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

The Galleries:

Photo Gallery
Silicon Zoo
Pharmaceuticals
Chip Shots
Phytochemicals
DNA Gallery
Microscapes
Vitamins
Amino Acids
Birthstones
Religion Collection
Pesticides
BeerShots
Cocktail Collection
Screen Savers
Win Wallpaper
Mac Wallpaper
Movie Gallery

Image Brightness
Interactive Java Tutorials

Numerical Aperture and Magnification Effects

In optical microscopy, image brightness is governed by the light-gathering power of the objective, which is a function of numerical aperture. Magnification also plays a role in determining the brightness as explored in this interactive tutorial.

Interactive Java Tutorial
ATTENTION
Our servers have detected that your web browser does not have the Java Virtual Machine installed or it is not functioning properly. Please install this software in order to view our interactive Java tutorials. You may download the necessary software by clicking on the "Get It Now" button below.

 

The tutorial initializes with the objective magnification set to 10x and a numerical aperture value of 0.15, which is adjustable using the Numerical Aperture slider. As the slider is translated to higher numerical apertures at a fixed magnification, the light cone size changes and the image brightness (intensity) values for transmitted and reflected light illumination modes are computed and displayed in red letters. To select another magnification, use the Magnification pull-down menu to choose an objective in the range between 10x and 100x. Note that at lower numerical apertures and magnifications, the transmitted illumination intensity is greater than that observed with the same objective in reflected light. These values reverse themselves at higher numerical apertures and magnifications with the reflected light intensities becoming much greater than those observed in transmitted light.

Image brightness is directly proportional to the objective numerical aperture and inversely proportional to the square of the lateral magnification:

Image Brightness µ (NA/M)2

where NA is the objective numerical aperture and M is the magnification. The ratio given in the equation above expresses the light-gathering power of the objective in transillumination (note: the case with epi-illumination is somewhat different, as discussed below). In general, objectives with high numerical apertures are also better corrected for aberrations. Thus, for the same magnification, higher numerical aperture objectives collect more light, produce a brighter and better-corrected image, and the overall image is better resolved.

When an objective is used in transillumination, image brightness decreases rapidly as the magnification increases. In contrast, utilization of a specific objective for epi-illumination produces increasingly brighter images as the magnification increases. The terms F(trans) and F(epi) refer to the light-gathering power of an objective and were calculated according to the following equations:

F(trans) = 104 • NA2/M2

F(epi) = 104 • (NA2/M)2

In theory, the intensity of illumination depends on the square of the condenser numerical aperture and the square of the demagnification of the light source image (in effect, the field diaphragm image becomes brighter as it is made smaller, according to the square law). The result is that brightness of the specimen image is directly proportional to the square of the objective numerical aperture as it reaches the eyepiece (or camera system), and also inversely proportional to the objective magnification. Therefore, when examining specimens in transmitted light, changing the objective without altering the condenser affects image brightness in response to changes in numerical aperture and magnification.

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.


BACK TO IMAGE BRIGHTNESS

Questions or comments? Send us an email.
© 1998-2013 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: Wednesday, Mar 26, 2014 at 01:23 PM
Access Count Since October 22, 2001: 18580
For more information on microscope manufacturers,
use the buttons below to navigate to their websites: