WB imaging on Vilber-Lourmat Fusion - (Nov/25/2017 )
We routinely use films to develop WB illuminated by HRP and are in the process of moving to digital. We have Vilber-Lourmat Fusion Solo S machine, which should be giving even maybe superior images, but so far I'm not that content. Our boss wants a very high quality images for each blot, so it's not just a question of "will do". And we do a lot of them all the time.
I'm looking for someone who has experience with digital imaging on Vilber or even different machines.
Not talking about the software, because that is once again one example of totally unintuitive weird work-flow ideas, but never mind. The biggest problem is the bands seem more blurry than from film. We are putting the membrane inbetween a common transparent office sheet, as for the film cassette, but the sheets have bit of mate finish.
Other option was to drop the substrate directly just on the membrane laying on a parafim or something, but when done in less wet conditions I would be affraid of membrane drying before I finish the capture, and when I pre-wet the membrane during handling then I could see the substrate flowing here and there and inconsistent band lighting then. Still it looks similarly blurry. But I can't decide if it actually is blurry or just not that intense since weak bands on film are also a bit blurry.
Also it allows to capture the image in white light to see the protein markers (the only way to label the band sizes later, when using digital imaging), but I can't seem to get it work other than in preview (in capture it is totally highligted). We could surely use some contrast enhancement on the marker image, but I don't know how to force it and modifications outside the program are not saved in the same color depth (see below)
The default TIFF is saved in 16-bit depth, which is even more than computer can display, so you can play with the dynamic range cuttofs but I see no point in doing that, other than just getting a darker picture. Having bigger dynamic range is usually better, but I can't seem to find out how to use that advantage in this case.
Maybe part of the problem is we are using the lowest tray to get the biggest field od view, but that is still less than capacity of film cassette, so maybe putting on the closer tray would allow better focus, but since we are new to that we are not sure, if we don't screw the preset options somehow (also the optional program setting are.. weird) by movin the aperture (it is manual as well as filter setting).
I would appreciate any comments on how you do your digital imaging and how are you satisfied with it.
I can't comment on the Vilber-Lourmat, other than I have used an old system of theirs many years ago, but I am sure such knowledge is outdated and probably only half-remembered.
I would check that it is a focus problem - put a blank/old membrane on there in the same sandwich as you would for imaging and check the focus of the sides of the membrane and markers using white light; if the image is blurry under white, it will be flurry under chemi too. Writing on the membrane can also give you some good information about resolution and focus.
I note that you are taking an image using the biggest field of view - are you then enlarging to look at your bands? If so, the resolution is dependent on the pixel size of the image - equivalent to a digital zoom on a camera- all you are doing is zooming in on the pixels and making them bigger. Optical zoom is always better, so if you can adjust this, the images should get better - either that or move the membrane closer to the camera.
With respect to the aperture, the wider open the aperture (smaller number) the narrower the depth of field and the harder it is to (maintain) focus on that point. There are also optimal ranges for different apertures with different amounts of zoom. For chemi blots, you will want the aperture wide open to capture as much light as possible, which means that if you have the blot a long way away from the lens, you need to keep the imager very still (no centrifuges running next to it, no bumping the bench etc.) to maintain consistent sharp focus. Another thing to note here is that light follows the inverse distance squared rule - if an object (membrane) is 2x further away from the lens, only 1/4 (1/(22) of the light is reaching the lens. This means that less light is reaching the lens = longer exposures and lower sensitivity. Moving the blot closer is the only solution here.
We use an Odyssey Fc, and it is very good, both for chemi and using IR-fluorescence-tagged Abs.