BPAE fluorescent cells

[Pau]

Bovine Pulmonary Artery Endothelial Cells (BPAE Line), a very classic triple stained fluorescence prepared slide from a lab culture.
I got a pair of very nice samples as a gift for participating in the Nikon Small World years ago (this kind of slides can be purchased at very high prices).
Now, with spare time and my custom arranged multi-LED fluorescence microscope finished I’m attempting to get good images of them, despite the net being full of better ones typically taken with much more modern and expensive equipment

Each image is a composite of three non-stacked images each one taken with a single color LED and the matched filter cube, processed in Canon DPP and PS and merged in PS as single color channels

The camera is the Canon 7D and the microscope stand the Zeiss WL with fluorocondenser IV
The 20X image was taken with Nikon CFI Plan Apo 20X 0.75 direct projection with bellows and Sigma LSA as tube lens, the others with Leitz Pl Fluotar objectives taken afocally with a Periplan 10X eyepiece and a Pentax 40mm pancake lens (1.6X secondary magnification) most them somewhat cropped to a more square form.

Red: Mitochondria / MitoTracker Red CMXRos
Green LED 535nm; filter cube: EX: ET545/25x //BS: T565lpxr //EM: ET605/70m

Green: Cytoskeleton actin microfilaments / Alexa Fluor 488- phalloidin
Blue LED 465-480nm; filter cube: EX: HQ480/40 // BS: Q5051P// EM: HQ 535/50

Blue: Nuclei (DNA) / DAPI
UV LED 365nm; filter cube: EX: 380x //BS: 410DCLP // EM: LP 410AELP + BP450-490

1. 10X/0.30

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2. 25X/0.60

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3. 50x/1.00 oel

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4. 20X/0.75

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5. 20X, 100% pixel crop

[houstontx]

wow looks great like a confocal image. Will these dyes lose their fluorescence over time or once its set in the mounting media its "locked in"?

[RogelioMoreno]

Pau,

Very nice set with excellent results!

I see that you are using ET filters (they are very good, hard coating).

Rogelio

[iconoclastica]

Best pictures of a cow I ever saw :-))

Are these combined images of three exposures with different excitators & filters?

[Adalbert]

very nice !!!

[Pau]

Thanks all for your kind comments!

...Will these dyes lose their fluorescence over time or once its set in the mounting media its "locked in"?

I got these slides in 2008 if I remember well, and they have survived flawlessly -stored in a dark not too warm place and with few brief expositions to fluorescence microscopes- although they can suffer some photobleaching degradation when exposed to the excitation illumination over the time (the zone of the slide where I did most of the setup tests under the blue excitation LED has lost at least half of its fluorescence)

Are these combined images of three exposures with different excitators & filters?

Yes, each shot was taken with one LED and the (almost) matched filter cube, then I combined the relevant channel of each shot as a channel of the composite image, for example the mitochondria shot excited with the green LED and the red channel of the image was pasted as the red channel of the final composite image, and so on

[ModelZ]

Kudos for beautiful work on a classic target! Indeed holds ground even next to confocal stuff.
Just curious, have you experimented with stacked pics? Stackers introduce almost by default alignment off-sets in different channels, how much extra difficulty/work does that imply?
-Karl

[rjlittlefield]

Stackers introduce almost by default alignment off-sets in different channels, how much extra difficulty/work does that imply?

I do not understand this issue. In all the stackers I know you can just turn off adjustment, so the images stay aligned exactly as they came from the camera.

Can you explain the difficulty?

--Rik

[ModelZ]

No such question if you just go by the frame, perhaps that is the way to go. What I was thinking is that the images in different color channels are really qualitatively quite different. Filament mess in one, organelle cloud in another, nuclei blobs in other etc. Especially in a bit thicker samples aligning separately could perhaps improve each individually (preference to top and bottom layers and such) but joining then together might get harder, no? As said just layman wondering. Would be fun to experiment with this eventually (but I'm not going to pay $250+ to play with this sample :-).
-Karl

[rjlittlefield]

No such question if you just go by the frame, perhaps that is the way to go. What I was thinking is that the images in different color channels are really qualitatively quite different. Filament mess in one, organelle cloud in another, nuclei blobs in other etc. Especially in a bit thicker samples aligning separately could perhaps improve each individually (preference to top and bottom layers and such) but joining then together might get harder, no?

Yes, for sure. But these problems are not unique to multi-channel fluorescence. At high magnification / large NA, it is common for different depth planes to look so much different from each other that computational alignment messes up. Best practice is always to turn off "corrections" for all problems that are prevented by the setup. When working with low mag macro setups, you almost always have to correct for scale and shifts because of optical issues and mechanical instability. But when working at high mag inside the frame of a commercial microscope, usually everything is stable enough that best results come from stacking images exactly the way they were shot.

--Rik

[Pau]

Kudos for beautiful work on a classic target! Indeed holds ground even next to confocal stuff.
Just curious, have you experimented with stacked pics? Stackers introduce almost by default alignment off-sets in different channels, how much extra difficulty/work does that imply?
-Karl

Thanks, Karl

The sample is so thin that I haven't done any image stacking (partially for avoiding long exposition to excitation light) although with the higher magnifications it could have been convenient.
The three color channels in my approach are so different that stacking between them seems to do not make much sense.

I've only had alignment issues with the nuclei of the 20x image due to a small displacement of the stage when changing the LED rail position. I've aligned it with the red channel manually in PS (it required few attempts).

[FotoChris]

Awesome!! You managed to capture the effect perfectly! It looks almost like a rendered image, really great work!!!

[KI4THC]

Absolutely gorgeous work. Actually better than a lot of Laser Scanning Confocal images that I've seen.

[hkv]

Beautiful work!