Microscopy Primer
Light and Color
Microscope Basics
Special Techniques
Digital Imaging
Confocal Microscopy
Live-Cell Imaging
Microscopy Museum
Virtual Microscopy
Web Resources
License Info
Image Use
Custom Photos
Site Info
Contact Us

The Galleries:

Photo Gallery
Silicon Zoo
Chip Shots
DNA Gallery
Amino Acids
Religion Collection
Cocktail Collection
Screen Savers
Win Wallpaper
Mac Wallpaper
Movie Gallery

Modulation Transfer Function
Interactive Java Tutorials

Test Target Intensity Scans

The optical performance of a light microscope is usually determined by its response when imaging high-contrast periodic line gratings that are employed to measure contrast transfer functions. An ideal test target (MBL-NNF) for this purpose was developed at the Marine Biological Laboratory in Woods Hole, Massachusetts in collaboration with the National Nanofabrication Facility at Cornell University.

In addition to the periodic line spacings, the MBL-NNF test target features both single and paired lines and dots and a Siemens test star (Figure 1), which allows a careful analysis of the diffraction patterns generated by these microscopic features as a function of illumination mode.

The interactive tutorial demonstrates intensity scans made from images of the periodic lines present on the MBL-NNF test target measured with a plan apochromatic objective. Gratings were imaged by a scanning laser microscope in the non-confocal transmission mode with a Nikon 40x/0.95 NA objective at a laser wavelength of 515 nanometers and a condenser numerical aperture of 0.82. Intensities were averaged over the dimension parallel to the grating lines. To view the entire range of intensities, move the slider to either the left (lower spatial frequencies) or right (higher spatial frequencies). Note that the intensity is dependent upon spatial frequency, being greatest at low frequencies and lowest at the higher frequencies.

Modulation of the output signal, the intensity of light waves forming an image of the specimen, corresponds to the formation of image contrast in microscopy. Therefore, a measurement of the MTF for a particular optical microscope can be obtained from the contrast generated by periodic lines or spacings present in a specimen that result from sinusoidal intensities in the image that vary as a function of spatial frequency. If a specimen having a spatial period of 1 micron (the distance between alternating absorbing and transparent line pairs) is imaged at high numerical aperture (1.40) with a matched objective/condenser pair using immersion oil, the individual line pairs would be clearly resolved in the microscope. The image would not be a faithful reproduction of the line pair pattern, but would instead have a moderate degree of contrast between the dark and light bars. Decreasing the distance between the line pairs to a spatial period of 0.5 microns (spatial frequency equal to 2000 lines per millimeter) would further reduce contrast in the final image, but increasing the spatial period to 2 microns (spatial frequency equal to 500 lines per millimeter) would produce a corresponding increase in image contrast.

The limit of resolution with an optical microscope is reached when the spatial frequency approaches 5000 lines per millimeter (spatial period equal to 0.2 microns), using an illumination wavelength of 500 nanometers at high numerical aperture (1.4). At this point, contrast would be barely detectable and the image would appear a neutral shade of gray. In real specimens, the amount of contrast observed in a microscope depends upon the size, brightness, and color of the image, but the human eye ceases to detect periodicity at contrast levels below about three to five percent for closely spaced stripes and may not reach the 0.2-micron limit of resolution.

Contributing Authors

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

John C. Long and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


Questions or comments? Send us an email.
© 1998-2019 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, scripts, or applets may be reproduced or used in any manner without permission from the copyright holders. Use of this website means you agree to all of the Legal Terms and Conditions set forth by the owners.
This website is maintained by our
Graphics & Web Programming Team
in collaboration with Optical Microscopy at the
National High Magnetic Field Laboratory.
Last modification: Thursday, Jun 14, 2018 at 03:35 PM
Access Count Since December 10, 2000: 21607
For more information on microscope manufacturers,
use the buttons below to navigate to their websites: