Hi Steemit,
Yesterday, I tried a new imaging technique.
I was using MitoTracker Deep Red on some bone marrow derived macrophages (BMDMs), trying to see if our WT and variants had any difference in mitochondrial shape, numbers, or whatever else we could see.
MitoTracker Deep Red is a far-red fluorescent dye that is used for mitochondrial localization (1). It is actually the only MitoTracker we have in the lab that can withstand paraformaldehyde fixation and permeabilization. I was really excited about imaging our BMDMs with this new dye; I had spent a previous afternoon Frankensteining a protocol from three different labs + the manufacturer's protocol.
On the Thermofisher site, there are some absolutely breathtaking images of cells stained with this dye. I was particularly excited about this image of A549 cells, as that is a line I have some experience with:
Needless to say, I was chomping at the bit to get taking pictures yesterday. I had reserved 5 hours on the scope, since my usual scope, a Nikon A1Rsi, was undergoing maintenance and I had to work my way around new equipment and new software.
To get to this point, I had worked close to 40 hours acquiring the cells, culturing them, developing new protocols, fixing, and doing both MitoTracker and surface staining on them.
I fired up my scope, applied exactly one drop of oil to the objective, laid down my slide, and began to focus.
What I saw was nothing like those gorgeous A549 cells. My cells looked more like this:
Honestly, the cells above look 100x better than what I saw yesterday. What I saw yesterday was failure, and defeat. It took a week to culture these BMDMs, and I wasted so much time and resources trying to mount them on coverslips so I could take some pictures.
I should have prepared more. I should have done this, or that. It's my fault that they're so damn ugly.
I exported a couple of tiffs at the beginning of my scope time and sent them to my boss. I asked her, "What do I do with these? I don't see a single discrete mitochondria."
My boss responded, "You take the median intensity from each, and compare."
The lesson to learn here is that experience and failure are both great teachers. Just like I can find meaningful data in images I didn't expect to get, we can make meaning in our lives and grow from things that disappoint us. It is a simple lesson, one most of us learn pretty young. It is one I am taught again, and again, and again every time I come into work and sit at my bench.
This, along with the data and the publications and the this and the that is what makes science worth doing. There is always a new lesson to learn at the bench!
Love always,
T
(1)Thermofisher.com. (n.d.). MitoTracker Deep Red FM - Thermo Fisher Scientific. [online] Available at: https://www.thermofisher.com/order/catalog/product/M22426 [Accessed 21 Dec. 2017].