RESOLUTION TEST PATTERNS

Measuring the resolution capability of a digital camera or film scanner for photomicrographs ideally requires a microscope resolution test slide.  The lowest magnification objective is commonly a 5X 0.10 NA (4X 0.10 NA used with a 1.25X tube factor in some Zeiss microscopes).  This lens should resolve 300 line pairs per mm in object space forming an intermediate image with 60 line pairs per mm resolution. A 3X3” chrome on glass resolution test chart with patterns from ¼ line pair per mm to 600 line pairs per mm is available and quite costly.  I previously used this test chart with a 0.10 NA Zeiss objective and verified the theoretical resolution with both the MegaPlus 1.6i/AB digital camera and recording on Polaroid Type 55 film.   Low cost test patterns with a small field size with line patterns covering a range of 300 to 3000 line pairs per mm could be made by electron lithography but are not available because of lack of market demand.  An alternative test method is to use a high resolution macro lens on the camera to image a pattern like the NBS Microcopy Resolution Test Chart in the 89X114 mm field size of a 4X5 Polaroid film print for a 1280X1024 pixel CCD sensor (89X133 mm field for a 1534X1024 CCD sensor or film scan of 35 mm format) as done for Figures 6 & 7.

click image to enlarge (295K) FIGURE 6 Comparison of digital camera resolution with Kodak Photo CD resolution using the highest resolution portions cropped from the original images.

click image to enlarge (201K) FIGURE 7 Resolution test results for the Canon CanoScan FS2710 film scanner.

DIGITAL CAMERA AND KODAK PHOTO CD RESOLUTION

The test result for the Mavica camera demonstrates the significant loss in spatial resolution due to the color mosaic filter over the CCD sensor when compared with the grayscale capture by the MegaPlus camera.  The pattern results with the MegaPlus camera demonstrate that line patterns oriented orthogonal to the pixel array are more difficult to record without an alias line problem.  The alias lines are a moiré fringe effect from close alignment of the pixel spacing with the line patterns in the optical image when the spatial frequency of the line patterns approach the pixel spacing.  The moiré effect was accentuated with the MegaPlus results because the original images were high pass filtered to improve the apparent resolution.  The position of the test pattern during imaging was purposely adjusted to avoid this effect for the orthogonal orientation of the 5.6 line pairs per mm pattern.  The orthogonal patterns from the Photo CD scans show less evidence of this effect, perhaps mainly because they were not sharpened. The solution to the alias line problem is to capture the same image with a larger number of pixels.  Since my home microscope and the older Zeiss Universal microscope I used before retirement have a maximum intermediate image field diameter of 18 mm, a 1534X1024 pixel array meets my requirements for resolution and field size. The 3000X2000 pixel resolution result indicates that the 35 mm film to digital method using this Kodak Photo CD file size cropped to 2048X2048 pixels should be adequate to cover an 18X18 mm portion of a 26 mm intermediate image field diameter for those fortunate enough to own a modern research microscope.  The 11 line pair per mm pattern just resolved in the 3000X2000 pixel file corresponds to 40 line pairs per mm in the film image.  The blurring effect of limited film resolution is showing up at this higher spatial frequency. 

CANON FILM SCANNER RESOLUTION

Film scanners for use with fast PC’s with 8 megabyte graphics cards, 1600X1200 pixel monitors and CD writers are now affordable for home use.  I have a large collection of family 35-mm slides and negatives and wanted to digitize them with storage on CD’s for distribution to the younger members of the family so their past will not be lost. The cost savings of owning my own film scanner versus having the film images scanned to Kodak Master Photo CD’s justified purchase of a Canon CanoScan FS2710 film scanner.  A maximum resolution scan for this scanner is 3888X2720 pixels. This pixel array size is not adequate to record the finest spatial frequencies in very high quality slides and negatives, but should achieve about 52 line pairs per mm resolution.  Some 35 mm film images exceed 63 line pairs per mm resolution.  The need to digitize the highest spatial frequencies on 35 mm film was met by the Kodak Professional Photo CD 6000X4000 pixel scans.  Film scanners with this resolution are now available, but quite expensive for home use.  Owning the CanoScan film scanner is a big advantage for my low budget, home microscopy.  I can record on 35 mm color negative film and use locally available 1 hour processing of the film prior to scanning it at home.  Scanning the negatives rather than photographic prints from the negatives avoids the problem of variable print quality, which the mass market probably would not notice.  The disadvantage is that I have to bracket my exposures to be sure of a good film image to scan.  Naturally I have compared the resolution of the Canon scanner, using the same resolution test slide, with the Kodak Photo CD results in Figure 6.  The finest pattern on the NBS test chart just resolved on the film is 14 line pairs per mm pattern corresponding to 51 line pairs per mm on the slide.  PhotoShop LE comes with the scanner software and was used to downsize the large file size to 1940X1360 and 1534X1024 pixel files.  A photomontage of the finest patterns in these files is shown in Figure 7.  It is important to note that the other than 2:1 downsizing used to obtain the 1534X1024 pixel file introduces noticeable alias line artifacts less evident than in the Photo CD result shown in Figure 6.  The 30% loss in spatial resolution resulting from use of a CCD camera with a Bayer color mosaic filter, such as the Nikon Coolpix 995, can be compensated by doubling the number of pixels used to capture the same image field; this is shown in Figure 8.  The bicubic resampling procedure of PhotoShop can then be used to maintain the spatial resolution using half the number of pixels. This step is equivalent to reducing the empty magnification.

click image to enlarge (270K) FIGURE 8 Resolution Test of the Nikon Coolpix 995 Camera.

SUMMARY

Film-to-digital and direct digital capture, with a digital camera, can clearly be acceptable methods for obtaining high quality digital photomicrographs that match the field size and resolution of traditional photomicrograph prints.  Unfortunately there are no national or international standards dealing with this important transition in technology.  The Photographic and Imaging Manufacturers Association (PIMA) is participating in an ISO TC42 effort to develop such standards. Their preliminary efforts concentrate on using a resolution test pattern of their own design and alias effects are considered. Alias effects are inherent in the digital capture, but should not be worsened in subsequent image downsizing and desktop publishing.  Understanding the operating principles of the light microscope and using them to obtain an optimum optical image for recording is vital for quality digital images.  Unfortunately the users who think software can “fix” the resulting defects in their images do not appreciate this requirement.

REFERENCE

Clarke, T. M., “Digital Imaging in the Materials Engineering Laboratory,” THE MICROSCOPE, 1998, 46(2), 85-100.

Clarke, T. M., “Building an Affordable Universal Student Microscope,” THE MICROSCOPE, 2000, 48(1), 19-39.

K. D. Moller, OPTICS, University Science Books: Mill Valley, Ca., 1988.

Edwards, Parulski, & Holm, “Setting Standards--Developing Standards in Electronic Imaging”, PEI, February 1998, 48-52.