In an article titled “Film Fades Out” (Photonics Spectra, March 2006:32), Hank Hogan makes an important point: the digital wheels of change are rapidly moving forward. Nikon Corporation’s decision to substantially decrease attention on film camera business and a similar announcement by Konica Minolta Holdings, suggesting their withdrawal from the film business by March of 2007, supports this common harbinger.

As can be expected, economic forces fueled by the capability of digital cameras now drive the market. This is not only true of the consumer sector, but is increasingly true of the scientific and biomedical imaging market as well.

The decision to move from film technology to that of the digital arena usually takes many steps to complete. It may very well start as a move to digitize an optical light microscope, or even to purchase an inexpensive scanner for digitizing film. However, these small steps will eventually lead to the ultimate decision to leave the darkroom and film technology behind and proceed with caution into the digital world. There are many questions one should ask during this transition. Often, the usual list of the advantages and disadvantages must be considered.

Of course, film has been around for a long time. While affording fine grain and allowing huge enlargement possibilities, film demonstrates a wide range of sensitivity suited for diverse specimen stability, electron exposure, and flexibility in processing. In additional film is inexpensive when considered on an individual, per sheet basis.

On the other hand, film is a very labor-intensive media compared with that of digital technology. It is truthful to say there are three major costs in any electron microscopy facility: 1) space/ overhead; 2) equipment service contracts; and 3) material and labor costs associated with the preparation, documentation, processing, and analysis of film based imaging. Number three is by far the greatest expense.

How can we qualify this statement? We all know fixed labor costs and variable material costs collectively make up a large portion of the budget. This includes the time involved in ordering supplies, making and disposal of processing chemicals, labeling and archiving negatives and prints, resulting in an overall reduction in productivity associated with labor and materials. Additional labor is required if one wishes to share film images with colleagues, thus requiring the labor of scanning images or printing additional images.

There are three primary benefits by which digital imaging can increase productivity. These include technical benefits, physical benefits, and fiscal or productivity benefits.

Technical benefits:

Digital imaging creates a seamless segue from the imaging components of the microscope to the through put process of examination, documentation, and analysis.

The use of digital technology reduces the necessity to break from the microscope interface, thus allowing a seamless flow of information. In combination with the physical components of the camera/microscope system, the software used to basically acquire, enhance, and save the images is a key consideration. Software drives the frame rate, clock speeds, binning rates, and an incredible array of functions required of the CCD sensor and other camera components. It is within this software wherein lies the unique qualities not found universally among camera manufacturers.

In addition to software driving the ‘the activities behind the scene’, options to save data in a variety of image formats and styles are increasingly important for multi-user facilities. The ability to view images on a computer monitor, transform the images into a user designed page layout, print as a hard copies, send to colleagues via email, share via digital streaming video, or post on a website are important considerations.

Another consideration falls in the category of simple measurements and image analyses, as well as saving this data in separate venues such as spreadsheets or written documents.

Physical benefits:

Space requirements are frequently evaluated and are either fiercely guarded or redistributed as an extremely valuable commodity. The physical space required for film and print processing, as well as storage, has rapidly become the new battlefield among facility operators and administrators looking at dollars per square foot cost returns.

The darkroom to digital transition removes the necessity for a dedicated film developing/ darkroom space. In addition, storage of processing chemicals, negatives and photographic prints is reduced or eliminated. The space regained from the darkroom can be converted to an image analysis work area or any number of possibilities.

Fiscal or productivity benefits:

For obvious reasons, the benefit of better utilization of facility space leads to fiscal benefits. However, what other fiscal benefits can be derived from the move to digital imaging?

Obviously, labor related activities such as loading and unloading film into the microscope, the purchase of film and paper, mixing, and disposal of photographic chemicals, film and print processing, and the labeling of glassine envelops and prints can be substantially reduced or eliminate with digital imaging. This reduction in fixed labor costs creates a more efficient means of productivity.

However, have you considered other, less tangible benefits? Digital imaging eliminates the need to vent the camera chamber and/or column, thus maintaining a high vacuum operating environment. This action translates into better imaging, cleaner apertures and specimen, and longer filament life. Fiscally, this translates to a reduction in maintenance, possibly encouraging a reduced service contract.

Now that we have discussed the benefits of digital applications, let’s discuss the process of digital imaging and indirectly re-evaluate the basic principles learned by every microscopist in the life sciences.

In transmission electron microscopic applications, as is the case in optical light microscopy, we in the life sciences are dealing with phase producing, low contrast objects or specimens. Consequently, we must go through the process of converting low contrast specimens to high contrast, amplitude-producing specimens.

Some processing steps provide the dual function of fixation and contrast, i.e., osmium tetroxide or en block uranyl acetate.

Other steps, such as using a small diameter objective aperture, low accelerating voltage, and thicker cut sections to improve contrast can actually reduce or have a negative impact on the resolution of the specimen.

Consider the following questions:
• Do you routinely use en bloc U.A. staining, as well as uranyl acetate and lead citrate to increase contrast of your sections? Why?
Within the parameters of fixation and contrast, the use of osmium tetroxide without the use of en bloc uranyl acetate can be adequate for imaging. Less staining translates into greater resolution or at least an increase in resolvability. In addition, less staining also translates into less work and less chance for stain precipitate.

• What is the routine thickness of your EM sections? Gold, yellow, silver?
Thinner sections mean better resolution in the z axis (the electron beam direction). This combination can be incorporated with the use of higher accelerating voltages.

• What is the importance of accelerating voltage?
Higher accelerating voltage means less electron scattering and better image resolution. However, increasing the accelerating voltage, for example from 60kV to 80kV, will create less observed specimen contrast on the fluorescent screen.

• What objective aperture do you routinely use to increase contrast?
The selection of a smaller diameter objective aperture can indeed increase contrast, but can also restrict the observable field-of-view at lower microscope magnifications and image resolution.

Consequently, the microscopist must take into consideration the dynamic range of the CCD digital camera. There is inherently more contrast observed on the monitor than can be appreciated on the fluorescent screen. It is this fact that allows the microscopist to reduce staining times, increase accelerating voltage, and increase objective aperture diameter (Figures 1 and 2).

Fig.1 (Left) Renal capillary loop: Low magnification image.
Fig.2 (Right) Medium magnification image. Unstained section taken at 80kV with a 90um objective aperture. (Gatan MSC600CW).