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How does one go about properly selecting an objective for a microscopic task at hand? The first consideration is the type and size of the specimen. What microscopic technique is to be employed and how large do you wish to magnify the specimen? Magnification is fairly simple and straightforward. We all know that 10X means that the objective lens has an effective magnification of ten times life size and when combined in the compound with a 10X ocular lens will give a final magnification of 100X (10 X 10). But what are all the other markings on the lens and how can they help us in selecting the objective lens suited to our needs?

This section covers this subject because knowledge of the markings on an objective will give you the information concerning its proper use and whether it is suitable for the microscopic task you have in mind.

    · Lens Type. The first thing that most lenses have is some lettering such as Plan-Neofluar, Plan Fluotar, Planapochromat, Plan or Achroplan. These are all different types of objective which have many glass, fluorite, or quartz elements for light path corrections. The types of lenses listed here are based on the Zeiss objectives as the Facility microscope is a Zeiss LSM 310. However, the names listed here should allow you to determine the type of objective from any manufacturer. If not, you will have to contact the manufacturer to explain the name and markings.
    · The term Plan stands for flat field. Lenses which are uncorrected for flatness of field will have the center of the field in focus and the outer edges out of focus (or vice versa depending on how you focus the lens). So Plan means the lens is corrected to allow the whole field to be in focus. Achroplans are best for transmitted light while Epiplans are designed for reflected light use. Some microscope manufacturers will list their flat field achromatic lenses as simply "Plan".
    ·
    Achromat lenses have good color correction for two wavelengths of light. They are budget priced lenses. Planachromats are achromats with correction for flatness of field as well as the aforementioned color correction.
    ·
    Plan-Neofluar or Plan-Fluotar lenses are semiapochromatic lenses. They have good color correction for at least three wavelengths and also have the all around flatness of field. They are excellent for polarization microscopic techniques such as differential interference. As they also transmit UV very well, they are excellent lenses for all types of fluorescence microscopy. Any lens with the term fluar in it has fluorite elements in it and all of these are very good for fluorescence work.
    ·
    Zeiss recently introduced a new line of semiapochromatic lenses named Fluar lenses. These are objectives without a flat field made especially to increase the brightness of fluorescence. The image from a fluar lens is approximately 10% brighter than the equivalent Plan-Neofluar. In the UV range, the light transmission increases by 25-50%. This line of objective lenses was introduced about two years ago.
    ·
    Apochromatic lenses (Planapochromat)are the most highly color corrected objectives: they are corrected for four wavelengths and are top of the line in objective lenses. These most often have the highest numerical apertures (see below). Be careful in using these lenses for fluorescence, however. They do not transmit UV light. They work very well for visible light excitation in the blue and green ranges.
    · Immersion. Lenses will be marked for the immersion medium in which they are designed to be used:
    · (Oel) or (Oil) for oil.
    ·
    (W) for water immersion.
    ·
    (Imm) Multi-immersion, for oil, water, and glycerin.
    · Phase marking. If the lens has a phase ring and can be used for darkphase illumination, the lens will be marked above the lens type with a "Ph" followed by a number corresponding to the manufacturer's phase ring number system for matching to a ring in the condenser. Phase lenses are generally not as good for fluorescence applications as the light transmission is reduced by the presence phase ring inside the lens.
    ·
    Magnification. As stated before, this is obvious and self-explanatory.
    ·
    Numerical Aperture. After the imprint of the magnification on any quality objective lens, there is usually a slash followed by a number which may be anything from 0.035 to 1.4. This number is the numerical aperture (N.A.) of the lens. This number is directly related to the resolution and second, for those of you doing fluorescence microscopy, it is related to the amount of brightness of the specimen brought into the lens (obviously very important for fluorescence microscopy!) The higher the N.A. of a lens the better its resolving power and the brighter the image it can produce. Resolution is defined as the ability of a lens to distinguish between small objects. Obviously, this differs greatly from magnification which is just the ability of the lens to enlarge the image of an object. It does not mean that you will necessarily be able to resolve details in the object.

      For a more detailed discussion of numerical aperture and resolution, click here .

    · Tube Length and Coverslip Thickness. The marks on the line below the the magnification and the numerical aperture are the tube length/coverslip thickness. The mechanical tube length (between the objective flange and the eyepiece seating face) is normally 160 (in mm) for older objective lenses or ( infinity for infinity-corrected objectives). The number after the slash is the thickness in millimeters of the cover glass for which the objective was designed and corrected. For most objectives for close working distance, this number is 0.17. This designation means that you should use No. 1½ coverglasses which range between 0.16 and 0.19 mm in thickness. No. 0, 1, and 2 coverglasses are not recommended. Some lenses will have a - sign. This means that the objective is meant to be used with no coverglass. LD (long working distance) objectives may go up to 1.5 mm so that one may look through slides or tissue culture flasks or dishes.
    ·
    Some lenses will also have a rotatable ring which allows one to correct for a coverslip thickness. They are sometimes labelled with "Korr."

Objective Lenses Available

     This is a list of the objective lenses which are available for your use on      the Zeiss LSM 310. They are all infinity corrected:

     Plan-Neofluars

    · 10X/0.3
    ·
    20X/0.5
    ·
    40X/0.75
    ·
    63X/1.25 Oil Iris
    ·
    100X/1.3 Oil
 

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