Re: More on A.pellucida

The TEM images are very interesting. Given the irregularities of the
pores, the absolute, perfect regularity of the finer pores is
surprising - almost suspicious...


On 25 Jan, 21:38, "NoSpam" <NoS...@xxxxxxxxxxx> wrote:
Steve,

You touch on important points.

First is the effect of the direction of the diatom's axis with respect to
the
direction of polarization upon achievable resolution in DIC observation.
In my case the diatom was as right angles to the direction of the polarizer
and parallel to the direction of the analyzer and at 45 degrees to the so-
called shear axis. The image and pertinent text is at:http://www.mikroskopie-forum.de/read.php?2,25394,25394#msg-25394.

I did try at one time to rotate the specimen, but this lead to loss of focus
and loss of good immersion oil contact.

Regarding the pores I have the following to say. Athttp://www.mikroskopie-forum.de/read.php?2,25440,25440#msg-25440
I reproduce TEM-images from the article "Fine structure of the Diatom
Amphopleura Pellucida" by E.F. Stoermer and H.S. Pankratz from the
Amer. Jour. Bot. 51(9): 986 - 990, 1964.

This article describes the punctae (pores or dots or pearls) as a series of
rounded openings of 60 millimicrons in diameter which in turn are
covered by porous plates with even finer pores of some 6 to 8 milli-
microns in diameter. I hypothesize that if these porous plates are not
removed by the cleaning process, they may obstruct the imaging of the
punctae (pores,dots, pearls) by light microscopy. The tiny pores in the
porous plates may not even be visible on SEM if the sputtering process
obscures them. Then they may also obscure the punctae on SEM.
Obscured pores are visible in some of Mr. K. Kemp's SEM samples.
It may be important to verify the existence and nature of these porous
plates.

I recommend a study of the mentioned article or at least of the pictures
in the above post of mine. One picture shows one horizontal row of porous
plates, while the other image shows the rectangular arrangement of the
punctae covered by porous plates.

Note that the 60 millimicrons quoted for the diameter of the punctae by
Stoermer and Pankratz does approximately, but not precisely agree with
Dr. Gaugler's determinations in the imagehttp://photoweb.net/micro/Apellucida-I-50kx-1.jpg.
Other images from Dr. Gaugler's FESEM are shown athttp://photoweb.net/micro/.

I hope the forgoing is of interest to you and the group.

G.R.

"justbeats" <steve_be...@xxxxxxxxxxx> wrote in messagenews:1169751013.605760.196180@xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx



Great thread!

Resolving AP ain't that tough (with enough NA). Doing so with
sufficient contrast to see it, is!

DIC has best resolution in one direction (orthogonal to the plane of
polarisation I think). I don't know if that's slightly greater than the
equivalent brightfield or not (as someone suggested). I have seen the
pores of AP appear and disappear at 90 degree intervals as the
frustrule is rotated.

Reckon Mr Kemp might get a few orders for AP 'cos of this thread ;-)
Might grab some mounted with no coverslip to try a few epi techniques.
Hadn't thought of that before.

NoSpam mentioned "plugged pores" which triggered a thought. Biological
sections are stained to increase contrast so could the pores (only) of
AP be plugged with an opaque material? They'd stand out in bright field
then! Or embed (cast?) them on a slide with that material and treat
like a rock section; done before?

Cheers
Beats

On 20 Jan, 19:05, "NoSpam" <NoS...@xxxxxxxxxxx> wrote:
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 isatwww.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 atwww.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 shownatwww.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 sitewww.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 Hoebelatwww.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.- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -- Hide quoted text -- Show quoted text -

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