Talk:Collaboration:Shriner

Reviewers' comments from the 1st stage of selection were:

1. How does this extend beyond what you have already done?

2. Please discuss how this study will affect human health.

These are quite important questions.

The answer to the first question was addressed in the past meeting. Thus, the answer to the second question is discussed in the following.

(A) More shot-term, direct impact in the clinical setting;

I will list up a few example, out of so many.

In the field of cardiac surgery, there are two famous operations; Maze Operation for atrial fibrillation, and Batista Operation for dilated myopathy. Both operations are about dissecting out some unnecessary/harmful portion of the heart either to improve the cardiac output, or to treat arrhythmia. Both operations are, however, extremely difficult and requires skill, especially when we try to evaluate what part to dissect. Clearly, no one wants to remove too much of the healthy part of the heart. So, question has always been, how one can distinguish the normal portion and abnormal portion?

If one can somehow "see" the focus of arrhythmia, it becomes so much easier to determine which portion to cut out by Maze Operation, based on such information. If one can visualize which part of the left ventricle has poor contractility, has more fibrosis than other parts, or has poor shedding of blood vessels due to such fibrotic changes, it becomes easier for one to decide the dissection area.

In the experimental setting, we are already at the stage where we are, on the regular basis, observing calcium waves in detail, measuring practically all the different parameters of microcirculation, movement of the myofibers, detailed morphology, cell death, autophagy, extent of NO production, ROS production, etc, all in the muscles of live mice.

It would be beneficial to extrapolate these techniques in the future to serve to improve those difficult cardiac surgery.

(B) Impact in the long-term

This grant proposal is about a completely new technology, and the potential application in the human health is virtually infinite.

The impact on the study of pathophysiology in the cardiac diseases with this new technology is enormous. This space is too small to list up all such diseases or potential experiments. However, the study of pathophysiology is more about basic science, though it is difficult not to imagine that such finding from basic pathophysiology impacting heavily on clinical science.

Very important things one have to remember are, MACRO-scopic blood flow measurement and analysis of MICRO-circulation represent completely different circulatory state. These two are, sometimes, discrepant. There will be many disease mechanisms or physiological regulation that can be very well overlooked, if one is only studying macroscopic blood flow. In vivo microscopy is thus indispensable. Secondly, we might, under the current medical technology, prefer to take biopsy from the tissue of the patients or animals and study the histology of those tissues. However, once we take the tissue out, all that is left there is structure, but not function. If functions of, let's say, microcirculation, calcium signaling, NO production, etc, were essential in the pathogenesis of some disease, histological analysis will not be covering such mechanisms no matter how beautiful slide staining one can make. Medical science will, soon in the future, will come to the stage where those "functions" play important roles in the explanation of many diseases. Such discussion goes on and on and on. The interaction between the tissue and the blood vessels, the interaction between the nerves and the blood vessels, the intercommunication between myocyte and the blood vessels, etc, etc. Those can only be studies by in vivo methodologies in the live animals. Cardiac diseases will be no exception. In vivo microscopy is one of such research tools that might fulfill these purposes.

(C) About the future NIH Grant proposal: We should study the cardiac complications in the Duchenne Muscular Dystrophy. We should also study ischemic heart disease. These will be discussed later on.

From the second stage:

Is the technology developed for too small size of motion correction for the mouse heart, and thus unsuitable for application for big-sized heart in the human setting?

The simple correct answer after good amount of deliberation is, NO. This technology can very well cover the range of movement of the human heart beat, at least in the x-y dimension.

Well, very often, when somebody for the first time hears about our motion control, he/she might, by mistake, think we are trying to motion-correct by moving the objective lens of the microscope. The truth is, we are not moving the lens, but we are moving the mirrors that send the illumination beam to the objective lens. Therefore, it is the angle of the beam, but not the lens that moves. If one can remember the basic optics, the angle at the oblective lens represents the position of the focal plane.

So, this will lead to the equation, the dynamic range of the motion-correction is a function of angle x focus distance.

We are using long-focal distance, sometimes, like >4cm. The moving range thus can be as large as >2cm. It can probably become much larger, if we design such optical light path.

Even under the current setting, this range of motion will be big enough to cover that of human heart beat.

Another point is, in the cardiac surgery field, the surgeons are using 'stabilizers' during the last couple of decades. These include devices such as, 'Heart Exposure' from Maquet, and many other 'positioners'. When these stabilizing devices are used, the motion range of the heart becomes so small, and the srugeons can perform operations without stopping the heart beat. The motion range is virtually <0.5cm. Our microscope can easily cover this dynamic range.

Can't the heart movement be oblique? We seem to be only discussing the image caption in the xy plane, and thus our technology might be useless for the actual volumetric observation of the human heart?

We are already aware of such potential problem, and have been discussing this issue intensely. To overcome this problem, we are already trying to install multiple PMTs (detectors) in order to attain multi-focus confocal microscope. This new setting will be capturing multiple slices simultaneously. Once the image is captured, the computer will reconstitute the image into 3D. Thus, by using this technique, we will be able to capture obliquely aligned cells or structures in the heart.

Can't we just take out the tissue from the patient and study the histology?

This was a wonderful question. Of course, histology is important. Histological diagnosis has many strong points over our in vivo microscopic method. Our method has, however, many strong points, too, that will compensate the technical difficulties of the tissue histology. For example, if one want to analyze the 'function' of microcirculation, instead of the structural information of the microvessels, histology cannot provide too much information. One has to study the RBC flux of a capillary, of an arteriole in order to understand oxygen supply to the local. The velocity and flux can be sometimes discrepant, and can be important in many physiological regulation or the cause of some diseases. The blood flow can vary from one capillary to another, an important discussion basis for studying the distribution of blood flow, or the functional capillary density. Such information can only be attained by in vivo microscopy. For example, in the liver or in the lung, we know from the physiology textbook that these organs can sometimes show a misterious status, so called 'functional AV shunt'. Functional shunt is a shunt where the arterial blood bypasses into the vein without any obvious structural AV anastomosis. Such elusive pathological status can theoretically very well explained by dysregulation of microcirculation. For example, it is known that when there is irregular distribution of functional capillary, oxygen supply to the tissue becomes dramatically so poor even when the MACRO-scopic total blood flow remains the same. This means, functional shunt can very well derive from the dysregulation of microcirculation. This is only one example among so many other reasons why in vivo microscopic diagnostic methods becomes more and more important in the future. Another example is, think about cases where one wants to detect and evaluate the complications of diabetes. Thus far, most common way has been either histological observation from biopsy, or MACRO-scopic methods. With these existing diagnostic tools, one can only detect abnormality when complication becomes so much, starts to affect the vascular structure, or becomes irreversible. By studying the 'function', or responsiveness of the microvessels by in vivo microscopy, it is likely that we can detect the complication at much, much earlier stages.

Does our technology compete with the endoscopic microscope?

It will not compete with endoscopic microscope. In fact, there is still no endoscopic microscope that incorporated the feed-back motion correction system to stabilize the image. Our technology will in fact have a synergestic effect on the improvement of the pre-existing endoscopic microscope, and vice versa.

Is our technology designed more for open chest surgery, or for endovascular catheter-mediated method?

At this moment, with the specific grant proposal, it is designed for open-chest surgery for mice, and in the near future, for humans. However, catheter-mediated method should have a great impact on the study of atherosclerosis and vascular biology. Further discussion is going on how we can extraporate our technology into such direction. Hopefully, we might be able to find an appropriate collaborator with a background in such field.