modality Research, Innovation and Diffusion Centers (Cepids); Coordinator Hugo Fragnito – IFGW, Optics and Photonics Research Center (CePof) at Unicamp; Investment R$ 1,000,000.00 a year for the whole CePof (FAPESP)

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