Daddy Long Legs
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Daddy Long Legs
Using a 5x objective and a stack of about 60 images using Helicon focus. Registration artifacts were not cropped.
Will
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rjlittlefield
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Will,
I'm glad to see you're getting some mileage out of that new scope. Arthropods are always fun -- it seems like the detail just doesn't stop no matter how much magnification you pile on.
I'm curious about the bright halos. Usually I see those when the lighting changes somehow during the stack. But I'm far from sure what is causing these. Do you have any idea?
--Rik
I'm glad to see you're getting some mileage out of that new scope. Arthropods are always fun -- it seems like the detail just doesn't stop no matter how much magnification you pile on.
I'm curious about the bright halos. Usually I see those when the lighting changes somehow during the stack. But I'm far from sure what is causing these. Do you have any idea?
--Rik
Rik:
I think I can explain the fringe. The sample was caught on a sticky bug catcher that was on the lab floor. The fringing was most likely the reflection of the adhesive. This is not a usual procedure we use.
I had placed this under scope to show a guest how the scope worked. It was a pinch choice. But I thought the head structure was really interesting. I also wanted to see how Helicon Focus would handle such a thick subject. The other problem is the sticky fly paper is not as stable as it needs to be, but once something is caught, there is no way to get it off.
The interesting thing is I have been helping faculty with stacking with fluorescence. Fluorescence presents a very difficult exposure problem. It is not only very dim, but also it is also very contrasty - seems to be a contradiction, but areas with a lot of photofluors are very much brighter than areas with few photofluors. This is further complicated because I am also trying to get a background image using DIC or Hoffman Modulation Contrast. I have been most successful with making separate fluorescence and background images and then combining them.
I think I can explain the fringe. The sample was caught on a sticky bug catcher that was on the lab floor. The fringing was most likely the reflection of the adhesive. This is not a usual procedure we use.
I had placed this under scope to show a guest how the scope worked. It was a pinch choice. But I thought the head structure was really interesting. I also wanted to see how Helicon Focus would handle such a thick subject. The other problem is the sticky fly paper is not as stable as it needs to be, but once something is caught, there is no way to get it off.
The interesting thing is I have been helping faculty with stacking with fluorescence. Fluorescence presents a very difficult exposure problem. It is not only very dim, but also it is also very contrasty - seems to be a contradiction, but areas with a lot of photofluors are very much brighter than areas with few photofluors. This is further complicated because I am also trying to get a background image using DIC or Hoffman Modulation Contrast. I have been most successful with making separate fluorescence and background images and then combining them.
Will
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rjlittlefield
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This sounds like a fascinating and challenging application. Whatever images you can share with us, I would appreciate seeing and hearing about.
With regard to combining images from separate stacks, be aware that stacking software may subtly distort the subject's apparent geometry as it works its way through the stack by lining up visible detail. I have no idea whether this will actually happen for your subjects, but it's something to keep in mind if details in your fluorescence and background images don't seem to register properly after stacking.
--Rik
With regard to combining images from separate stacks, be aware that stacking software may subtly distort the subject's apparent geometry as it works its way through the stack by lining up visible detail. I have no idea whether this will actually happen for your subjects, but it's something to keep in mind if details in your fluorescence and background images don't seem to register properly after stacking.
--Rik
Yes, I knew the stacking and background registration may present a problem, but so far the specimen is thin enough where it seems to be OK. The only issue is I have not been stacking the background and so some of the extent in the tissue is not imaged because of depth of field. Obviously two stacks would eliminate that problem.
My biggest problem is the very fine details in the fluorescence is still lost when the background is added - or the background is very dark. Bring the levels up on those small details makes retaining detail in the highlights a problem. It is like trying to store an 18 foot canoe in a 16 foot room; something has to go out the window or out the door. I could use a smaller canoe, but the results are not as good.
My biggest problem is the very fine details in the fluorescence is still lost when the background is added - or the background is very dark. Bring the levels up on those small details makes retaining detail in the highlights a problem. It is like trying to store an 18 foot canoe in a 16 foot room; something has to go out the window or out the door. I could use a smaller canoe, but the results are not as good.
Will