[CNC] [Request] Designing/cutting Lab equipment for the real time visualization of DNA strand exchange

[Lukas Lindorfer]

Hi Sebastian,

thank you for your interest! The difference between the flow cells commonly used and the ones you are probably seeing Right now is that we do not work with whole cells but only with DNA strands and proteins in solution, so a little bit of shearing would not be too terrible. The holes are 4mm wide since they are optimized for those FPLC screws to make a wider range of applications possible: https://www.fishersci.co.uk/shop/products/peek-finger-tight-connector/11390052

We would drill a M5 thread, pitch of 0.8 into the holes to lead a very thin tube (1/16 inch) through them. Due to this tubing, the smoothness of the hole is not really a factor.

What is critical is to have a really flat ground surface since TIRF microscopy is only practical in a range of about 200nm. I assume (but actually do not know) that the block to cut the cell out of would be rather flat to begin with. Is this the case?

The model I sent still has some (a lot of) room for improvement – Would you be interested in meeting at some point in the next weeks to talk about the cell? That way, I could give you some insights on what we know and need to know.

Best,
Lukas




________________________________
Von: CNC <cnc-bounces at lists.metalab.at> im Auftrag von Sebastian Bachmann <metalab at reox.at>
Gesendet: Monday, October 28, 2019 2:12:20 PM
An: Neigungsgruppe Zerspanungstechnik/CNC und Werkstatt allgemein <cnc at lists.metalab.at>
Betreff: Re: [CNC] [Request] Designing/cutting Lab equipment for the real time visualization of DNA strand exchange

Hi!

That sounds interesting!
I'm just now looking at some pictures in the internetz of those flow cells
and it looks like the cavity is in the micrometer range - usally like 400µm.
When looking at the screenshots, I suspect that the dimensions are in mm, hence
the hole should be 500µm in diameter - right?
So I would say, this should be do-able in the CNC using regular drills.
An important question is the required surface structure of the holes.
I would guess, that the surface must be very smooth, in order to not damage
cells and also provide a laminar flow through the cell?
This might be problematic when using regular drills, as they do not leave a nice
surface finish.
You can get 0.5mm reamers but they are horrible expensive: https://www.accu.co.uk/en/machine-reamers/444489-221450-0500?PageSpeed=noscript
If you break one of these ... also our CNC can not run with low speeds, but
maybe at this size the lowest rpm of 6000 might be sufficient.

So in theory, you could build this thing using a three step process:
1) mill the pockets on the lower surface
2) mill the first wedge and drill holes
3) mill the second wedge and drill holes

For steps 2 and 3 you need either some angle plate or a sine table - we dont
have either of those in the lab.
There might be working drills in the lab, I think they have 0.75mm diameter -
not sure how critical the dimensions are.
Drills are cheap - everything else is not. So depending on the surface, you can
get it cheap or horrible expensive.

Also note, that our CNC is right now in some undefined state where our spindle
drive has some undefined issues.

Sebastian



On Mon, Oct 28, 2019 at 12:00:47PM +0000, Lukas Lindorfer wrote:
> Hi all,
>
> my name is Lukas.
> The Metalab was recommended to me by my friend Juli („Juli wie August“) as a place to develop and share ideas. Right now I am sitting in your main room for the first time, typing up an idea that might interest some of you. I am a biology student currently working in a group doing research on on the generation and analysis of proteins involved in plant meiosis. We know what (some of them) do, but we have never seen it “live“ – And I have an idea to change that.
>
> I am seeking help in designing  and cutting a flow cell for TIRF microscopy.
> If anyone is interested in collaborating on this idea with me, I would be very grateful and happy.
>
> Attached you will find a screenshot of the prototype. If anyone could spare some time for advice in cutting and design and is interested in the project as well as a beer/dinner/whatever on me, please contact me!
>
> Cheers, Lukas
>
>
> More Infos:
>
> The „state of the art“ is applying the proteins to investigate at the lab bench and then going to the laser microscope. The Problem: Those proteins work fast – less than five minutes. When I arrive at the microscope, the reaction is already finished which makes visualizing/filming it rather hard considering sample preparation takes up to 3 hours.
>
> This is where the flow cell comes in. With this system, we could prepare the protein sample in the microscope itself – visualizing meiotic strand exchange in real time for the first time in history!
>
>
> Some vocabulary:
>
> Meiotic Strand Exchange is the exchange of DNA between two chromosomes in the generation of sperms/eggs --> The stuff that makes every human unique!
> https://www.yourgenome.org/facts/what-is-meiosis
>
> TIRF (Total Internal Reflection Fluorescence Microscopy) is a niche laser microscopy technique, being able to visualize single molecules linked to a glass plate. https://www.microscopyu.com/techniques/fluorescence/total-internal-reflection-fluorescence-tirf-microscopy)
>
> A flow cell is exactly what it sounds like: A cell stuff flows through. In our case, the glass plate to investigate would be rubber cemented on the bottom of the cell so it can be looked at with the laser.
>
>



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