RONNIE DAS

(last updated 02/01/2013)



BS Bioengineering - University of California, San Diego
MS Bioengineering - University of Illinois at Chicago
PhD Bioengineering - University of Washington, Seattle





CURRENT RESEARCH * PREVIOUS RESEARCH * BACKGROUND * CONTACT



CURRENT RESEARCH

Welcome to my homepage =)

I'm a researcher in the Human Photonics Laboratory, an optics lab led by Eric Seibel and his diverse group of researchers, engineers and scientists, located at the University of Washington here in Seattle. Currently, my primary research objective is to enhance the screening and diagnoses of pancreatic cancer, and provide possibly a new method to study the general disease state and its pathology. To achieve this aim, I'm researching and developing bioinstrumentation to handle patient biopsies and am coupling this engineering to OPTM, an imaging modality that stands for optical projection tomographic microscopy, one focus of this lab's many research areas. If you're unfamiliar with OPTM, then a good analogy is a CT-scan at a hospital: instead of using X-rays to image the entire body, plain white light is used to image cells in 3D on a microscope.

Right now, my specific research study/project is still in its initial stages and it still has many moving parts, which if you know me is a great place for me with respect to my personality and multidisciplinary interests. The project is also a perfect blend of benchtop instrumentation and human physiology, which is something I first look for in projects I take on. The study is also highly collaborative in that we work closely with the Departments of Bioengineering and Mechanical Engineering, the Departments of Pathology, Gastroenterology and Surgery at the University of Washington Medical Center, and VisionGate, a company and industrial collaborator that works in parallel with our research group.



PREVIOUS RESEARCH

My previous work consisted of my PhD research, which involved bioinstrumentation development and the fields of muscle contraction and cell water. In the later years, my main focus was centered on instrumentation and cell water. A basic summary of the project is given below...

Like the planet, cells are composed of as much as 75% water, with the remaining 25% consisting of proteins and other intracellular structures (figure 1). Most people think the water in your cells is like the water which comes out of your faucet, i.e. bulk water. This is not the case.



Figure 1: L) Cross sectional view of a typical muscle cell. The internal space of the cell consists of proteins and various intracellular organelles (i.e. sarcomeres, mitochondria, sarcoplasmtic reticula, etc.). C TO R) Water occupies the rest of the interstitial space and consists of as much as 75% of the cell volume. In some plant cells, water may occupy as much as 90% of the total cell volume.

Water at the microscopic level has been demonstrated to be mechanically, chemically and electrically distinct from bulk water. The water is so unique, that it is more commonly referred to as interfacial water by many researchers and investigators, including those in my previous lab led by my fearless advisor, Jerry Pollack. Over the last decade, Jerry has become one of the leaders in this exciting field and has proposed that this water is a fourth phase altogether (outside of solid, liquid, or vapor; see water on wikipedia, fourth sentence; see the 32nd Annual University of Washington Faculty Lecture hosted in 2009).

Understanding interfacial water is important on many levels for basic and applied research. For example, think about how difficult it is to pull apart two glass slides with a single drop of water between them, or the ability of giant red wood trees to pull capillaries of water to the very tops of their branches. Could you imagine a material that orients water in a fashion that makes it a super adhesive? Or an apparatus that transports water seemlessly, with no moving parts like pumps and valves? There is even research out there that is focused on fabricating materials and arranging them in such a pattern, that one could harvest drinking water right out of the dry, desert air, mimicking the evolved abilities of the Stenocara desert beetle (figure 2).



Figure 2: TL TO TR) The Stenocara beetle harvests drinking water right out of the dry Namibian desert air, an evolutionary advantage of its well-developed outer shell. BL TO BR) The ability to harvest water using different hydrophilic and hydrophobic surfaces has been an inspiration for groups to microfabricate surface features that copy the same design as the beetle's back, but on a scale to harvest drinking water for humans.

Interfacial water is water at an interface. At the interface, a water-loving material is known as a hydrophilic surface, while the opposite is known as a hydrophobic surface. Hydrophobic materials are commonly seen in cooking. Typically, cooking pans have a nonstick coating, which is simply a layer of Teflon, a superhydrophobic material. If we deposited a drop of water on a Teflon sheet, it would bead up, since the water wants to make the least amount of contact with the hydrophobic surface. This surface contact phenomenon is governed in general by 1) the surface's chemical make-up, 2) its roughness and 3) electrical charge.

Here are some basic experiments I've conducted illustrating this simple phenomenon. In the example, I compare a water drop on a superhydrophobic Teflon surface to (hydrophilic) glass, metal (which can exhibit various orders of hydrophilicity) and a leaf surface (which is typically hydrophobic due to its waxy outer surface). Click here for the video.

When Teflon is chemically modified such that it contains sulfonyl branches (figure 3), the resulting material is Nafion, a polymer frequently employed to study interfacial water due to Nafion's unique water properties. Click here for the video.



Figure 3: Nafion's molecular structure. Nafion is a copolymer of a Teflon backbone and sulfonyl groups.

(In reference to the previous experimental video...) Initally, when a water drop is placed on a Nafion sheet, the drop beads up and thus reflects the hydrophobic (Teflon) properties of Nafion's chemical make-up. In the video, a sudden mechanical buckling of the polymer is visualized because one side of the Nafion sheet is hydrating. This results in a difference of physical expansion between the top and bottom surfaces, and the subsequent bowing of the Nafion sheet. Nafion is unique because it not only absorbs water, but it also adsorbs it. Eventually, when both sides of the polymer are hydrated (not shown in the previous video), the water drop and the polymer sheet flatten out, and consequently demonstrate Nafion's hydrophilic properties.

Advanced spectroscopy of hydrated Nafion and the water along Nafion surfaces has demonstrated that interfacial water has a distinct molecular structure. These data show that the structure is in between liquid water and ice. In the field of spectroscopy, it's known as "ice-like" water. Ice-like water has also been observed spectroscopically at many kinds of hydrophilic surfaces and therefore may be a more general phenomenon found in nature. For biology, this possibility has significant implications since most biological surfaces are hydrophilic and interfacial water is assumed to exist ubiquitously.

On a mechanical level, interfacial water has also shown properties vastly different from bulk water. For example, on the microscopic scale at hydrophilic surfaces, interfacial water viscosity has been measured to be several thousands to hundreds of thousands of times greater than bulk water viscosity. Imagine that! If you were in a swimming pool of just interfacial water, you would not be able to swim one bit since the water viscosity would be hundreds of thousands of times greater than regular bulk water. For a real world comparison, molasses and putty are 10,000x and 100,000,000x more viscous than water, respectively. In diffusion experiments, significant reductions in the diffusion and partition coefficients have been directly measured from observations using basic light microscopy of the interfacial water layers at surfaces. In the cell, where the cytosol is consisted of 75-85% water and many internal surfaces are hydrophilic, similar results have also been measured and reported.

By using simple light microscopy in this lab, we've observed that at the Nafion surface, positive- and negatively-charged microspheres, dyes (i.e. methylene blue, pH-sensitive dye, etc.), and even some solutes have been excluded from the near water layers (see video data 1). We have also observed this phenomenon at many kinds of hydrophilic surfaces, such as hydrogels and biological materials (i.e., skinned muscle, plant surfaces, etc.). Because of the widely exclusionary nature of these water layers, our lab has named this region the exclusion zone.

The exclusion zone is very extensive, measuring hundreds of microns in thickness. Spectroscopically, it is distinct from the more distant bulk water. And electrical potential measurements have shown that this region is negatively-charged relative to the bulk. In subsequent investigations using pH-sensitive dye (see video data 1), it has been found that to balance this negatively-charged region, protons are released from the exclusion zone region and the Nafion polymer, leaving a net negative charge.

Now that I've built up the general story, its background and significance... given this separation of positive and negative charge, (for my PhD) I hypothesized there was enough separated charge that in water, a significant electrostatic force would be generated between the proton-filled outer bulk water and the negatively-charged exclusion zone water.

Testing this hypothesis was straightforward and is simple to describe, but conducting the experiments themselves required a meticulous sense and very steady hands. My previous work experience and background were therefore a perfect fit for this problem. Simply, I attached a Nafion ring to a custom-made force sensor, which consisted of a pair of cantilever beams I call microribbons. One microribbon was the reference beam, while the other (where the Nafion ring was attached) served as the sensing beam (figure 4-5).



Figure 4: Top view of the experimental setup. A Nafion ring was attached to a custom-designed force sensor. As the exclusion zone formed along the Nafion ring, we hoped the sensing microribbon it was attached to would block the formation of the exclusion zone and the subsequent release of protons on that side. In this fashion, we could test the hypothesis that there is enough separation of charge that the free protons in the bulk water would attract the negatively-charged exclusion zone and the Nafion ring this interfacial water was tightly bound to. Hypothetically, this would result in a deflection of the sensing beam in the direction of the orange arrow.



Figure 5: Real image of the experimental setup. To give you some dimensions, the length of the microribbon is 5 mm in this picture and its width (the height of the Nafion ring) is ~200 microns. The stiffness of the sensor was ~1000 pN/nm. It's hard to get a sense of how stiff this is, but let's just say the sensor is able to measure very small forces. Later, I will use a "roadmap" of force to help you figure out how large these measured forces really are.

When water was added to the chamber and the experimental setup (see video data 2), the microribbon which had the attached Nafion ring deflected in the direction we predicted and confirmed our hypothesis. Basically, we added water, the exclusion zone formed, charge was separated and enough charge was separated that electrostatic force developed and attracted the exclusion zone (which was tightly bound to the force sensor-coupled Nafion ring) to the proton-filled bulk water. I'll leave the extra control experiments and explanations for the paper my research was published in, but for now, see the main results in video data 2, video data 3 and figure 6 below...



Figure 6: TL) With a Nafion ring attached to the sensor, the sensor deflected after water was added to the chamber. Orange arrows point to time points 0, 10 and 20 min. BL) With no Nafion ring attached to the sensor, no deflection was observed optically, nor measured. R) After averaging point-by-point many experiments with and without a Nafion ring, the deflection data were converted to force via the stiffness of the force sensor. The experiment demonstrated that at the end of 20 min, the amount of force measured by the sensor was on average ~22 uN.

So what are the important take home messages?

1) The fact that the sensor deflected not only confirmed our hypothesis, but it opens a new avenue within the field of interfacial water. If interfacial water exists everywhere in biology, then it is possible that charge separation occurs all the time at biological interfaces and hence, long-range and significant electrostatic forces are constantly being generated, which could play a signficant role in protein folding, adhesion and perhaps some of the examples I mentioned earlier (i.e. the force between two glass slides, capillary action of trees, water harvesting, etc.).

2) The force is on the order of 22 uN, which is quite large. To give you some reference, see figure 7.

3) We had to set a time limit (20 min) for the experiment, otherwise, we wouldn't have been able to make multiple measurements to confirm our hypothesis. Nevertheless, even at 20 min, the force sensor was continuing to deflect, indicating the phenomenon had still not reached steady-state (equilibrium).



Figure 7: The road map of force. From left to right, the amount of force generated by your tibialis anterior muscle (opppsite your calf muscle), the amount of force generated by the same muscle in a bunny rabbit, the amount of force (weight) a tennis ball exerts on your hand when you're holding it, the weight of a coin which sits on the water surface, the amount of force you feel when a mosquito sits on your arm, the amount of force generated by 4-6 muscle cells (fibers) when activated, the weight (force) of a 1 mm square cut from standard printer paper, the weight of a single grain of sand 500 um in diameter, the weight of an average mammalian cell, the weight of a single microsphere. For the apparatus I designed, the sensor could measure forces on the order of several hundred uNs to single pNs (the dashed rectangle above). If you were holding the coin in the example above on your little pinky finger, then the measured 22 uN of force from figure 6 corresponds to a force that is 4300x less than the weight of that coin, or about 30x less than the weight of an average contact lens you put in your eye.

Much of my research presented here was conducted in my advisor's lab, where I learned, collaborated, synergized and partied with many talented individuals coming from different countries and representing a diverse range of backgrounds, such as engineering, physiology, botany, physics, medicine and materials science. I could not have done it without them =) This research was also generously supported by a fellowship provided by the University of Washington College of Engineering.



BACKGROUND

Since early on in college, I've always been working in one research lab, or another, trying to get real world, hands-on exposure to science and engineering, much to the suffering of my classwork and grades =( On the other hand, I learned a great deal, both professionally and personally, from my mentors and experiences, and looking back, I would never change a thing =) I'm very lucky and grateful to have mentors who stressed independence, being meticulous and working with your own hands, while still being a team player and having a good time with the research.

In terms of my expertise, I would say I'm 35% physiologist and 65% electrical engineer. I'm very much a benchtop experimentalist and truly enjoy working with my hands. When pursuing research projects, I try to keep a fine balance between taking a pure science versus a pure engineering approach as I believe good engineering comes from good physiological-based problems, and good science comes from good engineering/instrumentation. With my students, I always attempt to bridge classroom theory with practical experimentation, while emphasizing a broad mindset. Based on my own mentors' training methods, I make it a point to understand my students' own natural strengths, research-related, or not, and to help them take advantage of and capitalize on these abilities in their specific research projects.

On a daily basis, you can usually find me tinkering with various kinds of instrumentation. I'm always trying to learn new techniques, or technologies and to be creative with their applications. Specifically, I focus my abilities and attention on instrumentation directly involved with physiological, or bioelectrical phenomena, and experimentation on the microscopic level. My ultimate goal is applying novel instrumentation to neuromuscular research and related pathologies. This is why I have tried to fill up my background with as much experiences as related to bioinstrumentation and muscle/neural research. To give you some idea of the kind of work I've been involved in and projects I've been exposed to, here are a few references (1, 2, 3, 4, 5).

As for myself, I'm originally from the Bay Area and an Indian from a first generation Bengali family. I gravitate towards diverse individuals with humor, imagination and a strong work ethic. My other passions in life are cycling, general acrobatics, science fiction movies and just hanging out with friends exploring the city.

Thanks for visiting and I hope you found it interesting =)





Ronnie Das, PhD
University of Washington
Human Photonics Lab
204 Fluke Hall
4000 Mason Road
Seattle, WA 98195
rdas@u.washington.edu