Connections: Wide Field Virtual Array Imaging - Innovation at the Microscopic Scale

The Foundation of Disruption From A Scientific Imaging Perspective

Considering scientific imaging, the common application of microscopes for research can span a variety of imaging modalities to examine things ranging from whole organism to atomic level. For example, a light microscope, at best, can resolve an object that is 0.5 microns (5x10-7 meters) in width, approximately the size of a bacterium. Anything smaller (cellular structures, individual proteins, individual molecules) cannot be seen. This limitation is due to the properties of visible light and cannot be breached by improving the mechanics of the light microscope.

Electron microscopy, by its design, employs electrons at a wavelength much smaller than visible light thus enabling structures smaller than a bacterium to be made visible. Numerous software-based techniques are being developed to further push the resolving limits of light microscopy to achieve 'near molecular resolution'. But whether you agree or not, electron microscopy (EM) still reigns supreme for ultimate structural examination of cellular proteins and the cellular microenvironment by virtue of the imaging source wavelength.

However, like traditional light microscopy with fixed objective lenses, pre-defined magnification settings on an electron microscope result in increasing narrowing fields of view as magnification levels are increased. Many researchers not only want detailed structures, they also want to see a larger view, i.e., 'the bigger picture'. Currently, technology exists that enable overcoming several limitations of fixed focal length microscopic observation with a number of unique advantages. One modern application of technology to address and disrupt significant shortcomings to routine light microscopy is Digital Pathology. Likewise, traditional electron microscopic imaging is ripe for disruption and long overdue for change!

Biological EM: A Traditional Workflow

Invented in 1931 and developed for commercial use in 1938, the general workflow for the preparation and viewing of biological samples in a transmission electron microscope has been well-established. Areas of interest are identified and image capture is conducted at low magnifications in order to present general cellular architecture containing low resolution but having a wide field of view (FOV). Higher magnifications are used to focus on areas of interest which contain high resolution data but have a narrow FOV.

Historically, photographic films were used for image capture. These film negatives were processed and then printed via photographic darkroom techniques. Now, few labs still employ film methods and most have converted to digital imaging. However, the classical, and time-consuming workflow of capturing individual images continues and results in a cumbersome management of image files well into the hundreds for any given investigation. In addition to the mechanics of image capture and storage, the image file naming system must typically comply with a standardized nomenclature for ease of identification at a later time point, a process which is also cumbersome and time-consuming. The images are usually reviewed on an individual basis and select ones chosen for presentation purposes as 'representative' which introduces bias into the scientific process. While being sufficient for purpose (aka 'easy'), the resulting collection of image files are static, non-interactive, and any individual image file is disconnected from the others (see Figure 1A).

Moving from Static to Dynamic: Wide Field Imaging Array Methodology

"A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur"

-Frank G.A. Fass

A team of researchers led by Dr. Frank Faas went so far as to create a whopping 281 gigapixel (921,600 x 380,928 pixels) data set from a total of 26,434 images! Why would Dr. Fass do this you might ask? Because he understands that the nanometer scale at which molecular events are occurring are 10,000x smaller than a single cell. He sums this up very succinctly: "A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur". This ability to resolve smaller and smaller detail results in an increasingly smaller and smaller FOV. Providing both mechanistic detail and overall cellular context is an unspoken issue that generates both an associated effort of image file organizational overhead and an increasing level of research frustration.

As Dr. Fass discovered, the answer to the dilemma of context and detail is wide-field imaging technology! This elegant and highly efficient method produces not a collection of unconnected single images, but a single "image data set" comprised of an array of tens to tens-of-thousands of high resolution images each with a narrow FOV, which are then stitched together 'on-the-fly' in a tiled format and presented to the viewer as a single virtual image. A combination of toolbar menu items and computer mouse buttons allow for panning and zooming. The nature of these datasets provide context and connection, enabling the end-user to easily transition from low-resolution overview (at the 'top' of the image pyramid) to a detailed examination of specific features (at the 'bottom' of the image pyramid) (see Figure 1B).

Similar to Google Earth, a multitude of high resolution, albeit small and individual FOV image tiles are delivered to the end-user as a single virtual image which provides both overall context and structural detail, all in one highly manageable package.

Figure 1A. In light of modern technological advances, traditional methods of image capture and the laborious management of thousands of individual image files are highly inefficient yet continue to persist well into the 21st century.
Figure 1B. Automated array imaging of a single large area at high magnification (containing high resolution data) and reformatted into a pyramidal image format structured into groupings of image tiles at a variety of resolutions (aka 'Deep Zoom') which provides the end user with ease of both a low-resolution wide field view (providing context) and requisite high-resolution detail required for examining minute features.

This innovative imaging technology solves a number of problematic issues associated with traditional EM work processes including:

View examples of zoomable array imaging in my Zoomable Digital Art Gallery!