Achieving High Resolution Digital Photomicrographs Revision 10/10/10

Light microscopes use specialized glass lenses (objectives, eyepieces, glass prisms and other glass elements) to magnify tiny objects for observation by human eyes, and in some cases, automated inspection devices. They are called light microscopes because they employ light (most often generated by a bulb with a glowing filament inside, although LED lights are starting to be supplied by some manufacturers). Other more specialized light microscopes may use lasers as the light-generating source.

Man’s capabilities in producing high quality optical grade glass is well established however it is also well known that optical glass suffers from certain deficiencies (called aberrations) when it comes to faithfully rendering a high quality magnified image of the subject in question. Certain manufacturing and engineering methods have been successfully employed to correct for these aberrations, therefore the modern optical microscope is an unparalleled instrument for the investigation of the microscopic world. Further discussion of optical aberrations is unnecessary for the present purpose, but the reader is directed to examine the superbly detailed discussions provided by Florida State University’s Molecular Expressions website. See:http://micro.magnet.fsu.edu/primer/index.htmland also (for a discussion of optical aberrations):http://micro.magnet.fsu.edu/primer/lightandcolor/lenseshome.html

Until recently most photos taken through a microscope (called micrographs) have been taken using photographic film because the size of the silver halide grains on film are very small, allowing for very high resolution images to be made. However with recent advances in digital cameras and computer software (that allows images to be quickly “snapped” and imported onto the computer), digital imaging is quickly becoming an inexpensive alternative to traditional film. Digital camera resolution is now approaching the resolution of film (or maybe even better) and some of the many benefits of employing a digital camera include the ability to instantly “snap” and throw away as many images as one likes without the hassle (and cost) of developing multiple rolls of film. Digital images may also be quickly and easily labeled with arrows, scale bars and other notations while in the computer. Additionally most journal editors today prefer to receive submitted manuscripts and figures, including micrographs, via the digital method.

The question is, however, are digital images as highly resolved as film images?

One obvious answer is that the camera will only perform as well as the microscope that is delivering the optical image to the camera in the first place. If your microscope does not resolve images well, your camera (no matter how many mega-pixels it has) will do no better. Therefore a short discussion of microscope resolution is in order.

In short, the resolving power of a microscope objective lens is the ability of that lens to gather as much light from the subject under examination, and to clearly reproduce a magnified image of the tiniest details possible. A host of technical factors play into this

capability and our discussion could become highly theoretical very quickly. The reader is again directed to a marvelous and well-illustrated discussion of the factors that influence lens resolution at the Molecular Expression website: http://micro.magnet.fsu.edu/primer/anatomy/numaperture.html

However, to simplify the discussion – imagine two tiny dots on a microscope slide that are one-tenth the diameter of the period at the end of this sentence. The closer you can bring those two dots to each other while still observing through the microscope that they are indeed two distinct dots, the higher the resolution of your microscope lens. In other words, the smaller the distance d between two tiny dots, the higher the resolution capability (numerical aperture) of your lens. Numerical aperture (NA) is a number, usually engraved on the outer metal tube of your objective lens and it can range from 0.025 to 1.4 for very good (and usually expensive) lenses. The higher the NA, the better that lens will perform in resolving two distinct points just as they are. That, in simple terms, is a manageable definition for lens resolution.

Another obvious question is, how do I measure the resolving capability of my lenses?Can’t I just read the NA on my lens and know that it is good or bad?

The answer is, not really. The quality standards of microscope manufacturers vary widely. Just because your 50X lens may have 0.95NA inscribed on it does not mean it is a well-designed and high-resolution lens. The best way to be sure is to purchase a calibration standard such as that sold by Ted Pella Incorporated, for example (see: http://www.tedpella.com/metro_html/metrochip.htm) and take some images using your microscope lenses and various digital cameras.

For the Jenoptik C14+ high resolution digital camera, the flowing micrographs were made using several objectives and a metal foil calibration standard that has a 1.5 micron bar and gap on it (see arrows on the next figures).

DIGITAL MICROGRAPHS:

25X Carl Zeiss Jena Planapochromat Objective, NA = 0.65. Total magnification 250X.Note the small white bar at the bottom right of the “staircase” (see arrow). The white bar is 1.5 microns wide. The black gap just to the left of it is also 1.5 microns wide.

Another scale is here reproduced at 250X magnification. This scale has 3 micron wide bars.

50X Carl Zeiss Planapochromat Objective, NA = 0.95. Total magnification 500X. The white bar (at arrow) and black gap are each 1.5 microns wide.

Another scale is here reproduced at 500X magnification. Once again these bars are 3 microns wide.

100X Carl Zeiss Apochromat Objective, NA = 1.40. Total magnification 1000X.

Another scale is here reproduced at 1000X magnification. Once again the scale bars are 3 microns wide.

Note that with each of these objectives the Jenoptik C14+ camera clearly and easily resolves the 1.5 micron bar, and would probably resolve at least half of that size (at 0.75 microns) or even better. Therefore the combination of good quality lenses and the C14+ Jenoptik camera offer high quality resolution equal to or possibly even exceeding film resolution for microscope use.

Another standard tool used for calibrating each microscope objective for use in measuring small objects under the microscope is the stage micrometer (or stage graticule). A stage micrometer is a glass slide upon which is etched or painted a finely divided scale of lines set apart in precisely measured steps. The reader is directed to a commercial website which features a variety of stage micrometers for purchase: http://www.emsdiasum.com/microscopy/products/magnifier/stage.aspxAlso see: http://www.pyser-sgi.com/images/thumbnails/Graticules/Stage%20Micrometers%20web.pdf

For example, a calibrated distance measuring 1mm can be divided by 100 lines, thus rendering a series of finely calibrated spaces. In this case, each space between two lines is 1.0 divided by 100 = 0.01 mm or 10 microns. The next image is a high magnification micrograph taken with the Jenoptik C14+ and shows a series of 10 micron spaces.

1250X magnification on Carl Zeiss Jena microscope. These are 1.25 micron lines. The spaces between the lines are 10 micron spaces.

It is because of this example of the resolution capability of the Jenoptik camera, together with the very easy to use (and powerful software), that I employ Jenoptik cameras in my Imaging Laboratory at California State University , Northridge, Biology Department. In fact, I have just successfully installed a Jenoptik C14+ camera on my Carl Zeiss EM-10 Transmission Electron Microscopes. I am achieving superior resolution on my electron microscope to cameras that cost 5-10 times more than my Jenoptik camera.

Mark H. Armitage, M.S., Ed.SElectron Microscope LaboratoryDepartment of BiologyCalifornia State University, NorthridgeMark.armitage@csun.edu