Achieving high resolution digital photomicrographs
Achieving High Resolution Digital Photomicrographs Revision 10/10/10Light microscopes use specialized glass lenses (objectives, eyepieces, glass prisms andother glass elements) to magnify tiny objects for observation by human eyes, and in somecases, automated inspection devices. They are called light microscopes because theyemploy light (most often generated by a bulb with a glowing filament inside, althoughLED lights are starting to be supplied by some manufacturers). Other more specializedlight microscopes may use lasers as the light-generating source.Man’s capabilities in producing high quality optical grade glass is well establishedhowever it is also well known that optical glass suffers from certain deficiencies (calledaberrations) when it comes to faithfully rendering a high quality magnified image of thesubject in question. Certain manufacturing and engineering methods have beensuccessfully employed to correct for these aberrations, therefore the modern opticalmicroscope is an unparalleled instrument for the investigation of the microscopic world.Further discussion of optical aberrations is unnecessary for the present purpose, but thereader is directed to examine the superbly detailed discussions provided by Florida StateUniversity’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.htmlUntil recently most photos taken through a microscope (called micrographs) have beentaken using photographic film because the size of the silver halide grains on film are verysmall, allowing for very high resolution images to be made. However with recentadvances in digital cameras and computer software (that allows images to be quickly“snapped” and imported onto the computer), digital imaging is quickly becoming aninexpensive alternative to traditional film. Digital camera resolution is now approachingthe resolution of film (or maybe even better) and some of the many benefits of employinga digital camera include the ability to instantly “snap” and throw away as many images asone likes without the hassle (and cost) of developing multiple rolls of film. Digitalimages may also be quickly and easily labeled with arrows, scale bars and other notationswhile in the computer. Additionally most journal editors today prefer to receivesubmitted 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 isdelivering the optical image to the camera in the first place. If your microscope does notresolve images well, your camera (no matter how many mega-pixels it has) will do nobetter. 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 togather as much light from the subject under examination, and to clearly reproduce amagnified 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 isagain directed to a marvelous and well-illustrated discussion of the factors that influencelens resolution at the Molecular Expression website:http://micro.magnet.fsu.edu/primer/anatomy/numaperture.htmlHowever, to simplify the discussion – imagine two tiny dots on a microscope slide thatare one-tenth the diameter of the period at the end of this sentence. The closer you canbring those two dots to each other while still observing through the microscope that theyare indeed two distinct dots, the higher the resolution of your microscope lens. In otherwords, the smaller the distance d between two tiny dots, the higher the resolutioncapability (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 from0.025 to 1.4 for very good (and usually expensive) lenses. The higher the NA, the betterthat lens will perform in resolving two distinct points just as they are. That, in simpleterms, 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 varywidely. Just because your 50X lens may have 0.95NA inscribed on it does not mean it isa well-designed and high-resolution lens. The best way to be sure is to purchase acalibration 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 yourmicroscope lenses and various digital cameras.For the Jenoptik C14+ high resolution digital camera, the flowing micrographs weremade using several objectives and a metal foil calibration standard that has a 1.5 micronbar 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 baris 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 widebars.
50X Carl Zeiss Planapochromat Objective, NA = 0.95. Total magnification 500X. Thewhite 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 3microns 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 are3 microns wide.
Note that with each of these objectives the Jenoptik C14+ camera clearly and easilyresolves the 1.5 micron bar, and would probably resolve at least half of that size (at 0.75microns) 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 filmresolution for microscope use.Another standard tool used for calibrating each microscope objective for use inmeasuring small objects under the microscope is the stage micrometer (or stagegraticule). A stage micrometer is a glass slide upon which is etched or painted a finelydivided scale of lines set apart in precisely measured steps. The reader is directed to acommercial 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.pdfFor example, a calibrated distance measuring 1mm can be divided by 100 lines, thusrendering a series of finely calibrated spaces. In this case, each space between two linesis 1.0 divided by 100 = 0.01 mm or 10 microns. The next image is a high magnificationmicrograph 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. Thespaces between the lines are 10 micron spaces.It is because of this example of the resolution capability of the Jenoptik camera, togetherwith the very easy to use (and powerful software), that I employ Jenoptik cameras in myImaging Laboratory at California State University , Northridge, Biology Department. Infact, I have just successfully installed a Jenoptik C14+ camera on my Carl Zeiss EM-10Transmission Electron Microscopes. I am achieving superior resolution on my electronmicroscope 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.firstname.lastname@example.org