More on A.pellucida

Microscope Group,

I wish to present a light microscope image (LM) of A. pellucida,
with a determination of the distance between the striae. The re-
sult is compared with those obtained on various A. pellucida
frustules imaged by field emission microscopy (FESEM). Dr. G.
Gaugler, USA did this work and his SEM images are presented at
his site, www.photoweb.net/micro/ .

My LM image is at
www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 .

Pertinent details are as follows. The sample for the FESEM
images was specially prepared by Mr. Klaus Kemp, UK. It con-
sisted of nine A.pellucida frustules placed on top of a coverglass
in free air, no mounting medium was used. Dr. Gaugler used
Palladium to sputter the sample before doing SEM microscopy.

The frustule used for the LM picture is on one of Mr. Kemp's
test slides. It is mounted the conventional way in Zrax and
there is one A. pellucida diatom together with samples of other
species on the test slide.

All of the samples, SEM and LM show "inner views" of the
diatom, that is views with the concave side poining up.
Mr. Klaus Kemp's site is at www.diatoms.co.uk/ .

The LM picture was obtained using an Olympus BX51 micros-
cope using a planAPO objective 100x/1.35 using their universal
condenser with top oil lens and double immersion. DIC was
used to enhance contrast and the image was projected with
a 2.5 Olympus eyepiece upon the CCD of a Pentax *ist DS
digital, single lens reflex camera. The vertical dimension of the
CCD is 1.57 cm. JPG format was used with 2000 pixels along
the vertical. Illumination was by electronic flash and image
processing was limited to adjustment by Auto Levels, follow-
ed by Grayscale in Adobe Photoshop. No other processing
was done. The image horizontal extent was shortened, the
vertical dimension preserved at 1980 pixels from the full 2000
and that is what is shown at
www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394 .

While I have been able to resolve the striae I was unable to re-
solve the striae into punctae (dots) using my light microscope.

Since I do not have a stage micrometer I determined the distance
between the striae as follows: I used the magnification of
the objective of 100x and that of the photoeyepiece of 2.5x to
calculate a total magnification of 250 x. Counting the number
of striae in 150 pixels as 18, one obtains 150/18 = 8.33 pixels
per stria. Each pixel represents 1.57cm/2000 = 7.85 micrometers
on the CCD and with a magnification of 250 this corresponds
for 8 pixels to 8.33 x 7.85/250 = 0.26 micrometers for the distance
between striae.

Dr. Gary Gaugler used his Zeiss Supra 55VP FESEM instrument
to image and measure important distance parameters on the
samples for SEM microscopy. One needs to look at the images
at his site www.photoweb.net/micro/ to appreciate the signifi-
cance of the results. An overview showing a bird's eye view of
the samples is given under the thumb nail Apellucida-specimens.

Currently there are details for five of the nine A. pellucida dia-
toms posted. This allows a comparison of variability between
the parameters for individuals belonging to the same species.
It will be seen that the variability of distances between striae and
between the pores of a stria (dots) amounts to as much as 9 %.
This is probably more than has generally been expected. Please
refer to Apellucida-50kx-2.jpg, Apellucida-50kx-D.jpg,
Apellucida-50kx-E.jpg, Apellucida-50kx-F.jpg and
Apellucida-50kx-I.jpg for images and measurements.

For these diatoms the following results apply (all in micrometers):
-2 distance between striae 0.289 distance between dots 0.229
-D 0.272
0.176
-E 0.301
0.283
-F 0.284
0.181
-G 0.275
0.161.
For LM from above:
LM 0.26
............................

Even within the same diatom distances between pores may vary.
This is best appreciated at high magnifications. Also note the
thickness measurements of the frustule's bottom (Apellucida-
100kx-3). These were made at 25 degrees inclination. If a frustule
is inclined, the bottom layer may obstruct a fair portion of the pore
and have an effect upon the LM picture. The measured thickness
of the bottom was about 0.09 micrometers at the center and less
at the ends.

It will also be noted that some pores are plugged up, which might
make LM observation of details more difficult on some diatoms.
Particularly diatom E shows many plugged pores.

Now the question may be raised whether separation of the pores
within a stria, can be done by LM. I do not believe that I can do it
with the setup described above. I might be possible to do better
with a projection magnification of 5x or more.

However Peter Hoebel at
www.mikroskopie-forum.de/read.php?2,25384,25384#msg-25384
presents such a LM picture with dots separated for A. pellucida. He
had used a near UV LED and averaged 1000 images to obtain the
separate dots. Details can be found at the above link.

Regards
G.R.


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