[Lista-Algas] THE PERFECT MICROSCOPE AND IMAGING SYSTEM FOR DIATOM MICROSCOPY

[Andres Boltovskoy]

TOMADO DE ALGAE-L at LISTSERV.HEANET.IE
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Hello All! 

Apparently, the attachment didn't work for most. I am just going to paste the text into the body of the email. Apologies!

I had a number of people request that I post responses to my request for information on Hardcover KLB 2:1-4 and "the perfect microscope and imaging system for diatom microscopy", so here is my best attempt at condensing the wealth of information I received. 

Thank you to everyone who responded, I greatly appreciate and respect your opinions. I'm sure this information will help a lot people. Please see pasted document below for condensed responses concerning KLB, microscopes, and imaging systems.

Concerning the hardcover KLB references:
A number of people suggested checking Amazon.com, Ebay, and Koeltz. Perhaps if I
continue to check back I may happen across a set being sold used, but currently, to my
knowledge and those that responded, there is no supplier that has a stock of hardcover
copies.
A number of people also made suggestions, such as having the paperback books spiral
bound or bound in hardcover. This is a great idea, but the paperback copy that I saw
also seemed to have a lower quality, non-glossy paper, reducing the quality of the
photomicrographs in the plates. Hopefully this is something we can resolve in the future.

Concerning Microscopes:
In general, a number of things were recommended by many:
1. Differential Interphase Contrast or DIC (and/or phase contrast as a cheaper
alternative) to increase contrast
2. About Objectives:
a. The best available 100x oil immersion objective, generally 'Apochromatic'
(has Numerical Aperature or NA >1.4), to increase resolution. In order to use
DIC with this objective you will also need to purchase both an interference
contrast objective prism and a condenser prism.

As a note: this one objective may potentially be the single most expensive
piece on the microscope ($5000.00 US or more), but is also one of the most
important pieces of the microscope. It was suggested by many, that every
effort should be made to get a 100x objective with an NA of 1.4 or greater
(best if matched to the NA of the condenser) even if it meant it was the only
objective you could afford to purchase for the scope.

b. Although apochromatic is best, because the 100x objective is used most
frequently for identifying diatoms, in order to save money, a 'Semi-
Apochromatic' 40x or 60x objective was suggested (this should cost
significantly less, but is still somewhat expensive). Also, a number of people
recommended having the 60x objective be oil immersion as well (this
increases resolution). Again, in order to use DIC with this objective, you will
also need to purchase both an interference contrast objective prism and a
condenser prism.

c. 5x, 10x, and 20x objectives may be useful, but are not generally necessary to
identify diatoms to the species level. If these objectives are desired a simple
"Achromatic" objective can be purchased. For many companies, you could
get all three (5x, 10, and 20x) for under $1000 US. Generally, for diatoms, it
is not necessary to have the DIC slider or prisms for any of these objectives.

d. The final suggestion for objectives is that a diamond scribe (aka diamond
stylus) is purchase for the nosepiece position. This will allow you to circle
specimens (so you can find them later) as well as draw lines across the
coverslip (to aid in counting). This is helpful for a number of reasons: 1. You
can create a personal reference collection by circling species, then be able to
easily locate them in the future. 2. Circling specimens also allows for easier
collaboration. You can circle a specimen, then send it to a colleague for
verification, or help with identification. 3. It is also helpful for QA/QC (Quality
Assurance). For example, if you have a line drawn across the coverslip, you
can easily re-count a slide along the same path used before.

Conclusion: Start with the best 100x objective you can afford, fill in the rest
when you can. Remember that you can almost always, unless they are
discontinued, choose to add on objectives and prisms at a later date. As you
move from lower quality to higher quality objectives, you will have fewer
aberrations, essentially increasing resolution to the eye.

3. About the Condenser:
a. The NA (1.4) of the substage condenser should match the NA of the
objective, in order to get the best resolution.

b. Many suggested that the substage condenser should be oil immersion (just
as the objective is). This will increase resolution. Essentially, an oiled
condenser allows an oil immersion interface between the slide and the
condenser main element.
4. About the stage:
a. Some complained of faulty specimen holders, so be sure that the specimen
holder is of a good quality and very sturdy.

5. Illumination:
a. DIC creates a "light starved environment", so be sure to have, at the least,
high wattage (100w) halogen illumination. To upgrade to a tin halide
illumination, it is VERY expensive ($5000.00 US or more), so it is mostly
unnecessary. But be sure to replace bulbs often to get the best images.

6. About specific manufacturers:
a. Leica I had the most recommendations from those who owned Leica
microscopes. Many have the Leica DMLB, which is now discontinued, but the
Leica DM 2500 appears to be comparable. All of those who wrote to me
about Leica were very happy with the quality of the scope. One person
warned not to get a scope with preset illumination or programming because
you have better contrast and options when setting this manually. I recently
demoed a Leica scope and really appreciated the ergonomic features they
have added. As diatomists, we often spend hours in front of the scope and
these features made it much more comfortable. For example, the eyepieces
could be shifted up and down, allowing you to look straight-forward rather
than down into the scope. The focus knobs and stage movement knobs are
very low on the scope, allowing you to comfortably rest your hand on the
table and reducing wrist/arm fatigue. Also, you can switch the stage
movement from left to right in seconds by using a magnet. As a note: other
companies are advertising some of these ergo benefits as well, I just haven't
had the chance to try them yet. Bottom Line: Leica offers high quality
scopes and optics at a good price.

b. Zeiss Nearly as many responded about Zeiss scopes as did about Leica, and
they were all very positive. More than one person said that the Zeiss optics
cannot be beat, but they are often a bit more expensive. The only complaint
about Zeiss was that they only had a one-year warranty (compared to as
much as 5 years elsewhere). If you aren't investing in automated stages and
other pre-programmed equipment, this probably isn't a problem, though.
Botttom Line: Zeiss offers what some say are the highest quality optics
and great scopes, but it may cost you a bit more.

c. Nikon A few people wrote about Nikon scopes, all were happy with the
quality of the optics. The current recommended scope is the 80i (manual) or
the 90i (automated). Apparently there were a number of glitches with the
automated stage during the first month, but all problems were solved with a
bit of back in forth. Now the scope comes highly recommended. Nikon was
said to have a 5 year warranty, which could prove to be helpful, especially if
you choose to go with automated parts. Bottom Line: Nikon also offers
high quality scopes and optics, but fewer researchers seam to end up
with these scopes.

d. Olympus A couple of people also wrote about Olympus scopes. Both had the
BX51 with DIC, one also had phase contrast. Both were extremely happy with
their scopes. According to others that wrote, Olympus used to be regarded as
not quite as good as other companies, but they are apparently stepping up to
the plate in recent years. Bottom Line: Olympus may not have always had
the highest quality optics for DIC, but more recently are more than
capable competing with other companies, providing both high quality
optics and scopes.

Concerning Cameras and Imaging software
1. There is a bit of debate on whether or not a cooled camera system is necessary or
not. Many have cooled cameras and many don't. I haven't decided which is best
for our purpose, yet. I do, however, know that cooled cameras cost significantly
more than uncooled.
2. Although this is coming from knowledge garnered from different dealers and I don't
completely understand it myself, I thought I would pass along this information, you
may take it for what it is worth: when taking pictures at 100x, you will not get better
images with a higher megapixel (MP) camera. The optimal would be a 2MP
camera. It has something to do with the number of times that the area is "sampled"
and the amount of "noise" you will get in images if a 8MP camera "over-samples" a
highly magnified image. Like I said, I'm no expert on this, but the bottom line is that
a 2MP camera is what you want. If you have information otherwise, please share it.
3. I got very mixed reviews on imaging systems from Zeiss, Leica, Nikon, and
Olympus. One company, Diagnostic Instruments with SPOT imaging systems
came out on top in the end.
a. Zeiss One user felt quality was okay, while another said the software was
horrible, difficult to understand and essentially a waste of time.

b. Leica One user said there was certainly a learning curve with software, so
they export to photoshop to manipulate photos, but it does the job. Another
user said the software was difficult to use with erroneous functions that don't
allow you to take the pictures you want and also that the camera breaks
easily and can be a pain. When I had a demo here, the system crashed and
we were unable to use any of the functions of the $5000 US software
package he was trying to sell during the demo. I took it as a sign to run, if the
Leica dealer couldn't get it to work, I don't know what I would do.

c. Nikon One user has the Nikon DS-U2 5mp with Nikon NIS Elements D
documentation and measuring software, they consider it to be user friendly
and of good quality. Another user is not satisfied with images captured with a
DS 5M camera from Nikon.

d. Olympus I only heard from one user that had an Olympus camera, he was
very satisfied with the camera and the microsuite software.

e. Diagnostic Instruments SPOT I heard from numerous users about how
great SPOT cameras are. One great thing is that you do not have to
purchase software separately; it comes free with their cameras. Some have
the much cheaper, but uncooled Insight 1800 series of cameras. You can
choose to have these in color or monochromatic. Still others have the much
more expensive, but higher quality and cooled RT2500 series. Again, you can
choose to have monochromatic or one with a slider allowing high quality color
images. One user said that they found the Insight to be more user friendly
and preferred it to the "better" RT series. Overall SPOT cameras and
software were said to offer:
i. Great measuring and scale-bar technology
ii. Fast focusing
iii. Simple brightness/contrast manipulation is simple
iv. Easy use, with hardly any learning curve
v. Great photos all the time even with limited computer
knowledge
vi. Photoshop brand software is integrated into their software
package. Many people are already familiar with its capabilities.

Good Luck with finding the scope of your dreams. I hope that at least some of this can
help you. It certainly helped me.


 
-- 
Emily K. Hollingsworth
Benthic Phycologist
Aquatic Biology Section
Dept of Health & Environmental Control
2600 Bull Street
Columbia, SC 29210

Work: 803.898.4331
Cell: 616.516.9901
HollinEK at dhec.sc.gov
EKBiofilm at gmail.com 
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