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A side view of cells with Galena

September 22nd, 2009 Posted in How To, Microscopy, Science

Choanofla­gel­lates are a bit of a chal­lenge to image in sev­er­al ways.  Putting aside the fact that many of them swim about a lot, even the ones that stick to sur­faces tend to attach to the sur­face of the dish and point direct­ly upwards—so they look like lit­tle cir­cles under the micro­scope, their col­lars and fla­gel­la not vis­i­ble because they are point­ing direct­ly towards you. What we need is a flat sur­face at right angles to the bot­tom of the dish, that the choanos can attach to and be in the per­fect ori­en­ta­tion for us to image them.  We also need the sub­strate to be thin—too thick, and it’ll cut off part of the cone of light that forms the image. 

I was think­ing that the ide­al solu­tion would be (very) tiny cubes that would sit with one face on the sur­face of the dish, and the four adja­cent faces pro­vid­ing good, flat, orthog­o­nal sur­faces for the choanos to attach to.  What is small and cubic?  I thought of salt—a nat­u­ral­ly occur­ring cubic-lattice crys­tal, which wouldn’t work of course because it would dis­solve, but lots of oth­er crys­tals have cubic lat­tices!  After order­ing a few dif­fer­ent min­er­als with cubic lat­tice crys­tals from Wards, and try­ing to crush them in a pestle and mor­tar to make tiny lit­tle cubes, I found one with the right properties—cheap, soft enough to crush into tiny crys­tals eas­i­ly, and insol­uble in water—Gale­na (Lead Sul­phide).

The sec­ond chal­lenge was to get a (rea­son­ably) uni­form size, which is actu­al­ly real­ly easy.  Just start by grid­ing up some gale­na in a mor­tar and pestle (feel­ing like an old alchemist).  I did this with water to pre­vent dust because I didn’t want to inhale lead pow­der, and wear­ing gloves—small things tend to be rather bio­log­i­cal­ly active, and I don’t want to absorb any lead through my skin if I can help it).  Then I iso­lat­ed a size range—putting the gale­na slush first through a large (150 µm) fil­ter, keep­ing the flow-through (<150 µm), then through a small (30 µm) fil­ter (nylon mesh) keep­ing the reten­tant (30–150 µm) and wash­ing to remove all of the very small par­ti­cles.  I stored this under ethanol, to try to pre­vent the gale­na dis­solv­ing too much.

Galena crystals covered in attached choanoflagellates

Gale­na crys­tals cov­ered in attached choanofla­gel­lates

It works quite well (see Figure—click on it for a larg­er view). The crys­tals aren’t per­fect cubes, but there are lots of flat sur­faces orthog­o­nal to the bot­tom of the dish, and the choanos attach all over them, their cell bod­ies lin­ing up in their the­cae a few microns from the sur­face.  The crys­tals are thin enough not to dis­rupt the imag­ing too.  The only down­side is that they tend to slide over the bot­tom of the dish if you’re not care­ful, plough­ing through the choanos on the sur­face and pil­ing them up on the edge of the crys­tal.  For long-term growth, I might be a bit con­cerned about lead dis­solv­ing into the solu­tion, but I’m not sure how much of an issue this is—galena is not sup­posed to dis­solve very much. 

I’m not includ­ing any high res images of choanos tak­en this way, because we’re not yet sure which ones we might want to pub­lish, but I may add some lat­er.

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