Bovine Pulmonary Artery Cells-Revisited

[Walter Piorkowski]

Top Image:
Leitz 40x Apo, 0.95 NA dry objective
Tiyoda Wide Field Cardioid condenser, NA 1.40
7 images at 1micron increments

Middle image:
Leitz 70x Fl. Oil 1.30 Na objective
Funneled for use with a 1.20NA darkfield condenser
Leitz darkfield condenser NA 1.20
single image

Bottom Image:
Leitz 95x Fl. oil objective, aperture iris set to NA of 1.10
Ziess (cardioid?) darkfield condenser NA 1.40
7 images at ½ micron increments

Leitz Ortholux
5x projection eyepiece with 1/3rd x relay lens
Canon 10D
All images processed in Combine ZM and Photoshop.

I have been doing a lot of experimenting with darkfield to improve upon my first images of this subject. I have also adapted several non-Leitz condensers for use on my old Ortholux to experiment with cardioid and possibly other high NA condenser designs.

Working at high magnifications with high numerical aperture condensers has been challenging but I believe the results are better than my earlier efforts. The blue arc in the top image is a flair I was unable to avoid. Image 2 is provided to show how an aperture funnel can make a very high NA objective usable for darkfield

I invite comments on the Macro and Micro Technique and Technical forum where I have posted some more info and questions.

Walt

[Mike]

Hello Walter,

Maybe I missed it, but aren't these fluorescence shots?

All the best,
Mike
_________________
"Nil satis nisi optimum"

[Walter Piorkowski]

Hi Mike. No UV light source or filters were used here, straight incandecent bulb and a darkfield condenser. I know nothing about how the slide was dyed by the maker. Are there fluoreccing dyes?

Walt

[Mike]

Hello Walter,

Have to admit I can't figure it out; your shots look very much like those obtained with fluorescence setup, perhaps not as bright on the various fluorophores, but very close!

From the NikonMicroscopyU website: "Applying a popular triple fluorophore technique, the BPAE cell culture presented above was stained with MitoTracker Red CMXRos, DAPI, and Alexa Fluor 488 conjugated to phalloidin, which target the mitochondrial network, DNA, and F-actin, respectively."

Is the slide an old one? Perhaps it wasn't prepared with the Alexa Fluors, that are brighter and more stable than previous dyes.

Take a look at http://www.microscopyu.com/galleries/fluorescence/cells/bpae/bpae.html
to see how similar your shots are.

Maybe someone brighter than I can explain how this is possible with phase.

All the best,
Mike
_________________
"Nil satis nisi optimum"

[bernhardinho]

Hi Mike

isn't it correct that fluorescence dyes need to be excited with well defined wavelengths to show the effect? Piotr's bulb couldn't possibly deliver that and I think that these darkfield (NOT phase!) shots look like fluorescence ones by mere coincidence.


Bernhard

[Walter Piorkowski]

Hello again Mike. I find this most interesting. After looking at the Nikon site images, which they state were shot in black and white, then colored to match the narrow band pass wavelength of the filter pack used. My images come remarkably close without all the high tech. And by the way no phase contrast optical configuration was used.

Walt

[rjlittlefield]

I'm betting on fluorescence.

As described by Wikipedia

[Cyclops]

wow fantastic shots! These are nerves cells from a cow yes?
_________________
Canon 10D | EOS 300 (Rebel-film) | Panasonic FZ-7 EB | Vivitar/Cosina 100mm f3.5 macro lens | EF 75-300 f4.5-5.6 USM III | EF 50 f1.8 II | Vivitar Series 1 19-35 f3.5-4.5 | Slik 88 Tripod. | My macro shots:
http://stumm47.deviantart.com/gallery/#Macro-and-Close-up

[discomorphella]

Hi All--
These are in fact, images of fluorescently labeled BPAE cells, looks like the labelling was properly described by Mike's reference. The darkfield transmitted setup Walter is using will have more than enough blue and green wavelength ranges to excite the Alexa and the MitoTracker. The DAPI is typically excited by mid-UV, e.g., 360 nm (see the absorption spectrum here for example http://omlc.ogi.edu/spectra/PhotochemCAD/html/dapi(H2O).html or here http://probes.invitrogen.com/servlets/spectra?fileid=1306dna ) but if you look at the spectrum you will see that there's some absorption at 400 nm, more than enough to give a weak DAPI image, which was nicely recorded. The darkfield illumination is providing all the appropriate wavelengths for the 3 dyes, and their fluorescence emission is going back into the objective to give those nice shots. Not as efficient as a newer epi-fluorescence scope, but perfectly adequate to see all the structures in a heavily labeled slide.
FWIW--

David

[bernhardinho]

Hi David

that is very interesting information indeed. And encouraging too, I'm sure I'll give it a try.


Bernhard

[rjlittlefield]

[discomorphella]

Hallo Bernhard--

You probably have a few slides stained with Eosin Y, which is chemically somewhat related to Fluorescein (the fluorophore in the famous FITC stain for labelling proteins). It absorbs green light (ok, its peak abs is ~525 nm and its peak emission is > 550 or so) and emits green. If you look at a standard transmitted light micrograph of an H and E (hematoxylin and eosin) stained slide, you will see the cytoplasm of most cells stained crimson by the eosin. Try this in darkfield, or throw it under a fluorescence microscope, and you will see the eosin fluoresce yellow. The hematoxylin usually quenches things, you probably won't see anything but dark nuclei. It's the same phenomenon Walter has beautifully captured, but the labeled endothelial cells in his slide are stained with much brighter fluorochromes, and they are mounted in low-fluorescent background medium, most likely with an anti-fade ingredient or two to help keep the dyes from fading out while they are being illuminated. Fading is not a big problem with the low flux from a darkfield condenser and a halogen lamp (it is with an Hg arc and a serious epi setup). With dark-adapted eyes in a darkened room (how they used to do fluorescence microscopy before CCDs...) you can see quite a few fluorescent dyes using your DF setup....of course, with a decent CCD you can see far far more...

--David

[Walter Piorkowski]

Thanks for the compliment Rik, David and Cyclops. And thank you also David for such a thorough review of what I was recording, wavelengh wise, without even knowing it. To answer Mikes question on the age of this slide I am afraid that I have no idea. It was provided to me as a bonus for entering the 2007 Olympus Bioscapes contest. As I stated in my original post, it was a total disappointment in bright field but most interesting in darkfield.

Now that I know that the subject is excited by UV light I must tell you guys that my first posting was done with a 50 watt quartz track light bulb that I have been experimenting with to get more illumination for darkfield work. It just so happens that this bulb has a UV blocking filter on it and as such was retarding my ability to record the BPAE subject. I of course did not realize this at the time. Exposure times were ridiculously long or as Rik stated phenomenally inefficient. Boy Rik how right you were. They were much shorter with the incandescent bulb for my revisited posting. I know that the quartz bulb of the quartz halogen lamp will pass more UV so I should find a way of removing the filter and see what improvement I get.

Now I will have to start all over with my review of dark field condenser types because the contrast improvements I was realizing on this slide were likely a result of light wavelengths and not condenser design. Thank you all, this has been most interesting and informative. Oh and Bernhard don’t mistake me with Piotr, I believe that is the handle for someone else posting on the forum.

Walt

[discomorphella]

Ooops, should have said Eosin absorbs green and emits YELLOW light. Hopefully it was clear from the rest of the paragraph...editing late at night is hazardous...

--David