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<p class="MsoNormal">Hi,</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">I need the cell by January only – Ideally, we could meet between the 4th of November and the beginning of December. Do you have time at some point in this period?<br>
<br>
Thanks for the tips about getting it flat! My Problem is that I never used a CNC mill or any comparable tools so I would need someone to mill with me in order to not die.</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">I would be eternally grateful if that was possible with you.</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal">Best, Lukas</p>
<p class="MsoNormal"><o:p> </o:p></p>
<p class="MsoNormal"><o:p> </o:p></p>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>Von:</b> CNC <cnc-bounces@lists.metalab.at> im Auftrag von Sebastian Bachmann <metalab@reox.at><br>
<b>Gesendet:</b> Monday, October 28, 2019 2:38:11 PM<br>
<b>An:</b> Neigungsgruppe Zerspanungstechnik/CNC und Werkstatt allgemein <cnc@lists.metalab.at><br>
<b>Betreff:</b> Re: [CNC] [Request] Designing/cutting Lab equipment for the real time visualization of DNA strand exchange</font>
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<div class="PlainText">On Mon, Oct 28, 2019 at 02:35:36PM +0100, Sebastian Bachmann wrote:<br>
> Hi!<br>
> <br>
> On Mon, Oct 28, 2019 at 01:26:03PM +0000, Lukas Lindorfer wrote:<br>
> > Hi Sebastian,<br>
> > <br>
> > 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:
<a href="https://www.fishersci.co.uk/shop/products/peek-finger-tight-connector/11390052">
https://www.fishersci.co.uk/shop/products/peek-finger-tight-connector/11390052</a><br>
> <br>
> Ohh okay, 4mm is not a problem and you can use plenty of tools to get a very<br>
> smooth finish even!<br>
> <br>
> > 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.<br>
> > <br>
> > 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?<br>
> <br>
> No, you can not assume that the blocks are flat. I would assume that you use<br>
> aluminium? In that case, the flatness is usually in the range of hundrets of µm.<br>
<br>
Or less, obviously!<br>
There might be suppliers which sell precision stuff - but usually that is steel,<br>
which we can not handle in the CNC.<br>
<br>
> Milling the block flat, should reduce it - ideally you would surface grind it,<br>
> or lapp the block using a master plate (which we dont have).<br>
> What is the approximate surface flatness you require?<br>
> (see also here <a href="http://www.engineeringessentials.com/gdt/flatness/flatness.htm">
http://www.engineeringessentials.com/gdt/flatness/flatness.htm</a><br>
> for a definition of this measure)<br>
> <br>
> You can produce very flat surfaces by lapping three plates against each other.<br>
> Oxtools has some videos on that: <a href="https://www.youtube.com/watch?v=rHmsQEAx16o">
https://www.youtube.com/watch?v=rHmsQEAx16o</a><br>
> <br>
> <br>
> > <br>
> > 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.<br>
> <br>
> Next week is very bad for me... I have time this week, and also wanted to visit<br>
> metalab and the CNC and see whats wrong with the VFD.<br>
> <br>
> Sebastian<br>
> <br>
> > <br>
> > Best,<br>
> > Lukas<br>
> > <br>
> > <br>
> > <br>
> > <br>
> > ________________________________<br>
> > Von: CNC <cnc-bounces@lists.metalab.at> im Auftrag von Sebastian Bachmann <metalab@reox.at><br>
> > Gesendet: Monday, October 28, 2019 2:12:20 PM<br>
> > An: Neigungsgruppe Zerspanungstechnik/CNC und Werkstatt allgemein <cnc@lists.metalab.at><br>
> > Betreff: Re: [CNC] [Request] Designing/cutting Lab equipment for the real time visualization of DNA strand exchange<br>
> > <br>
> > Hi!<br>
> > <br>
> > That sounds interesting!<br>
> > I'm just now looking at some pictures in the internetz of those flow cells<br>
> > and it looks like the cavity is in the micrometer range - usally like 400µm.<br>
> > When looking at the screenshots, I suspect that the dimensions are in mm, hence<br>
> > the hole should be 500µm in diameter - right?<br>
> > So I would say, this should be do-able in the CNC using regular drills.<br>
> > An important question is the required surface structure of the holes.<br>
> > I would guess, that the surface must be very smooth, in order to not damage<br>
> > cells and also provide a laminar flow through the cell?<br>
> > This might be problematic when using regular drills, as they do not leave a nice<br>
> > surface finish.<br>
> > You can get 0.5mm reamers but they are horrible expensive: <a href="https://www.accu.co.uk/en/machine-reamers/444489-221450-0500?PageSpeed=noscript">
https://www.accu.co.uk/en/machine-reamers/444489-221450-0500?PageSpeed=noscript</a><br>
> > If you break one of these ... also our CNC can not run with low speeds, but<br>
> > maybe at this size the lowest rpm of 6000 might be sufficient.<br>
> > <br>
> > So in theory, you could build this thing using a three step process:<br>
> > 1) mill the pockets on the lower surface<br>
> > 2) mill the first wedge and drill holes<br>
> > 3) mill the second wedge and drill holes<br>
> > <br>
> > For steps 2 and 3 you need either some angle plate or a sine table - we dont<br>
> > have either of those in the lab.<br>
> > There might be working drills in the lab, I think they have 0.75mm diameter -<br>
> > not sure how critical the dimensions are.<br>
> > Drills are cheap - everything else is not. So depending on the surface, you can<br>
> > get it cheap or horrible expensive.<br>
> > <br>
> > Also note, that our CNC is right now in some undefined state where our spindle<br>
> > drive has some undefined issues.<br>
> > <br>
> > Sebastian<br>
> > <br>
> > <br>
> > <br>
> > On Mon, Oct 28, 2019 at 12:00:47PM +0000, Lukas Lindorfer wrote:<br>
> > > Hi all,<br>
> > ><br>
> > > my name is Lukas.<br>
> > > 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.<br>
> > ><br>
> > > I am seeking help in designing and cutting a flow cell for TIRF microscopy.<br>
> > > If anyone is interested in collaborating on this idea with me, I would be very grateful and happy.<br>
> > ><br>
> > > 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!<br>
> > ><br>
> > > Cheers, Lukas<br>
> > ><br>
> > ><br>
> > > More Infos:<br>
> > ><br>
> > > 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.<br>
> > ><br>
> > > 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!<br>
> > ><br>
> > ><br>
> > > Some vocabulary:<br>
> > ><br>
> > > Meiotic Strand Exchange is the exchange of DNA between two chromosomes in the generation of sperms/eggs --> The stuff that makes every human unique!<br>
> > > <a href="https://www.yourgenome.org/facts/what-is-meiosis">https://www.yourgenome.org/facts/what-is-meiosis</a><br>
> > ><br>
> > > TIRF (Total Internal Reflection Fluorescence Microscopy) is a niche laser microscopy technique, being able to visualize single molecules linked to a glass plate.
<a href="https://www.microscopyu.com/techniques/fluorescence/total-internal-reflection-fluorescence-tirf-microscopy">
https://www.microscopyu.com/techniques/fluorescence/total-internal-reflection-fluorescence-tirf-microscopy</a>)<br>
> > ><br>
> > > 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.<br>
> > ><br>
> > ><br>
> > <br>
> > <br>
> > <br>
> > > _______________________________________________<br>
> > > CNC mailing list<br>
> > > CNC@lists.metalab.at<br>
> > > <a href="https://lists.metalab.at/mailman/listinfo/cnc">https://lists.metalab.at/mailman/listinfo/cnc</a><br>
> > <br>
> > <br>
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