How fluorescence transforms what you see in the microscope

Stack of 20 pictures showing a rotifer in a shell. Hybrid image of fluorescence and phase contrast.

  In this traffic heavy post I'm describing how fluorescence is used in microscopy and  showing with transition animations how what you see in a "regular" light microscope matches with fluorescent pictures.

  Notice that it can take some time for animations to start as they take time to load. Animations require flash, no Apple mobile devices support. Also, viewing this page might be very slow and glitchy with Internet Explorer, try other browsers if it happens.

  Briefly, fluorescence is light that is emitted by substance that is exposed to ultraviolet (or any other kind of light). (If you are into science don't take it as a definition=) )

  Normally, in biology it's used to reveal internal structure of cells. Fluorescence is observed under UV light and turned off "regular" lamp.

A ciliate Colpoda with acridine orange dye, transition fluorescence - phase contrast

  Basically, a fluorescent substance is required (a dye). Today gene engineering allows integrating natural fluorescent proteins into cells and marking specific structures, or even more precise, specific proteins, and it is possible to observe the dynamics of those.

A collection of images showing the same species of Colpoda stained with various dyes and under various conditions and in various stages. I did not use GFP (fluorescent proteins) in my work.

  Technically, you need a UV filter to protect your eyes, and the same filter is supposed to allow light only of certain spectra to pass, but I use a crap filter that's only blocking UV light, and all other kinds of light are visible.

A ciliate with acridine orange and propidium iodide under normal and UV light transition animation. Ciliates like birds like sitting on branches and shitting on your heads. That's the only picture in this post where I did not remove the background in the phase contrast image.

  With some patience and craploads of dye it's possible to colour entire multicellular organism   This is a tiny rotifer that hides itself in the shell when scared

The only acceptable fluorescent shot of rotifer out of the shell that I managed to make. They are very fast and to take a fluorescent picture you need long exposure. Basically, only one out of hundred shots is sort of successful.

  Notice that blueish light is not technically fluorescence in this case, it's reflected and diffused from mercury lamp that is used as a UV source, in other words, the visible part of light that illuminates the cover of the rotifer.   When it hid itself in a shell:   I managed to get 10 fluorescent and 10 phase contrast pictures that I combined into z stacks and then in the animation above   With more patience it's possible to dye big organisms, like oligochaets, which I mentioned in my previous post.

  Only when it finally stopped moving after an hour of torture I managed to get matching pictures to combine into an animation:   To me the worm looks much better under fluorescence, sorry for the poor quality of the "regular" light image.   All images made with Canon PowerShot G10 That's it for now, I'll be experimenting more in the near future

Bring my apologies for incompatibilities with Apple mobile devices, there is no other way of making this sort of animations on web pages. Also, sorry for Internet Explorer users, the browser is really heavy and does not display properly loads of interactive content.

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