Microscope Technical Info

Numerical Aperture (N.A.), Condenser Lenses and Immersion Oil

 

This is a somewhat complicated subject and the discussion that follows goes well beyond the entry in the glossary.

Numerical Aperture (N.A.):  This is a number that expresses the ability of a lens to resolve fine detail in an object being observed.   It is derived by a mathematical formula (n sine u) and is related to the angular aperture of the lens and the index of refraction of the medium found between the lens and the specimen.  The physical size of the lens is important in determining the N.A. of the lens and to get the most from the lens, a light condensing system should be employed which will completely fill the back lens of the objective  with light.   To do this, the Numerical Aperture of the condensing system must equal or exceed the N.A. of the lens.

Now, to state this in simple terms, it means that you must have a lens between the light source and the slide (in or under the stage) that will "condition" the light for the appropriate objective lens.  The higher the power, the more important this condenser lens becomes.  A value (N.A.) is associated with both the condenser lens and the specific objective lenses.  For the sharpest images, the N.A. value of the condenser lens should be a higher (or equal) number than the N.A. value of the specific objective lens.  Even with these N.A. values there are other variables to consider.  The thickness of the slide and coverslip used and the media (be it glass, air or oil) between these two lenses

Immersion Oil is a special oil used in microscope work with the highest power objective lenses (ie 100X lens).  There are two basic types of immersion oil, Type A and Type B.  The only difference between the two is the viscosity.  Either will work well.  One or two drops of oil are placed on top of the coverslip and the 100X objective lens is brought into position so that it touches the oil and creates a "bridge" of oil between the slide and objective lens.  It has a refractive index very close to that of glass.  This allows very little refraction of the light rays as they go through the slide, specimen, coverslip, oil and through the glass objective lens of your microscope.  

Most condenser lens systems for research grade microscopes (1000X) have an N.A. value of 1.25.  The 100X objective lens is also rated at 1.25.  The medium between the 100X lens and the slide can be air or oil.   Without getting too technical, the only way to get a Numerical Aperture greater than 1.0 is to use a material with a refractive index greater than 1.0.  Oil (1.5) is such a material.  So to get the best resolution at 1000X (N.A. 1.25), a drop of this special oil is placed between the lens and the slide.  This is why most 100XR objective lenses are called oil immersion lenses.   They work fine without oil but will give a sharper image with it (see image comparisons below).  The oil can get messy and must be wiped up when you are finished.  As a result, most people prefer to use their 100X objectives dry.  If you try using immersion oil, clean up with lens tissue or a KimWipe on the lens.  Avoid solvents.

Now, back to our discussion on Numerical Aperture.  What does this all mean for you?  Well, the condenser lens system should have an N.A. greater than or equal to the largest NA of the objectives.   For a microscope with 400X max power, you need a condenser lens with an NA of 0.65 for best possible resolution.  An in-stage condenser lens (0.65) works perfectly for these magnifications.  At 1000X, you now have an objective rated at 1.25 N.A., so your condenser system should match or exceed that value.  You will now want to switch to an Abbe condenser system that can match those values.  Without oil, you will never exceed an NA of 1.0 regardless of what the numbers are on the lenses.  So to get the best possible resolution at 1000X, you must use oil. 

Some entry level microscopes do not have condenser lenses at all but still work quite well, even at 400X.  See a comparison of images below.  What you see below are images from a National model 109-L (without a condenser lens at 400X) and a National model 138 (with an 0.65 condenser) at the same power.  These images have been purposely reduced in resolution for the web but you should be able to compare and see greater resolution with the 138.  You'll also note that the image is brighter and whiter.  This is a result of the fluorescent vs the tungsten illuminator.

     

What we see above is (left to right) 1.  The image at 400X through a model 109-L (no condenser lens) using a tungsten illuminator  2.  The same slide on a model 138 (0.65 N.A.) with fluorescent illumination  3.  The same slide at 1000X on a National Optical model 163 without oil  and 4.  The image as seen with oil.  The specimen is an allium (onion) root tip and illustrates the five phases of mitosis (you are looking at the chromosomes splitting).  These images also illustrate the relative magnification increase you will get when moving up to 1000X.  You can verify the magnifications by doing the following with a metric ruler.   Measure the width of the "spiders" on the left and right.  The one on the right should be 2.5 times bigger (ie: 1cm on left, 2.5 cm on right is the same ratio as 400 to 1000)

Objective lenses are color coded and have numbers written on them.  Let's look at an example.

The yellow band tells us that it is a 10X objective lens.  Red is 4X, Blue is 40X and White is 100X.  The first number "10" is the power (10X).  The 0.25 is the Numerical Aperture.  The 160 is a standard DIN measurement in millimeters of the tube length of the microscope required for this lens to work properly. Finally, the 0.17 is the thickness in mm of the cover slip that you should use.  0.17mm cover slips correspond to a number 1 cover slip.
At left, a 0.65 N.A. condenser lens mounted in the stage of a model 138 and an Abbe condenser system (1.25 N.A.) mounted on a rack and pinion focusing mechanism with variable iris (on a National Optical model 162).

Learn more about Immersion Oil and N.A. here (Florida State Univ.)

 
home forums links

Copyright 2014, Microbus, All rights reserved