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
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.
Immersion. Lenses will be marked for the immersion
medium in which they are designed to be used:
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
wavelenths of light. They are budget priced lenses. Planachromats
are achromats with correction for flatness of field as well as the aforementioned
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
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.
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
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.
(Oel) or (Oil) for oil.
(W) for water immersion.
(Imm) Multi-immersion, for oil, water, and glycerin.
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
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
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
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:
63X/1.25 Oil Iris