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.
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".
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
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
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
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
Lenses will be marked for the immersion medium in which
they are designed to be used:
or (Oil) for oil.
for water immersion.
Multi-immersion, for oil, water, and glycerin.
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.
As stated before, this is obvious and self-explanatory.
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.
a more detailed discussion of numerical aperture and resolution,
click here .
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.
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
is a list of the objective lenses which are available for
your use on the Zeiss
LSM 310. They are all infinity corrected: