MicrobeHunter.com Microscopy Forum

Hey everyone! Here is my first ever attempt at photographing botanical subjects. Earlier today I received a group of slides that I bought from John -- They took just 8 days to arrive from England and I'm very happy to have them. So, I took a bit of time and looked over them and attempted to take some photos. I noticed that they are so thin that I only needed to stack just a few images for most of them, which is really quite amazing when you think of it. And so, because I'm definitely on the learning curve for this subject, I welcome all critiques, suggestions, etc. from anyone who has something to say. They really are beautiful slides -- To say I'm impressed would be an understatement.

One thing I need to admit: Because I did these so fast I'm not entirely sure what I put down for the magnification was right. I think it was but of course I neglected to write it down before I processed the images. The photo of the Lily Anther pollen was indeed shot at 200x, I'm just not quite as sure as the fungus photo. Memory... It's always the second thing to go.

Hi Pat,

These are very, very good!...

BillT

Pat!
Well my friend, it's great to see your first images!
I can tell you this right now - these are really good images for starters - these thin brightfield slides are a blighter to photograph - the coincident focal-points plus the bright background always give my cameras, both the 5mp Toupcam and the Canon 1200D, nightmares!

I'm really pleased you like the slides, great to see these 'old friends' again.

Well done Pat, keep up the good work my friend.

John B.

I hope to be taking some more photos later today of John's slides. Stay tuned!

Go-get em Pat! Great to know you like them my friend.

Right! Here's a few more images from the slides I bought from John. I know I keep saying I need to get better organized and write stuff down and all that... Well, needless to say I had one of those slides thay I would totally remember (I have a mind like a steel trap). And of course I didn't (a rusty steel trap). So, perhaps John can remind me what it is we're looking at on that slide. Otherwise, I'll figure it out once I get a chance.

As always let me know what you think of these images. Because botanical stuff isn't really my specialty I would especially welcome any suggestions anyone would feel inclined to give. Thanks much!

Aha! Hi Pat! More old-friends!

The first image is of what is a 'weed' here in the UK, the Sonchus.arvensis. The section is a transverse section of a leaf that is infected with a fungus, probably a 'rust'. This fungus, like many, has several stages to it's life-cycle and here is on stage that invades via stomata and starts to thrive beneath the epidermis - between the epidermis and the mesophyll in fact.
Here the fungus has progressively pushed the epidermis away from the mesophyll as it grows - forming it's own little environment in a sort-of 'chamber' as the image show. Ultimately it will burst through and stand free and proud of the leaf surface before releasing it's spores to begin again!

Here are a couple of images of the actual (infected) leaf-parts from which the slide was made, in fixative immediately after collection....

incident-lighting trans'-lighting

Hi again Pat,
your 2nd image looks like a vascular-bundle from a Sunflower's stem in TS my friend - one of my favourite sections - Sunflower sections exhibit so many of the different tissue-types all in the one section.

The third & fourth images are a very thin (2µ?) Lily-pollen section, sectioned and stained specifically to reveal the nuclei (2 it seems) of the pollen-grain at the stage at which the anther opens to release the pollen. This number can be a taxonomic clue and may be 2 or 3 depending on the stage at which the (smaller) sperm-nucleus-to-be divides.... (the mature pollen will have 3 nuclei)

The surrounding cellular integrity is sacrificed in order to get the 2µ pollen-grain section.

The darkfield I find, as seen here, helps to differentiate the nuclei of the pollen-grains.

Keep up the good work my friend!

John B.

Thanks John! It was the second image in the new group that I forgot what it was; thanks for letting me know! I was kind of pouring over them rather quickly and as much as I thought I'd remember of course I didn't.

I'll try to get to some more of them early next week. It is quite fun and interesting to explore these slides. The fungus one I think is my favorite so far. So, for that slide, I assume I photographed a spot where the fungus has invaded the leaf. Are those roundish things spores? And I see what you mean in how it creates its own environment within the leaf structure. Mold is a rather destructive thing!

I also really like the pollen slide as well; it is very interesting to see it contained within the flower. And I still can't get over your 2 micron sectioning skills!


Plants are fun! More photos to follow.

Those Lily Pollen images are out of this world! Also, idk if this is just me or my computer or what, but the colors are so pretty that I want more exposure (for example, on the Lily Anther LS pollen with 2 nuclei; 20x Plan 0.45, 3 image stack, phase contrast lighting). I have neither imaged botanicals, nor have I used phase contrast, so this is only an ignorant opinion and I could be entirely incorrect.

The image "Unknown Subject (for now), 10x / 0.25, 5 image focus stack, phase contrast, Nikon d810, Photoshop CC" is extremely appealing.

Thank you for sharing such beautiful images! Please, keep'em comin'!

SunshineLW wrote:Those Lily Pollen images are out of this world! Also, idk if this is just me or my computer or what, but the colors are so pretty that I want more exposure (for example, on the Lily Anther LS pollen with 2 nuclei; 20x Plan 0.45, 3 image stack, phase contrast lighting). I have neither imaged botanicals, nor have I used phase contrast, so this is only an ignorant opinion and I could be entirely incorrect.

The image "Unknown Subject (for now), 10x / 0.25, 5 image focus stack, phase contrast, Nikon d810, Photoshop CC" is extremely appealing.

Thank you for sharing such beautiful images! Please, keep'em comin'!

Thanks -- I'm happy you like them! These are really my first serious attempt at photographing botanicals. As for the darkness of the image: I tend to like things darker and so I do need to watch for that. But on my screen they look fine, and on my other monitor they are even lighter. So, I'm not sure. I use a color calibration tool and so they should be fairly accurate as far as color and shade goes. I'm not sure if it is still an issue, but I know at one time a Mac (assuming that's what you have) had a different color space than a PC and pictures made on a PC would look different on a Mac. I can't remember if it was darker or lighter. So, I'm not sure. Is anyone else having trouble viewing these -- Do they look dark to anyone else?

I do think the dark-field photos may be a tad dark, though. I'll try to give them a bit more light next time.

Very beautiful color and sharp image. You can go on for more


Suphot

I know all microscopes and digital microscope softwares are different, but here are several things I fiddle with when assessing my color options for brightfieqld images (I know NOTHING about fluorescence! or DIC!). You probably know all of this, but I always use the same systematic process so I'll list it all for completeness sake. Also, I am a noob at this so this could be completely wrong in terms of how and what you are imaging. Besides, the photographer is the ultimate judge of exposure/ contrast/ color/ etc., and if you like what you are seeing, don't change it to please somebody else! I offer this list as a gesture of kindness and not to insult your technique because your images are stunningly beautiful. At this point, I would begin nitpicking every tiny detail to perfect every shot!

1. Place specimen on stage and focus image

2. SOFTWARE ADJUSTMENTS:
(A) If using "Auto-exposure", define the area of autoexposure to encompass the entire image.

(B) Look at the Red, Green, and Blue (RGB) histogram to ensure that the histogram is relatively evenly distributed from left to right. In general, if the RGB histogram is too far to the left, the image is underexposed. If the RGB histogram is too far to the right, the image is over exposed. Start with a histogram that has a nice spread (from left to right) and superimposition of Red, Green, and Blue humps. Ultimately, you are the judge of exposure, but the histogram can guide you to a starting point.

(C) Maneuver to an area that is clear and white and set white balance.

(D) IF NECESSARY, weak the above steps for each new objective and area of observation. I fight with my white balance the most. White balance and I have a love-hate relationship because we fight a lot, but in the end, we always compromise for a "whiteness" that makes us both equally satisfied.

3. MICROSCOPE ADJUSTMENTS:

(E) Find a spot to image, focus specimen, and begin preparations for imaging (do not change objectives or area of observation after this point)

(F) Set the aperture iris diaphragm of the condenser to 60-90% of the objective numerical aperture (may not be possible with every microscope) (do not touch the aperture iris diaphragm after this point).

(G) Use condenser focus knob and field diaphragm to put the image into Kohler illumination (may not be possible with every microscope) (do not touch the condenser focus knob or field diaphragm after this point).

(H) READY TO IMAGE!


Maybe this helps and maybe I am vomiting out a complete noob post, but these are some of the things that I found out on my own long after taking hundreds of images that could have benefitted from minor tweaks. I hope this was helpful and/or gave you ideas for improving upon that which you are already extremely proficient. Like I said, this is purely a gesture of kindness, and not to insult your technique. I hope you would do the same for myself should I ever post images that you would adjust differently.

Please, keep sharing with us your beautiful work!

EDIT: Hobbyst46, "IMHO, it would be better to set the condenser iris last, (reverse steps F and G) since the correct condenser aperture (whatever value you decide for it, I e.g. prefer towards the 60% not 90%) depends on the brightness of the illumination, which in turn depends on the centration of the image of the field aperture. I would start with centration and do it with the condenser iris fully open, or nearly so."

SunshineLW wrote:I know all microscopes and digital microscope softwares are different, but here are several things I fiddle with when assessing my color options for brightfieqld images (I know NOTHING about fluorescence! or DIC!). You probably know all of this, but I always use the same systematic process so I'll list it all for completeness sake. Also, I am a noob at this so this could be completely wrong in terms of how and what you are imaging. Besides, the photographer is the ultimate judge of exposure/ contrast/ color/ etc., and if you like what you are seeing, don't change it to please somebody else! I offer this list as a gesture of kindness and not to insult your technique because your images are stunningly beautiful. At this point, I would begin nitpicking every tiny detail to perfect every shot!

1. Place specimen on stage and focus image

2. SOFTWARE ADJUSTMENTS:
(A) If using "Auto-exposure", define the area of autoexposure to encompass the entire image.

(B) Look at the Red, Green, and Blue (RGB) histogram to ensure that the histogram is relatively evenly distributed from left to right. In general, if the RGB histogram is too far to the left, the image is underexposed. If the RGB histogram is too far to the right, the image is over exposed. Start with a histogram that has a nice spread (from left to right) and superimposition of Red, Green, and Blue humps. Ultimately, you are the judge of exposure, but the histogram can guide you to a starting point.

(C) Maneuver to an area that is clear and white and set white balance.

(D) IF NECESSARY, weak the above steps for each new objective and area of observation. I fight with my white balance the most. White balance and I have a love-hate relationship because we fight a lot, but in the end, we always compromise for a "whiteness" that makes us both equally satisfied.

3. MICROSCOPE ADJUSTMENTS:

(E) Find a spot to image, focus specimen, and begin preparations for imaging (do not change objectives or area of observation after this point)

(F) Set the aperture iris diaphragm of the condenser to 60-90% of the objective numerical aperture (may not be possible with every microscope) (do not touch the aperture iris diaphragm after this point).

(G) Use condenser focus knob and field diaphragm to put the image into Kohler illumination (may not be possible with every microscope) (do not touch the condenser focus knob or field diaphragm after this point).

(H) READY TO IMAGE!


Maybe this helps and maybe I am vomiting out a complete noob post, but these are some of the things that I found out on my own long after taking hundreds of images that could have benefitted from minor tweaks. I hope this was helpful and/or gave you ideas for improving upon that which you are already extremely proficient. Like I said, this is purely a gesture of kindness, and not to insult your technique. I hope you would do the same for myself should I ever post images that you would adjust differently.

Please, keep sharing with us your beautiful work!

Thanks for taking the time to write all this out -- I'll definitely keep it in mind and probably refer back to your suggestions regarding kohler illumination (which still gives me a bit of trouble every now and then). In my general photography I find that I seldom use bright field; I'm much more inclined to use phase contrast or darkfield (like the photos I posted above). Of course, this isn't to say I won't use bright field. And when I do I'll look into the illumination to make sure it's set up correctly.

Because I shoot camera RAW I don't do anything with the white balance in camera; it's all done in Photoshop's ACR utility. For the most part I don't really have any trouble with white balance; at least on my screen it looks pretty good. But no two screens (or eyes for that matter) are the same. Mine should be fairly close as I do use color calibration on my monitor. But it certainly doesn't hurt to pay attention to all that as well.

Thanks again for your post; I really do appreciate you taking the time to write it all out and share your own experience and knowledge. Be assured that I never took it as an insult -- I know I'm not that good!

Hi, I've had a look on the web and suspect the fungus may be Miyagia pseudosphaeria which seems to be a known enemy of Sonchus - even to the extent that it's being studied as a possible bio-control for Sonchus 'weeds'....

John B.

mrsonchus wrote:Hi, I've had a look on the web and suspect the fungus may be Miyagia pseudosphaeria which seems to be a known enemy of Sonchus - even to the extent that it's being studied as a possible bio-control for Sonchus 'weeds'....

Wow! Awesome find!

SunshineLW wrote: ...
(F) Set the aperture iris diaphragm of the condenser to 60-90% of the objective numerical aperture (may not be possible with every microscope) (do not touch the aperture iris diaphragm after this point).

(G) Use condenser focus knob and field diaphragm to put the image into Kohler illumination (may not be possible with every microscope) (do not touch the condenser focus knob or field diaphragm after this point).

(H) READY TO IMAGe

IMHO, it would be better to set the condenser iris last, (reverse steps F and G) since the correct condenser aperture (whatever value you decide for it, I e.g. prefer towards the 60% not 90%) depends on the brightness of the illumination, which in turn depends on the centration of the image of the field aperture. I would start with centration and do it with the condenser iris fully open, or nearly so.

Same here,the field iris needs to be set first then the aperture iris closed until the FOV shows a tiny darkening-effect just noticeable (through the eyepieces) - this almost certainly results in the 80%-ish desired. Anything past the exact (100 %) match of aperture to objective, where resolution is optimal at the full n.a. of the objective (and the image is not contrasty-enough really) is a balancing-act to get a nice contrast but not too much of the accompanying resolution-drop.

G then F I think too.

When I turn the condenser to phase contrast or darkfield the iris is automatically opened up. I don't usually use brightfield; I prefer the results from the other methods better. And to be honest, when using brightfield I'm more than likely doing it completely wrong. That's why the guide here is of really good use for me. Thanks!

Hobbyst46 wrote:IMHO, it would be better to set the condenser iris last, (reverse steps F and G) since the correct condenser aperture (whatever value you decide for it, I e.g. prefer towards the 60% not 90%) depends on the brightness of the illumination, which in turn depends on the centration of the image of the field aperture. I would start with centration and do it with the condenser iris fully open, or nearly so.

Thank you so much for the advice! Part of the reason I wrote that whole thing out was because I was hoping somebody could help me make improvements. I have added an edit with your advice to my original post above. I will take your advice next time I'm imaging. I am very grateful for your suggestions.

SunshineLW wrote:

Hobbyst46 wrote:IMHO, it would be better to set the condenser iris last...

You are most welcome, happy to help.

Bonjour
Très belles images
Merci pour le partage
Cordialement seb