Assignment 10, Part 1: Measuring the osmotic shock response of yeast

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Assignment 10 is the culmination of a lot of hard work. You've built a two-color epi-fluorescence microscope, including computer controlled flow and illumination. You've made a microfluidic device and tested its flow properties. It's now time to make some biological measurements!

As you may know, this is a new experiment that we implemented for the first time in Fall 2018. There are still many avenues for exploration and improvement, so treat the following instructions as suggestions. If you think there is a better way to do something, try it! Then tell us what you did when you turn in your assignment.

Assemble a microfluidic device

Follow the instructions in Assignment 8 to assemble a PDMS device and cut the needed tubing. Note that you should still have one or two cured PDMS devices left over from Assignment 8 that you can use.

Tips for a successful microfluidics experiment

As you know from Assignment 8, microfluidics can be finicky. Here are some tips to help maximize your likelihood of success.

  1. After first loading liquid into the device, do everything you can to avoid introducing air - ever. Air is pesky and hard to remove once it's introduced. It disrupts the flow, and can rip cells from the coverslip. Consider starting with a fresh device, rather than trying to flush air out after it's been introduced.
  2. Tilt the device when loading it, so that bubbles have a chance to float up and out of the device.
  3. Use positive pressure to load the device. For example, it's better to load the syringe with fluid and push it through the device and tubing, rather than using the syringe to suck fluid from a reservoir into the device. This is because the PDMS is gas permeable, so applying negative pressure can actually pull air from the PDMS into the fluidic channels.
  4. Degas solutions (i.e. place them under vacuum) for 30 minutes to an hour before starting your experiment. This helps to remove dissolved gasses from solution, and reduces the likelihood that bubbles will nucleate inside your device.

Incubate device with ConA

We need to immobilize yeast cells to the coverslip of the flow device in order to image them over long times. A standard method of immobilizing yeast cells for microscopy is to coat the coverslip with concanavalin A (ConA) - a lectin that binds to cell surface saccharides.

  1. Thaw 1 aliquot of ConA (250 μL of 1 mg/mL in PBS), preferably slowly, on ice.
  2. Load the ConA solution into a 1 mL syringe. Without introducing any bubbles, slowly push the entire aliquot of ConA into the device. Leave the syringe connected an let incubate for at least 20 minutes (or until ready to load the yeast cells).

While you're waiting, it may be a good time to set up you microscope, check its alignment, and verify that your circuits are still working.

Wash extra ConA from device and fill tubing with media


Global Tree.gif Please follow these safety tips when working with biological materials:
  • Wear a lab coat and gloves when handling yeast cultures and media. Clean up any spills by wiping with a paper towel sprayed with ethanol.
  • Wear safety goggles when loading microfluidic devices to avoid unanticipated spray from getting in your eyes.
  • Remove your gloves when not handling biological materials. For example, do not wear gloves when typing on the computer, or using hex wrenches and pliers. Have one teammate control the computer commands while the other handles the biological material.


Keeping to our philosophy of only using positive pressure, we'll fill the microfluidic device channels and tubing with media by pushing from the outlet. We want to load each side with the appropriate medium (high or low salt).

  1. If you haven't already, connect the inlet tubing by twisting the luer fittings onto the pink inlet needles.
  2. Use a binder clip to pinch closed one of the two inlets.
  3. Load 1 mL of high salt medium into a syringe.
  4. Disconnect the conA syringe from the outlet of your device. Without introducing air, connect the medium-filled syringe to the pink outlet needle.
    • One strategy is to drip some medium from the syringe into the needle to fill it up before connecting the two - making a liquid-liquid connection with no air in between. This strategy tends to overfill the needle and can lead some medium to spill out. That's ok; it's better than introducing air into the device. Wipe up any spills with ethanol.
  5. Slowly (seriously!) push the medium from your syringe into the device. You should be able to see the meniscus traveling through the open side of the tubing until droplets form at the other side. Hold the inlet tubing over a waste container, and push out almost the entire remaining solution in your syringe.
  6. Making sure there is a small droplet at the edge of the tubing, submerge it into the 15mL high-salt reservoir. It may be wise to tape the tubing to the edge of the tube so it doesn't accidentally get knocked out of place.
  7. Remove the binder clip and pinch the now-full high-salt inlet tubing.
  8. Fill a syringe with low salt medium and repeat the loading and washing steps above for the opposite inlet tubings.
  9. Leave the syringe attached for now, and remove the binder clip.

Mount the device on your microscope stage and load yeast cells

Mount your device

  1. Carefully carry your device and tubing reservoirs over to your microscope, making sure to keep the inlet tubing submerged in their reservoirs. This is likely a two-person job.
  2. Insert the 15 mL high- and low-salt media reservoirs into the 50 mL conical tube holders, and carefully mount the PDMS device to the stage holder.
  3. With the solenoid valve disengaged (i.e. foo.OpenLowSalt), feed the low salt-filled silicone tubing into the open slot of the pinch valve. Check that the tubing is completely seated inside the valve.
  4. Engage the solenoid valve (i.e. foo.OpenHighSalt), then feed the silicone tubing into the now-open slot of the pinch valve. Again, check that the tubing is completely seated inside the valve.
  5. Close the pinch valve (foo.OpenLowSalt) and keep the syringe connected while you prepare the cells.

Record a background image

Before you load your cells, record a background image.

  1. Focus the image on the side of your flow channel (it's hard to focus on the coverslip when the channel is empty, so focusing on the side wall will help you get close to the right focal plane).
  2. Move the stage to so that the field of view is roughly centered within the flow channel.
  3. Adjust the blue and green LED currents to their recommended maximum (1A) and set the blue and green exposure times to 5 s.
  4. Run the command foo.TakeTwoColorPicture;, which save a single two-color image into foo.OutputData.
  5. Save the data to a workspace variable so that it doesn't get overwriten. For example: backgroundData = foo.OutputData.

Prepare yeast cells

  1. Remove 0.75 mL of the cell culture into a microcentrifuge tube.
    • The cells are grown in synthetic-complete medium (SC) which has a lower autofluorescence than other types of medium like YPD.
    • The ideal yeast concentration for log-growth is for it to have an OD600 between 0.4 and 0.6.
  2. Add 0.75 mL of YPD medium to the culture tube. (YPD medium helps the cells form a better pellet when centrifuged.)
  3. Put the tube in the microcentrifuge (along with a second tube to act as a balance) and spin at 800g for 2 minutes.
  4. Pipette off the supernatant and resuspend the pellet in 200 μL of SC medium.

Load the yeast cells into fluidic device

  1. Double check that the tubing and device are bubble free, and that the pinch valve is in the low salt state.
  2. Disconnect the syringe at the outlet of the PDMS device, and fill it with the yeast culture.
  3. Without introducing any air bubbles, re-connect the syringe to the outlet needle.
  4. Slowly inject the yeast cells into the PDMS device. Avoid introducing air into the tubing by pushing the plunger only 2/3 of the way.
  5. Leave the syringe connected, and let the cells settle and adhere to the coverslip for 10-15 minutes. (You can jump ahead to Find the focus and set the imaging parameters if you wish, while you wait.)
  6. When ready, disconnect the syringe and connect the outlet tubing to the pink outlet needle. Put the other end of the outlet tubing in a waste reservoir.
  7. After a few minutes, you should see the meniscus of the media flow through the outlet tubing, ultimately forming droplets into the waste reservoir. If you can't see any flow, try raising the inlet reservoirs up higher on the post.
  8. Toggle the pinch valve several times to ensure that both media reservoirs are flowing as expected, then leave the cells to equilibrate at low salt.

Record movies

Once you're sure the flow is behaving as expected, let the yeast flow under low salt conditions while you align your microscope and prepare to record data.

Find the focus and set the imaging parameters

  1. Use the room lights (or a high gain setting) to bring the yeast cells into focus.
  2. Cover your flow channel with a box to block out background light (careful that the box doesn't pull on the tubing).
  3. Turn the blue light on, and using a high gain setting, focus on the cells.
  4. Turn on the green excitation, and again using high gain, adjust the focus. The cell nuclei are harder to focus on, so make sure to always use the green illumination as your final focusing step. If you don't see a good nucleus image, there's no hope for collecting good data.
  5. Lock the stage in at least the z-direction using the locking screws. This is extremely important for long experiment times to prevent the stage from drifting out of focus.
  6. With the green illumination on, use the SetGreenImageParameters command to set the exposure time to 5 s (5,000,000 μs) and adjust the gain to get the best image.
  7. Turn on the blue illuminator and set the exposure and gain. In both cases, exposure times longer than 5 or 6 seconds start to become impractical, so we can only increase gain at that point.

Start recording data!

Each group will record one oscillation movie and one step response. Check this google doc to find out which oscillation periods have been assigned to your group.

The options are:

  1. T = 4 min. Record at least 6 periods, and acquire 8 images per period.
  2. T = 8 min. Record at least 4 periods, and acquire 8 images per period.
  3. T = 16 min. Record at least 2 periods, and acquire 8 images per period.
  4. T = 32 min. You will be awarded extra credit if you successfully complete this one. Record 2 periods, wait for 5-10 minutes at low salt before starting this experiment.
  5. Step response. Record for 2 minutes at low salt and 25 minutes at high salt. Acquire an image every 1 min.

Tips:

  • These experiments are tricky and will only work if you are careful and vigilant. It's tempting to walk away while your data is recording, but check in frequently to make sure that everything is still behaving as expected. There's no use taking a 30 minute movie if your cells drifted out of focus in the first frame!
  • Lock the z-axis of your stage when taking data to prevent drift in the focus of your microscope. THIS IS VERY IMPORTANT.
  • Re-check your focus and camera settings after each measurement. Use high gain settings when focusing, long exposure times when taking data. You may also want to move your FOV to image fresh cells if the current ones seem dim.
  • Don't forget to save all of the output data to a variable in the workspace (e.g. OscMovie4min = foo.OutputData) and SAVE YOUR WORKSPACE after every movie!
  • The following are some useful commands:
% Initialize the software
foo = OsmoticShocker;
foo.Initialize;

% Turn on and off the illumination (only one will be on at a time)
foo.BlueOn
foo.BlueOff
foo.GreenOn
foo.GreenOff

% Set imaging parameters
foo.SetBlueImageParameters(2,5000000) % for Gain = 2, exposure = 5s
foo.SetGreenImageParameters(15, 5000000)

% Set parameters for oscillation experiment
foo.SetOscillationParameters % will prompt you for inputs
foo.StartOscillationExperiment % will start oscillating the valves and will start recording images after the specified equilibration time

% Set parameters for step response experiment
foo.SetStepResponseParameters % will prompt you for inputs
foo.StartStepExperiment

Upload your movies to Dropbox

It may be challenging to directly compare the amplitude and phase responses of Hog1 to osmotic shock when each each individual's analysis code is slightly different. So the instructors would like everyone to upload their movie data into dropbox, and we will run them through a consistent analysis.

Once you've collected all your data, please save three variables to the Fa2019 Assignment 10 Movies folder on the lab computer dropbox:

  1. In MATLAB, enter the Fa2019 Assignment 10 Movies dropbox folder, making it your working directory.
  2. Set the following three variables equal to the corresponding data that you recorded:
    1. OscillationMovie (if you have recorded multiple movies, choose the best one to upload),
    2. StepResponse, and
    3. BackgroundImage.
  3. Please save the entire struct provided by foo.OutputData to each of these variables, then save them using the command:
save A10_Firstname1Firstname2Firstname3 OscillationMovie StepResponse BackgroundImage

using the names of your group members. This command will save the three specified data structures to the filename 'A10_Firstname1Firstname2Firstname3.mat'.

Clean up

When you're sure you have collected and saved all your data, follow these steps to clean up.

  1. Wearing gloves, remove the two inlet tubings from their reservoirs.
  2. Remove the silicone tubing from the pinch valve, and let the fluid drain through the device into the waste container.
  3. Disconnect the tubing from the PDMS device, dispose of the tubing in a biowaste container, and dispose of the device in the biological sharps bin.
  4. Aspirate the remaining liquid from the reservoirs and waste container, and dispose of the empty tubes in the biological waste bin.
  5. Discard any opened syringes and needles in the biological sharps waste.

Analyze data

Use your code from assignment 9 to analyze your movies. Upload your measured amplitude and phase for the oscillation movie to the google doc.

To find the 95% confidence interval of each fit parameter, the following commands may be helpful:

    [fitValues, residual, ~, COVB, ~] = nlinfit(x, y, modelFunction, initialGuess, fitOptions);
    CI = nlparci(fitValues, residual, 'covar',COVB);


Pencil.png

For each oscillation movie that you recorded:

  1. Use your code from Assignment 9 to extract the Hog1-response vs. time.
  2. On one set of axes, plot the Hog1-response vs. time and the best fit sinusoid.
  3. Report the best-fit amplitude, phase, and offset for your measured oscillation frequency.
  4. Pool your amplitude and response data with other groups, and make a Bode Plot (amplitude and phase) of the Hog1-response as a function of frequency.
  5. How does your data compare to the amplitude and phase found in the paper? If your measurements do not agree, describe two or three reasons why they might differ.

For one step response movie:

  1. Use your code from Assignment 9 to extract the Hog1-response vs. time.
  2. Fit the response to the expression for the second-order underdamped step response of this system: $ y(t) = u(t) \text{e}^{-\sigma t} \sin(\omega_d t) $, where $ u(t) $ is the unit step function.
  3. Report your fitted values for $ \sigma $ and $ \omega_d $.
  4. On one set of axes, plot the Hog1-response vs. time and the best fit step response.
  5. What are $ \zeta $ and $ \omega_0 $ for this system? (Hint: use the definitions of $ \sigma $ and $ \omega_d $.)