EXPERIMENTAL PLAN

Principal Investigator/Program Director Williams,Robert W.

1. For streaming video by web microscopists, we are leaning toward use of a single x40 dry objective (NA 0.65) with proven optical resolution of 0.8 µm and pixel resolution as high as .1 µm/pixel. We will experiment with higher-NA dry and water immersion objectives for microscopes intended specifically to acquire very high resolution Z-axis image stacks. The higher the NA, however, the more difficult the implementation and the greater the maintenance problems.

2. For streaming video, two magnifications will be provided simultaneously via dual 3-CCD DV cameras, one with a field of view of 60 x 80 µm, the second with a field of 200 x 00 µm.

3. DC servo motorized stage and motorized Z axis adapted for the carousel slide delivery system.

4. Differential interference contrast optics.

5.Autofocus.

6.Alternative light sources: a low-voltage tungsten bulb with a very extended life for streaming web microscopy and a high-pressure Xenon-Mercury system (1000 h bulb life) for acquiring Z-axis image stacks.
Justification for DIC optics of Nissl-stained tissue. Standard brightfield microscopy of Nissl-stained specimens primarily conveys information about the optical density of the strain. DIC optics reveals differences in the refractive properties of the tissue. DIC optics of stained tissue is thus a multimodal imaging method and as such provides more information about the tissue (Farkas et al., 1995; Glasbey and Martin, 1996) than simple brightfield microscopy. A number of other advantages of DIC microscopy have been enumerated in Williams and Rakic, 1988a,b). One of the most important has to do with the unique modulation transfer function of DIC images (Inou, 1986). In short, DIC optics enhances contrast of high-spatial-frequency image content and suppresses contrast of low-spatial-frequency components of the imagebasically, DIC acts as a high-pass optical filter and edge enhancer. Furthermore, the depth of field of the high spatial frequencies is remarkably shallow, making it possible to optically section DIC through-focus series far more effectively than corresponding non-DIC images. DIC can be thought of as a pseudo-confocal system in which the out-of-focus blur from tissue above and below the focal plane is significantly attenuated (Oldenbourg et al., 1993). It is still not perfect, and many of the problems in high-resolution reconstruction of single cells discussed by Hibbard et al. (1996) remain. ABRUPT CHANGE HERE> The contrast enhancement of DIC also makes it possible to fully open the condenser aperture to match the maximum aperture of the objective. The usual compromise between high numerical aperture and low contrast is thereby avoided. There are no significant countervailing disadvantages of DIC except possibly the cost involved in adding it to the microscope.

Autofocusing the microscope.

Developing efficient autofocusing methods for the iScope is critical both when the microscopes are being controlled during a streaming video session and during the capture of through-focus series (Aims 3 and 4 below). Focus would not be quite so critical if the slides were not so large and if the sections were perfectly flatly pressed to the slide surface. Unfortunately, and the difference in the z-axis coordinate of the top of different sections on a single slide can be up to 100 µm. Thus, without automatic z-axis focusing it will be very difficult for our clients to remain in focus when leaping from one section to the next. What are some possible solutions? Connecting the XL-1 cameras internal autofocusing system with our motorized fine-focus may be an inexpensive and elegant solution. This is particularly practical with the Canon XL-1 because all lens controls, including those that drive the automatic lens focus, are readily accessible (the autofocus lens dismounts on this camcorder). Focus in the XL-1 is driven by the image quality itself, not by an infrared system. John Zemek, president of Applied Scientific Imaging, has offered to help us test the feasibility of this approach. A standard fallback is to use image analysis utility programs to access image quality and independently dive the z-axis. For example, Wu et al. (1996) at Mt. Sinai School of Medicine have developed efficient methods to drive an autofocus mechanism. More than 10 autofocusing algorithms that we will consider are succinctly reviewed by Santos et al. (1997).

DIC and focusing.

One of the minor advantages of DIC optics is that even in completely unstained parts of the section there will be ample phase contrast to generate the high-spatial-frequency signals of a well-focused image. When the image is not over tissue, but is over a blank part of the slide, we do not want the focusing mechanism to hunt forever for focus or to focus on the top of the cover glass. A possible solution to this problem is to force the focus control through one cycle of the maximum z-axis travel permitted for the stagesay 200 µmand if no z coordinate with adequate focus is detected, rest the microscope at the last focused z-axis coordinate. If the lag in control of the fine-focus can be reduced to a sufficiently short interval of < 1 second between command and response, then it may be easier simply to disengage the autofocus mechanisms entirely.

Z-axis calibration.

The PI has spent a considerable amount of time working on this problem. There are several vertical calibration standards in the form of series of polymer films of very precisely known thickness. However, these films have refractive indices that differ from glass and oil (~ n = 1.52), adding complications to the vertical calibration process. Boddeke et al. (1997) have developed a simple and elegant method to calibrate the z-axis using a "tilted slide" preparation. The slide can be a standard calibration slide with a series of closely spaced parallel lines. Moving the tilted slide changes the position of the line that is in best focus as a function of the tangent of the tilt angle. Their protocols permit estimates of backlash in the focusing block, long-term stability, and absolute precision.

Relocation accuracy.
 
Several factors affect how accurately a given coordinate on any slide can be acquired and then reacquired. One factor is the adequacy of the database that describes the slide and the section coordinates. Assuming that the database is correct, then there are a series of physical factors that affect relocation accuracy: how precisely the slide is positioned on the stage, the consistency of encoding on different microscopes, etc. Tucker et al. (1994) achieved a remarkable relocation precision of less than 17 µm between two different microscopes and a precision of less than 7 µm on the same microscope. R. Williams has constructed an encoded stage using a trio of Heidenhain digital length gauges that can consistently reposition a manually placed slide to within 5 µm radius. The ASI DC servo-motor stage that is now fit to the iScope has a relocation accuracy of better than40 µm even when the microscopist wanders all over the 2 x 3 inch slide prior to centering.
For the purposes of this research project, we will initially be satisfied if a client can select a point on one of our low-resolution slide images (25 µm/pixel) and then be delivered to that coordinate within 100 µm on any of the microscopes. This will require a database with an entry for each slide. This Slide-Coordinate database (described in Project 1) will be used to navigate from images in the MBL to points on particular slides. Slide registration on the microscope stage must be precise, and this registration will be improved by the use of the holders that will be designed with this problem in mind. We ultimately hope to attain relocation accuracy of better than 00 µm. The main purpose is to deliver users to single regions that were previously used to generate through-focus series. Relocation accuracy of 20 µm would be required to put the user in a field that overlaps the initial images. Having this level of precision will be useful to confirm and extend datasets generated using only MBL image resources. With the use of absolute slide coordinates and the ability to rezero each slide after it is loaded, we should be able to achieve good relocation precision.

Objective centering.

A single objective will be used in each iScope, simplifying relocation accuracy because we will not have to worry about nosepiece position and centering multiple objectives. However, we will be using multiple iScopes, and it will be to measure offsets of each objective on each microscope so that systematic bias in location among microscopes is minimized. Using a single objective per microscope obviously simplifies this procedure. Each microscope may require its own offset.

Software used in conjuction with the iScopes.  

The current user interface for the iScope is inadequate in many ways. The principal problem is that it is very difficult to navigate across a very large slide (50 x 75 mm) at a magnification at which the field of view is less than 80 by 100 µm. We have three ideas that should greatly improve navigation and which for the most part will be easy to implement:

1. Simultaneous display of a low-magnification image of the entire slide (taken from the MBL) with easy-to-read coordinates, or better yet, a cross-hair that corresponds to the site of the high magnification site being imaged by the iScope.

2. Greatly improved response time between initiating a movement and a video refresh that reflects that change.
 
iScope use arbitration.  

The current implementation is a simple first come, first served system. Any user can work or play with the microscope for a 10-minute period. Registered scientists actively using the MBL and iScope will be able to sign in for periods of 2 hours or more, depending on demand. We will come back to the issue of access in Section 4, but in short, we do not expect access problems because two microscopes will be on reserve specifically to acquire high-power image stacks at locations requested by our clients. It should be possible to define these locations using the 4.5-µm pixel images. Thus, even heavy users should not need long periods of uninterrupted access to the microscopes.

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