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Illuminating Remote Corners: Microscope with Self-Reconstructing Laser Beams

Better contrast, higher resolution: The Physicist Alexander Rohrbach Is Developing a New Type of Microscope with Self-Reconstructing Laser Beams That Enables Better Images
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Less scattering: Alexander Rohrbach’s self-reconstructing laser beams remain stable in the center longer when they penetrate an object. Foto: Universität Freiburg

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What do driving a car in the fog and viewing thick objects through a microscope have in common? – The two situations both present the same problem: The light is scattered. In the first case, fog droplets prevent the car’s headlights from penetrating through to obstacles and thus from sufficiently illuminating them. In the same way, thick collections of thousands of cells scatter the light from the illuminator of optical microscopes such as those used in modern cell biology. In fact, the scattering is so strong that cells of the object under observation located further from the light source are hardly visible at all.

Alexander Rohrbach, professor of bio- and nanophotonics, has succeeded in clearly reducing the undesired deflection and scattering of the light inside the object. Together with his research group at the Department of Microsystems Engineering of the University of Freiburg, he is developing a new and innovative microscopy method based on self-reconstructing laser beams.This development is a boon for scientists studying comparatively large specimens with a thickness of up to one millimeter. Up until now, microscopy has been unable to properly illuminate objects like cancer cell clusters, insulated skin, or animal embryos, which scatter the light too strongly due to their size. The laser beam loses its original concentrated form to scattering due to a multitude of tiny particles or is deflected, making it almost impossible for the microscope to illuminate anything at all on the far side of the object. Rohrbach, a trained physicist, has been investigating biological systems for many years but has not always had success in extracting all of the desired information from them. For example, it is not yet clear when and how various forms of energy are built up inside the cell. “We will need to develop new microscopy techniques, approaches, and analyses to find the answers to these questions,” explains Rohrbach – and these prospects excite his passion for research again and again.

Like several earlier research groups, the Freiburg professor has taken up the over-a-century-old idea of ultramicroscopy. Ultramicroscopy, or light sheet microscopy as it is called today, only illuminates the objects at a particular level: the level to which the focus of the microscope’s objective lens is set. This is made possible by a light sheet, which is irradiated into the object from the side. All parts of the object outside of this level remain unlit and thus dark. In order to create the light sheet, it is possible to use a special cylinder lens alignment or to move a laser beam back and forth quickly at the level of focus in order to obtain an even thinner sheet. However, the light radiated from the side is scattered and deflected by numerous cells and boundary surfaces – in other words by the layers between the various materials. This is where Rohrbach’s idea comes in: He wants to reduce the scattering by using new self-reconstructing beams.

 

  • Bessel Beams Penetrate Deeper

In a series of experiments, Rohrbach and his team demonstrated that specially formed laser beams can approximately reconstruct their original profile when various obstacles, such as light-scattering biological cells, repeatedly destroy the profile of the beam. This self-reconstruction works because scattered photons, i.e. quanta of light, are continuously replaced in the center of the beam by new ones coming from the side. “It is an astounding phenomenon that almost all of the photons coming from the side meet at the center almost simultaneously to form a new beam profile despite massive delays caused by the scattering cells.”

In order to create these special laser beams, the Freiburg researchers converted conventional laser beams to so-called Bessel beams. The most flexible way of doing this is with a computer-controlled hologram that changes the trajectory of the photons depending on their position over the cross-section of the beam. It is known that the profile of Bessel beams in scatter-free space remains largely stable, but until recently it was completely unclear whether and to what extent they also can also revert to their original form in inhomogeneous material, i.e., where there is a lot of scattering. Rohrbach first succeeded in predicting this on a theoretical level with computer simulations and then verified it with experiments. He was thus able to demonstrate that holographically formed self-reconstructing laser beams are especially well suited for microscopy since they are more robust against disruptive scattering. The Bessel beams can penetrate more deeply into the objects under observation, such as pieces of skin or cancer cell clusters.
Better contrast, higher resolution: more details of cancer cell clusters.

However, even the functioning of Bessel beams is not completely trouble-free, because only around 20 percent of the light particles are located in the central main beam; the rest are transported around the center in a ring system. While this extensive ring system surrounding the main beam helps the beam to reconstruct itself, it also leads to poor image contrast in a microscope.

 

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Schärferer Blick: Mit den neuen Mikroskopen lassen sich einzelne Bereiche von Krebszellhaufen detailreicher darstellen. Foto: Universität Freiburg
  • Higher Image Contrast, Higher Resolution

However, Rohrbach has also succeeded in solving this problem: He has developed a method for exploiting the stability of the beam in penetrating the object in which the latter is not illuminated all at once, but rather line by line – similar to the movement of a windshield wiper that travels over the entire surface of a windshield. At the same time, a camera also captures the object line by line as through a single-slit diffraction. This masks the light from the ring system. In comparison to traditional light sheet microscopy with conventional laser beams, this leads to a 50-percent increase in image contrast and an almost 100-percent improvement in the axial resolution – the smallest resolvable distance between consecutive image points – of the three-dimensional image.

In addition to providing new insight into the physically complex processes of light scattering, the optical microscopes developed by Rohrbach’s group also enable researchers in biology and medicine to perform new analyses. For example, the beams can penetrate around one and a half times deeper into human skin samples than conventional laser beams. The new method also allows scientists to observe processes like the cell movements within various layers of skin following contact allergies or sunburns in four dimensions – with 3D images that change in time. “The new method is no magic bullet, but in light sheet microcopy it’s the best we are currently capable of in physical terms.”

Rohrbach is planning on teaming up with colleagues from the Freiburg research cluster BIOSS, Centre for Biological Signalling Studies, to conduct further research with his microscopes, among other things on the dynamics of cancer cell clusters. With an eye to such future projects, he and his team will continue to work on improving the image quality of microscopes with self-reconstructing laser beams and computer holograms: “The future of modern microscopy lies in the use of lasers and computers to optimize the interaction between light and cell – and that goes for each individual beam position.”

 

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Prof. Dr. Alexander Rohrbach

Alexander Rohrbach has served as professor of bio- and nanophotonics at the Department of Microsystems Engineering of the University of Freiburg since January 2006 and as a member of the Faculty of Physics and the research cluster BIOSS (Centre for Biological Signalling Studies) since November 2007. After graduating from the University of Erlangen-Nuremberg with a degree in physics in 1994, Rohrbach earned his PhD in Heidelberg in 1998. While writing his dissertation he conducted research on optical microscopy and cell biology at the Kirchhoff Institute of Physics and the Max Planck Institute of Medical Research in Heidelberg. After conducting various studies on optical forces and cytological applications, he completed his habilitation in physics at the University of Heidelberg. His research interests include optical traps with interferometric particle tracking, molecular motors, cytoskeletal mechanics, and new methods in laser microscopy. (Foto: Manfred Zahn)

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Florian Fahrbach

has been working on his PhD under Prof. Dr. Alexander Rohrbach at the Laboratory for Bio- and Nanophotonics of the Department of Microsystems Engineering of the University of Freiburg since May 2008. He studied physics in Freiburg and at the Imperial College in London starting in 2001.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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How does the light in a microscope penetrate through to the most remote corners of an object? And what motivates a researcher to work on this problem? Surprising Science discussed these questions with Florian Fahrbach, doctoral candidate at the Laboratory for Bio- and Nano-Photonics under Prof. Dr. Alexander Rohrbach. The two researchers discovered a method for improving microscopic images with self-reconstructing laser beams.

Surprising Science: How did you arrive at this topic for your dissertation? Did you have experience with microscopy and the problem of light scattering before you began?

Florian Fahrbach: Light sheet microscopy with holographically formed self-reconstructing beams was already the topic of my diplom thesis in physics. I came up with the topic by chance: During my studies I had been more interested in atomic physics. However, optics play an important role in this area as a tool. My interest in the topic was initially sparked by a lecture Prof. Rohrbach held on the use of optical traps for studying biophysical questions. At the time, I didn’t yet have any experience in the area of microscopy – only the memory of an experiment in my beginning physics practical course, an experience that served more to put me off than to get me interested. While working on my diplom thesis, I only had time to become acquainted with the topic, build a functioning microscope, and make a few initial measurements, but my interest in the development of modern microscopy techniques was finally aroused: I wanted to keep working in this area and study the self-reconstructing beams in more detail.

Surprising Science: What problems have researchers met up with so far in studying relatively large objects like cancer cell clusters through a microscope?

Florian Fahrbach: Studying large, strongly scattering objects with an optical microscope is a special challenge – and will remain so in the future. However, illuminating objects with holographically formed self-reconstructing beams is an interesting approach that enables more information to be collected from scattering objects. A good image with high information content has a high contrast – meaning that the microscope measures a high signal with a low background and a high spatial resolution. Moreover, the microscope should deliver an accurate and artifact-free 3D image of the object on a PC. Ideally, a microscope should be capable of illuminating particular areas of interest with great precision. If the light is scattered by the object, however, this precise illumination is impossible because the photons in the object do not follow the desired direction. As a result, other parts of the object are also illuminated and the contrast of the image falls off until it is only possible to draw false conclusions about the structure of the object or none at all.

Surprising Science: How did you conduct the experiments in which you demonstrated that specially formed laser beams can even approximately reconstruct their original profile when it is destroyed repeatedly by various obstacles?

Florian Fahrbach: We conducted an entire series of experiments and studied a lot of parameters, such as the strength of the scattering or the composition of the scattering objects. In addition to studying biological objects we also used artificial specimens made of fluorescent microparticles. These latter objects had several advantages: They enabled us to replicate the scattering properties of biological material very accurately. Due to the fact that their dimensions were known, they also allowed us to measure the optical output of the microscope and compare the simulation with the measurement. The best way to visualize the self-reconstruction is with simulation data, because one then has access to the cross-section of the beam at all positions as it propagates through the medium. When it is compared to the profile of the unscattered beam, the self-reconstruction of the beam becomes apparent. In making measurements, one necessarily does not have direct access to the beam profile within the object but can take an image from the side with a light sheet microscope. This allowed us, for instance, to measure the self-similarity of the beam’s image for various positions in the sample at which the beam was scattered to various degrees. We discovered that the beam profile is considerably less dependent on scattering with self-reconstructing beams – a clear indication for the self-reconstructing properties of the beam in scattering media.

Surprising Science: How is it possible to form beams with computer-controlled holograms?

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Better contrast, higher resolution: The central main beam of the laser illuminates the object line by line. At the same time, a camera also captures the object as through a single-slit diffraction. This masks the light coming from the ring system. (Graph mit Laser)

Florian Fahrbach: Unlike a normal image, which only reproduces the light intensity or the number of photons, a hologram – which basically means “complete image” – contains information on the number as well as the direction of the photons. A computer-controlled hologram can thus be used to influence the number as well as the direction of the photons with a high resolution. In order to comprehend how a computer-controlled hologram changes the flight path of a photon, it helps to think of the device as a mirror that one can bend and twist with extreme precision. For example, one can use the computer to make a hologram that looks like an image seen through a normal lens, but one can also adjust the focal length in very precise increments. In addition, a conical optical element called an axicon can depict holographs and can be used to create Bessel beams of almost any shape or size. If you have the right software, all you need to do is push a button on your PC.

Surprising Science: What influence does the thickness of the laser beam have on image quality?

Florian Fahrbach: In light sheet microscopy, the thickness of the laser beam determines the thickness of the area within the object that is illuminated. The detection objective displays the entire illuminated volume. However, you only obtain a sharp image for a narrow area around the level of focus of the objective lens. The objective can’t select between the foreground and the background, and one thus sees a superimposition of the sharp image from the level of focus and blurry images of the surrounding levels. The thicker the laser beam is, the harder it becomes for the observer to extract relevant information from the image.

Surprising Science: What are the next steps of your research? Will you – within the context of or after completing your dissertation – solve or need to solve further problems concerning the Bessel beam and its ring system? Is it possible to improve on your method anymore at all?

Florian Fahrbach: My dissertation is as good as finished. From an optical standpoint, confocal light sheet microscopy is currently the optimum and delivers the best image quality. But there is certainly still room for improvement, particularly in imaging even more challenging objects. Besides, continuing to test this method is a matter of personal importance for me. It would be nice to be able to convince a lot of biologists and other potential users to build one of these microscopes themselves and use it for purely biological research – in situations in which a normal microscope just isn’t good enough.

 

Gallery

 From the idea to the converted laser beam

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The Freiburg professor took up the old idea of ultramicroscopy for his development. Ultramicroscopy, or light sheet microscopy as it is called today, only illuminates the objects at a particular level, the level of focus to which the microscope’s objective lens is set. This is made possible by a light sheet, which is irradiated into the object from the side. The Gaussian beam: Thousands of cells scatter the illumination lamp of an optical microscope. The laser beam loses its original concentrated form to scattering due to the multitude of tiny particles or is deflected, making it almost impossible for the microscope to illuminate anything at all on the far side of the object. The Bessel beam: Rohrbach and his team succeeded in demonstrating that specially formed laser beams can approximately reconstruct their original profile when it is repeatedly destroyed by various obstacles, such as light-scattering biological cells. In order to achieve this, they converted conventional laser beams into so-called Bessel beams.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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