MicrobeHunter.com Microscopy Forum

Hi,

This is my second attempt at darkfield. I extracted some coltsfoot pollen by cutting a flower off and leaving it on a glass, head down, for about half an hour. Coltsfoot has evolved into an "early bloomer", to avoid competition for pollen-bearing insects in early spring.

I did a hand-held "burst shot" of some coltsfoot pollen with my Nexus 6P. I then did a 12 image average stack in Photomatix. I also did a trick I sometimes apply in Astro photography. A quick look at the channels in Photoshop revealed that the blue channel contained more detail than the other two. I Selected the blue channel and blended it in on top as a new layer at around 30%. This increase the noise a bit, but reveales more detail in the fuzzy areas.

I have ordered a Universal microscope & telescope adapter from Amazon that I hope will arrive by the end of next week. That will make things a lot easier.

Thanks for watching.

Very interesting subject. You have some spots of tantalizing sharpness in there which would suggest a focus stack might be a good way to attack this subject.

There is always a struggle with depth of focus in photomicrography which is not a big consideration in Astrophotography. You can manage it to some degree by subject selection, sample preparation, isolation, stacking, etc.

I know some of these are nearly impossible while handholding, so just add them to your bag of tricks when you get your mount.

It sounds like you know your way around digital imaging and editing pretty well, so I look forward to your postings when you get your gear together. Keep em coming.

Rod

Another shot, while still on the flower

rnabholz wrote:Very interesting subject. You have some spots of tantalizing sharpness in there which would suggest a focus stack might be a good way to attack this subject.

There is always a struggle with depth of focus in photomicrography which is not a big consideration in Astrophotography. You can manage it to some degree by subject selection, sample preparation, isolation, stacking, etc.

I know some of these are nearly impossible while handholding, so just add them to your bag of tricks when you get your mount.

It sounds like you know your way around digital imaging and editing pretty well, so I look forward to your postings when you get your gear together. Keep em coming.

Rod

Thanks a lot, Rod! I noticed the depth of field "issue" with photomicroscopy right away, yes. I will certainly read up on stacking for the microscope while I wait for the adapter. I guess getting the subject as "flat" as possible also helps.

Both pictures are taken with a 10x Eyepeice and the 10x objective by the way.

If you haven't found it this is good starting point on stacking from a thread on the forum.

viewtopic.php?f=27&t=2500&hilit=stacking

Very nice Glen. Good sharp exine ornamentation (spines)...

I collected some Colt's-foot about a week ago and transplanted it (it is a perennial with nice hardy rhizomes) to a trough. It's taken OK and is in flower at the moment. I'm waiting for the flowers to fertilize and the ovaries swell before taking some for sectioning - still be a few weeks 'til the ovaries have grown yet though. It's a lovely little 'weed' that happens to grow in a rather large colony in a grassy-bank next to a local Asda where we live!

I think I'll put some of it's pollen under the 'scope tomorrow and have a look; your super picture has got me interested - pollen is a particular interest of mine, especially of British wildflowers - my main interest.

Thanks for the interesting post Glen. The wildflowers are really starting to show now in the UK!

Bonjour
Very nice photo
Cordialement seb

Nice images of an interesting subject, with pollen grains in the plane of focus rendered very sharp. I agree with Rod though that a focus stack would be in order here.

I finally have an adapter for my Nexus 6P now, making things a lot easier. I purchased a Gosky Universal Cell Phone Adapter Mount. (http://www.amazon.com/Gosky-Universal-P ... B013D2ULO6). The only problem with it is that the Nexus has the power button and volume buttons right where you need to attach the adapter. I can make it work by attaching the adapter so that it presses both volume buttons down at the same time.

Okey, so back to my Pollen. I located more pollen by looking on the flower itself rather than putting it on a slide. So, i changed tacktis a bit.

Antoher thing I noticed, is that the field is not evenly illuminated, a concept I am familiar with from astro photography. So, after taking 7 RAW shots with my Nexus 6P @ ISO 100 and auto shutter, I took 11 new "empty" shots with no slide, and at the same ISO.

Both the "flat frame" and light frame were averaged in photomatix.



As you can see, the "blank" or "flat frame" has a strong vignette that is desirable to remove. I subtracted the image from my 7 image stack, using photoshop, and you can see the result here;



On my todo list now is to get my head around z stacking

Bonjour
Très belle la dernière image
Cordialement seb

So, after taking 7 RAW shots with my Nexus 6P @ ISO 100 and auto shutter, I took 11 new "empty" shots with no slide, and at the same ISO.
Both the "flat frame" and light frame were averaged in photomatix.

Does this technique have a name?

Good progress.

As one who did a good deal of afocal imaging with a Nexus 6 (not 6p) phone, a couple of observations for you to consider.

First, it looks to me like your phone may be too close or too far from the eyepiece. You are using a very small part of the sensor and sacrificing a good deal of it to vignette. Not sure if your mount permits this maneuver, but if possible, try changing the spacing. I was able to get the image to entirely fill the short dimension of my screen without zooming. I was using a 10x wide field eyepiece.

I did not find it necessary to do darkframe subtraction. You should be able to manage any brightness in the field with exposure settings and post capture levels correction.

I used a third party app, Camera FV-5, to give me more control of cameras settings than the native camera app offered. Take a look at it. Worth the few bucks they charged for it.

This is all offered with the acknowledgement that the 6p is a new phone from a new manufacturer, so my experience may not translate, but for what it is worth.

Keep shooting!

75RR wrote:

So, after taking 7 RAW shots with my Nexus 6P @ ISO 100 and auto shutter, I took 11 new "empty" shots with no slide, and at the same ISO.
Both the "flat frame" and light frame were averaged in photomatix.

Does this technique have a name?

It is very common with DSLR astro photography where you get uneven illumination across the sensor. Mr rnabholz is probably on correct in that I can work with light levels to avoid this step. A good description and examples are given here; http://photographingspace.com/how-to-cr ... otography/

rnabholz wrote:Good progress.

As one who did a good deal of afocal imaging with a Nexus 6 (not 6p) phone, a couple of observations for you to consider.

First, it looks to me like your phone may be too close or too far from the eyepiece. You are using a very small part of the sensor and sacrificing a good deal of it to vignette. Not sure if your mount permits this maneuver, but if possible, try changing the spacing. I was able to get the image to entirely fill the short dimension of my screen without zooming. I was using a 10x wide field eyepiece.....

Keep shooting!

Thanks for the tip and encouragement. I have no WF eyepieces for my microscope, but I have a couple from some Row Rathenow 60's and pre-60's school microscopes that have a wider FOV.

I am not sure that helps. I think the eye piece, wide field or not, projects a 2mm image (exit pupil size) where your retina sits when you use the microscope. How large this image will appear on the camera will depend only on the sensor size. A wide field eyepiece will just squeeze more of the scene into the same picture.

This is the disadvantage of afocal photography. Idealy I would have liked to put a CCD sensor without any optics, at the location of the bottom lens of the EP. That's a larger image plane.

(The camera is as close as practically possible to the eyepeice)


This is how I understand it anyways, and might be wrong

Well, I was wrong. My Row Rathenow 1960-ish EP projects a larger image on the sensor than the original 10x ones. Not sure if it is actually more data, just the same image as with the 10x, but enlarged...

I think the best eyepiece to use for afocal may be the one with the highest (farthest from the eyepiece) exit pupil.