HyperCyt and Python Scripts

Today we had a long day in the robotic room. Yesterday Arik from Neotec came to calibrate the find details of the liquid handling arms on the Tecan which solves some issues we encountered on Tuesday.

Today Shai, who is responsible for "smart" robotic application came. We had a general discussion about different ways of integrating software to the robot controlling software. Afterward he, Avital and Assaf sat down and implement some pythons scripts that call the robot and others that the robot can call.

It seemed that this was very fast pace study as they reported success, and managed to fancy pipetting procedures on the fly.

In the meantime, Ariel and Jenia tried to get the HyperCyt to work in the new location. We decided to move the peristaltic pump onto the hypercyt deck. This however resulted in the arm pushing the pump off when the device was initialized. This was annoying, especially since the pictures of the device showed the pump sitting exactly where we put it. 

We searched the manual high and low, and finally realized that one of the figures mentions an L-shaped piece that serves as a stopper in such a configuration.  We used this as evidence that this is the right solution and installed the stopper, which indeed solved the problem.

However, now the coordinates of the arm were totally off. And so we learned how to "teach" it where the different locations are. This involved moving the tip of the needle very slowly with mouse controls until it was in the right location.

In the end the system was working, and we even managed to film it. As you can see the sampling needle goes into each well in succession. This means that it creates alternating bubbles of media (+ cells) and air in the tube. If you look carefully in the movie you can see these tubes.

After lunch break and group meeting Jenia run his nifty analysis software to break the long FACs stream of events to specific wells. It worked like a charm and immediately gave him detailed summary of each well. The investment in this software was definitely worth while.

Busy busy day (Robots, FACS, cabinets and sinks)

So another one of these crazy days. So what did we have today.

* David and Ariel from Merkel Technologies came to move the FACS and HyperCyt into the new robotic room (see movie).

* Udi and Shy from NeoTec continued installing the KiNEDx robotic arm. They managed to move a plate from the Tecan to the microscope and back (see movie again).

* We had our first wet-lab group meeting in the room next to robotic room. The furnishing need some work, but the room is usable.

 

* Yoel, the carpenter from "Wooden Horse" (סוס עץ) brought the new storage cabinets/lockers for the corridor. He also brough cabinets for the robotic room, which made it interesting to work there for a while. The workmanship of the new cabinet was impressive.

* The plumbers came to install a water heater in small yeast preparation room. They also finished the sink in the robotic room.

* We had a regular group meeting in the CS building as well :-)

* And most importantly, Yael came to visit the lab to approve it.

And movie of some of the installation around the robot.... Note the sequence at the end where the arm moves the plate from Tecan to Microscope.

HyperCyt on the run

Last week I reported on the Flow Cytometer and its new upgrades. These allow to start testing the combination of the HyperCyt loader with the Flow Cytometer. We finally have some preliminary results, but they are exciting.

Recall that the Flow Cytometer "sucks" cells from liquid sample and pass them through a flow chamber where they hit a laser beam which allow to measure both fluorescent signal and the size of the cell. The HyperCyt loader is a machine that allow to collect samples from a multi-well plate and send them to the FlowCytometer. You can see a movie in the original blog entry. Thus, the FlowCytometer receives a sample that consists of 1.5 seconds of a "sip" into a well, followed from a 1 second sip from air. At the end of a 12-well row, there is another step of sipping water to clean the pipes.

Today Ariel (our Ariel, not to be confused with Ariel from Merkel who did the Flow Cytometer upgrade), performed a test to see the whole thing in action. Now that we have upgraded the Flow Cytometer acquisition hardware/software we can start examining the results. To see how good they are, we plot the time a cell was collected vs. the measured value. In situations where there were no cells we will not see points. 

When Ariel ploted the time vs. Forward Scatter (a measure of the extent to which the laser beam was scattered by the cell, which is a proxy of cell size), we see a band for each row of wells.

Zooming in to look at a particular band, we can see that it consists of intervals that correspond to a sample separated by blank intervals (air bubbles).

We can observe several things here. 

First although each "sip" is of 1.5 second, some of them are spread out on longer measurement time. This is probably due to the fact that the bubbles are spread out by the transition in the tube, and the first bubble might have more space to spread out. 

Second, although air bubbles are easily detectable by the lack of any events (cells), the empty wells Ariel added (marked by Medium and water) do contain some cells. These are probably carry over cells that got temporarily stuck on the tubing and then released by the flow. We can see that by after the wash by one well, the subsequent water well has much fewer events. This will give us a sense of how much carry over we can expect.

If we look at the GFP measurement for these cells we see similar bands. Here Ariel used strains of different GFP intensities, and we can see from the plot that they differ. 

For example, the "no-gfp" strain shows very low level of GFP (basically auto-florescence), while some of the other strains (e.g., Sod1, Rpl25A) show very high levels.

This is a very first run out of many we are going to perform. Clearly there are issues to deal with, such as how to automatically parse the output into wells, how to reduce the amount of carryover, how reproducible are the results and so on.  However, I am very excited to start seeing all these different things start to work together.

HyperCyt!

We got the HyperCyt robotic FACS loader almost six weeks ago (wow, time flies when you are busy), and after some delays we are finally starting to see it getting into working conditions. The last few weeks David and Ariel (from Merkel Technologies) came several times to install the various components and checked the software installation. They also replaced the old Mac computer that runs the FACS with a newer G5 machine.


So, why I am writing about this today? Because, today was the first "wet test" for the HyperCyt. Ariel (ours, not Merkel Tech's, this is very confusing) and Ayelet seeded two strains of yeast for overnight growth. In the morning they organized them on a 96-well plate.

We then moved to the HyperCyt. The machine consists of a shaker on which we put the plate, a robotic arm that moves a sample needle in three degrees of freedom (X-Y and up/down), and a peristatic pump.

 

The idea is that we can in a fast succession sample successive wells on the plate. First, however, we need to program the control procedure to define which wells to sample.

Then, we can run the program and see the needle go from one well to the next.

You can see the needle going into each well in a succession. During the interval between wells the pump sucks an air bubble, which sepereate the samples from the previous well to the next one. Moreover, at the end of the row it goes to a wash station and shakes the plate to resuspend the cells. We can program the HyperCyt for different schedule of sampling, speed of the pump,  needle washing schedule, shaking schedule, and so on.

The test was the first step to get this system working. We are still missing a clean interface for connecting the cable into the FACS and for seemless integration between the HyperCyt controller and the FACS controller. However, the first test shows that the system does work and allow us to measure on the FACS the cells from multiple wells.