Multi-Color FACS

As you may remember, we are using the Flow Cytometer (FACS) to measure the fluorescence levels of individual cells. Our FACS is an older BD FACSCalibur that has gone through several updates. Almost exactly a year ago, we had all the electronic boards refurbished. At the same time we already had plans to replace the old 488nm laser with two lasers at different colors. These lasers arrived with the electronics, but we decided to hold off on their installation that until we stop using GFP markers (that need 488nm excitation). 

After few months we realized that this will not happen so fast, and ordered a third laser with 488nm capabilities. Due to many reasons the delivery of this upgrade was postponed for a long while. Finally, last week, all the necessary parts arrived. Last week Ariel and David from Merkel Technologies installed these upgrades and today we had a training session to go over the capabilities of these devices.

As part of the installation, Ariel removed the old (big and heat generating) laser, and put in a stack of three lasers instead. These required a panel of one-way mirrors to focus the beams onto the light path of the flowcytometer.

You can see the lasers here (with Ariel explaining) on the left side as small boxes. They are really tiny, but sit on aluminum blocks to disperse heat they generate.

The new light path involves mirrors that take the beams from the lasers

More schematically, the new light path configuration is shown below, where each line segment is either a bandpass filter (that allows only small "window" of wavelengths to pass through) or a half-mirror (that reflect certain wavelengths and passes others). As a result each sensor (FL) measures different area of the spectrum.

The consequence of this design is that we now can measure either GFP and mCherry  (a type of RFP) together, or YFP and mCherry. We cannot operate measure together both GFP and YFP since the YFP excitation is very close to the GFP emission.

As before, when the FACS is serviced all the covers come off, and it is a great chance to take pictures of the light beams going through the prisms and mirrors

 

and finally hitting the target area in the flow cell

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.

The Naked FlowCytometer

The last two days Ariel from Merkel Technologies Ltd installed an upgrade to the logic boards of our Flow Cytometer (FACS). The new logic boards, made by Cytek, will allow us to collect data using a newer acquisition station, which will solve compatibility issues with the HyperCyt loader. They also are the basis for the next upgrade of the lasers and the sensors (that will come in few weeks, depending when we are ready).

The upgrade involved replacing all types of boards inside the FACS, and for a whole day the lab looked like an electronic workshop rather than a biology lab.

In the process, Ariel also uncovered the lid off the laser part of the unit, which gave us a chance to peak into the working of the machine. The main lightpath

involves a laser on the left. The laser beam passes through two prisms and then onto the flow chamber on the right, and then from there to sensors (The cylinders beyond).

The flow chamber, has a two input the first is a sheath fluid and the other is the sample. The sheath fluid is flowing much faster and thus creates a thin stream of cells form the sample.

(source: http://www.abcam.com )

In our configuration we have the same setup but the flow is from below:

At the end of the day the device was closed up, assembled, and working.

Tonight was the beginning of the independence day celebrations and so we got to see some fireworks.

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.