Back to Basics

by Howard Webb (St. Louis, MO, USA)

Introduction

My problem with amateur microscopy is that I can go for a month at a time without touching my microscope, and along with disuse of equipment comes a forgetting of techniques.  I was reminded of this recently, while reviewing some of the entries to the Nikon photo-microscopy contest, and asking myself why my results were not like theirs.  Hardware is a significant factor, but technique can be equally important.  Average equipment combined with good technique are better than great equipment with poor technique.  What started off as a serious look at my equipment turned into re-looking at my techniques and what I had forgotten. Good images require good technique.

Optics and Equipment

While an understanding of optics is useful, much of the mathematics and details are more important to people designing optical systems (the microscope), than those who use existing systems.  Most of us fall into this latter category.  We obtain a microscope with existing optics, and only occasionally are we even likely to acquire a new objective or eyepiece.  For us, the issue is how to maximize the capabilities of what we have through good technique.  I will skip the theory, and present some simple 'how to' instructions, and show the difference these make.  For those who would like more detail of theory, I include several excellent references at the end of this article.  Exploring these web sites and tracing their bibliographies will take you as detailed as you care to go.

I don't want to denegrate the newest optics, but those of us who are amateurs shouldn't make too much of them either.  When we understand their intent, a quality $50 achromatic objective can meet our needs, and we don't need a $3,000 apochromatic.  Most of my work (with daphnia) is 40x to 100x, with occasionally 400x.  I am not doing fluoresence of inner cell structures.  If we are not doing cutting edge research, we don't need cutting edge equipment.  Look at the winning entries in the photo-microscopy contests, there are images from high-end equipment: the fluoresence, DIC and polarized images, but there are also common objects at low magnification.

The biggest issue is quality, not cost.  However, a cheap 'department store' microscope is no bargain at any cost, you will not get quality optics.  By matching our interests to our equipment we can avoid a lot of issues and expense.  Know your intentions and limits.  If we are willing to put up with a monocular scope, and used equipment, there are some wonderful microscopes available, particularly from schools and businesses trading in old equipment for newer technology. Check microscope dealers for used equipment, or possibly on-line auction sites. 

Equally important to the optics is a condenser.  The formation of the light before and as it passes through the specimen and enters the objective can be as important as how the light is handled by the objective and eyepiece.  I think the sales literature is thin on condensers because they are relatively simple compared to objectives, but the condenser is the one place where you have a lot of control and manipulation of the light (and subsequently your image).

The rest of this article is an exploration of what changes to the condenser can do to an image.  I want to take one slide, and show the effects of a few simple changes.

Abbe Condensers and Kohler Illumination

Ernst Abbe (1840-1905) worked with Karl Zeiss, and established much of the theory behind good microscope optics. In 1870 he developed the two lens condenser. The purpose of a condenser is to create a uniform cone of light converging at the same plane where the subject is in focus.  This is important, if you want to know why, you need to understand the science of light, which is covered in the articles I reference. The ability to optimise the condenser's cone of light to that of the objective in use is also important, thus most condensers come with a diaphragm.

My microscope came with a nice Abbe condenser, I didn't know much about it at the time I got the microscope, but I have come to appreciate how good it is (if treated right).  If you don't have one, seriously consider getting one.

Diaphram and Condenser

Diaphragm and Condenser

The lever on the side of the diaphragm is used to control the cone of light.

Diaphram and Condenser, bottom image in parts
Bottom of Diaphragm with filter holder.
Condenser in two parts

If you look closely at the neutral density filter, you will realize it was a square Cokin camera filter, which I cut to fit the microscope.

Diaphram and Condenser on the microscope
Diaphragm and Condenser mounted on the microscope, with the light (black box).

This is the complete assembly.  The diaphragm and condenser screw together over the bracket, which also holds the light.  The condenser then slides into the mounting ring (held in place by a small set-screw knob).  This whole assembly can be raised and lowered on a rack & pinion via the knob to the right of the picture.

Achieving Kohler illumination with an Abbe condenser is quite straight-forward and simple, assuming the lamp and bulb has been correctly set-up according to the manufacturer's instructions.

  1. Using 100x (a 10x objective), bring the subject into good focus.
  2. Close down the lamp field diaphragm.
  3. With the condenser assembly in a low position, slowly raise it till the edge of the lamp diaphragm is in focus. (If no lamp iris, homemade card apertures can suffice, see this Micscape Topical Tip.)
  4. Open the lamp diaphragm to just clear the image area for glare free lighting.
  5. Adjust condenser iris by inspecting the back of objective (look down eyepiece tube with eyepiece removed). Setting the iris initially to two-thirds of field is about right to start but can be adjusted while inspecting effect of iris setting on image quality when eyepiece reinserted. Slight focus adjustments of the condenser can also be made while inspecting back of objective to maximise illuminated aperture.

 
Diaphram in focus
Diaphragm closed down and in focus.

My diaphragm has a light coating of grease, which over time picks up bits of dust, which you can see on the left side of the image.

Raising the condenser to the proper level can be important.  Depending on equipment, if the condenser is too low, it can create image abberations.  On  the other hand, a microscope does not show you the actual object on the slide, it is an image of manipulated light, so if you are aware of the trade-offs, what may be a less than accurate image may be either artistically pleasing or reveal other information.

Raising the condenser too high can give you an image of the light filament, or in this case, the frosted glass cover of my light box.  With the wrong setting, you can get dust and other artifacts in the image that can be avoided with proper adjustment.

Focus on frosted glass
Focus on the frosted glass of the light box


Condensers are usually made for a 10x objective and higher magnifications,  and you will find at lower power (4x) a vignetting of the field.

4x vigneted by diaphram
40x with condenser vignetting the image

As I discovered with my condenser, the top lens unscrews to give a bigger (but less focused) cone of light.  This makes it usable at lower magnifications.

Condenser with one lens removed
Condenser with one lens removed
 
Just as the condenser loses effectiveness as you lower the objective magnification, they are also not as critical for higher magnification.  Adjusting the diaphragm seems to have little effect for my work at 400x.  There are however, special condensers for higher magnification, complete with oil immersion (for contact with the bottom of the slide).

400x jaw
400x close up of jaw surface
 
I have not talked about depth of field, how much of the image is in focus at one time, because this is something set by the design of the microscope.  There is nothing you can do about it.  You just need to be aware that the higher the magnification, the less depth of field you have.  Thus, in the above image, you can see how quickly focus is lost.

There were several adjustments that I thought would be important, but turned out to have little effect (at least with digital photography).  Many books talk about cutting the voltage to the light for visual observation, but avoiding voltage regulation with photography.  Lowering the voltage lowers the 'color temperature' of the light.  However, digital cameras usually have 'white balance' correction built into the software (either adjusted automatically, or via a menu).  I found that by shifting the white balance, I could get rid of the sepia tone from low voltage.  With digital photography, not only is the microscope manipulating light, but the digital system is also not a direct image, but a manipulation.
White balance adjustment
White balance adjustment

Another factor that proved to have little effect was the use of a neutral density filter.  To reduce light intensity, instead of lowering voltage, a filter can be introduced to the light path.  Often there is a filter holder built into the diaphragm, or another part of the microscope.  I found with the auto-adjustments of my camera that I got almost the same final image, whether I used a filter or not.

While not a filter, definitely consider using a patch-stop for dark field work.  This is a subject of its own, and there are many good articles on this site covering this specialization.

40x dark field
40x Dark Field


Technical Details - side note on PVA

I started to use some old slides for this article, but swapped out a new slide at the last minute (for no particular reason).  The slides I had considered using were made about four months ago.  While relatively short term (relative to how long slides are often kept), it has provided the opportunity to observe how well PVA-G mountant holds up.  So far, the slides show little change from when they were made.  There is no deterioration of the speciments, and no noticable change of colors.  The only evident distortions (due to osmotic pressure) are to the volvox and stentors, and this was evident within a day or two of when the slides were created.  Unlike when I used jellied glycerin, I do not seal the edges of the coverslip, yet there is no indication of drying out or shrinkage of the mountant.  Though (or because) the slides are kept in boxes, there was a haze or film on them when I went to take these photographs.  I suspect this may be caused by some evaporation and condensation as the slides 'set up'.  This is not a significant problem, as the film easily cleaned off of the slides.  I am glad that I gave PVA-G a try, and will continue to use it as my primary mountant.

An alternative to PVA mountant formula has also been suggested, which contains lactic acid.  I have yet to try it, though do have one suggestion.  If you have difficulty finding lactic acid, try a wine and beer making supply shop.  Lactic acid is used by these folks to adjust pH, and you can get several ounces for only a few dollars (USD).  This is a much easier source for small quantities than a chemical or scientific supply company.

Slide Used: Shed exo-skeleton of a Daphnia magna (from my cultures).  The darkest object is part of the grinding jaw.  When daphnia shed, the exo-skeleton usually comes apart in two pieces.  What you do not see in these images is this other part, the 'head shield'.  You are seeing the main body, antenna and legs.

Microscope: Bausch & Lomb monocular, 10x ocular, 4x, 10x and 40x objectives.

Camera: Canon A70

Software: Photoshop Elements

Conclusions

An Abbe condenser and diaphragm offer significant control over the image, they are well worth the extra investment in equipment.  On the other hand, traditional image manipulation via filters seems to be less significant with digital images, which lend themselves for manipulation at many points in the process, from when you take the image, to post-processing.

Get to know your equipment, and the basic techniques that will give you the best possible results.  If my budget was not a consideration, I would like the improvement that infinite focus, flat-field apochromatic objectives would give; but the incremental improvement they provide is not proportional to the cost.  Working on good technique will likely give me greater relative improvement.

References

 

Comments to the author Comments to the author sent via our contacts page quoting page url plus : ('hwebb','')">Howard Webb are welcomed.
 

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