Re: Pix of A. pellucida

On 15 Jan 2007 11:29:11 -0800, "justbeats" <steve_beats@xxxxxxxxxxx>
wrote:

I think that it is beyond the limits of classical LM. Much is placed
on NA for resolution, and for good reason. However, in the AP
respect, Lamda (wavelength) is more important...not to denigrate NA.

Hmmm. I understand resolution as a function of N.A. *and* wavelength. I
isolated the shortest wavelength I had a detector for (soft UV) *and*
used the highest N.A. I could get (1.4). The rest was about contrast.
Can wavelength be MORE important? Maybe from a SEM perspective...?

Let's be clear. For A. Pellucida under SEM or LM specifically. SEM is
the only way to get useful images of the pores themselves. It is
waaaaaaaaay beyond the limits of classical LM. You win that one hands
down!

But once the pore morphology is known (we establish they are uniformly
round for example) then LM can achieve measurements accurate to 10nm or
better.

I used astrometry s/w. Given very well calibrated images (bias, dark
and flat frames are essential) the location and 'diameter' (magnitude)
of a star in a digital image can be calculated to 1/100th of a pixel! I
found that it works equally well on diatom pores and the delta
positions yield direct measurements of pore centre spacing to near-nm
resolution. However, I'm not sure if 'magnitude' can be scaled as pore
diameter to the same resolution. Your images and measurements could
establish that. Of course, proper confirmation would need the same
samples as you - before you ruin them with (what I perceive as)
horribly destructive observation techniques ;-)

Note: there's probably dedicated s/w catering for all this and more,
but I already had the astrometry s/w (for it's intended purpose) and
didn't look further; so I know nothing about microscopy equivalents.

Anyway, this just shows the near-nanometre scale is not the *sole*
preserve of you SEM jockeys! Resistance continues. We are not yet
absorved [sic] ;-)

Cheers
Beats

PS. Do you put specimens 'under' a SEM? Is that the phrase you use? If
so; "hahahahahahaha" ;-)

Right...not "under" but "in" a SEM.

The pores are not circular but rather rectangular at 106nm x 132nm.
The other factor to consider is that the pitch of the pores is uneven
between horizontal and vertical (whichever one defines as horizontal
or vertical). I got 192nm vertical and 272nm horizontal. Another
interesting measurment was the thickness of the bottom portion of AP
looking through the holes at 25 degrees tilt--89nm thick, +- perhaps
5%. So, being so thin, too high of KV and probe current will drive
the beam through the specimen. EDS measurment and simulation proved
this by showing up the material way underneath the diatom.

I don't see how LM can achieve measurment of these ranges much more
than achieve measurments accurate to 10nm. Maybe it can.

If I recall, Gregor or someone did a nice posting about resolution.
Perhaps we can re-visit that and see how it applies here. What I
found interesting and puzzling was that at 1300X, the pores were not
resolved but at 1400X and higher, they were. So besides resolution
based on NA and lambda, there must be some other relationship that
says that for size and pitch of the specimen, some minimum
magnification is required.

gg


Kiss French. Drink California.
.

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