All posts by Ashley Cannaday

Fixed Cell Measurement Protocol

  1.  Put coverslip with adherent cells in the sample chamber and wash with PBS. This involves gently spraying the PBS on the coverslip with a pippette, and gently rocking the sample chamber back and forth. This should only require about 0.5 mL of PBS.
  2.  Aspirate PBS and fill chamber with 1 mL of 4% formaldehyde. Let sit for 15 minutes at room temperature (in cell prep box, covered with a chem wipe).
    1. There are variances in both time and temperature (some incubate rather than using room temperature), so this is something that could be varied if desired.
  3.  Aspirate formaldehyde and wash twice with PBS. Aspirate PBS.
  4. For measurements, fill with HBSS at 37C as is typical with live cell measurements.

Throughput vs angle correction

To our knowledge, the throughput of our system versus angles has not previously been accounted for. To determine it, we placed the teflon cap in the sample chamber, submerged it in water and put a square coverslip on top, just as we would for a bead sample. The scattergram for the cap is shown below.

Teflon_scattIf the throughput did not change versus angle, then the intensity should be about constant (with noise) throughout the entire scattergram.

Teflon_cutHowever, we can tell from the cut-throughs that there is indeed a falloff. This falloff can be well fit to a cos(theta) function:  Throughput(θ)=I(θmin)cos(θmin)cos(θ)

ThroughputHere is the least squares fit to a 5um bead without any throughput correction:

5um_nocorrectionAnd here is the fit with the throughput correction. The theory cut-throughs were multiplied by the above throughput equation and then least square fit to the data (with an offset and stretch term).

5um_thrucorrectionThe throughput correction did not have much of an impact on the quality of the fits. If instead you LSF the theory cuts to the data, like normal, and then multiply by cos(θ):

5um_LSF_thenthrucorrThe amplitudes of the peaks are still to small, but the falloff trend seems more correct.

Throughput vs Angle

Reference beam setup/alignment

  1. If pellicule beamsplitter is out, put it in between the CCD and the last lens of the 4f system. The engraved side should be facing away from the CCD. It should be at as much of a 45 degree angle as possible, given the space constraints. Make sure the beamsplitter is not clipping the sample beam.
  2. Direct the reference beam towards the CCD using reference mirrors 1 and 2.
  3. With all ND filters out, adjust reference mirror 1 so that the reference beam transmitted through the pellicule and the sample beam reflected off of it hit the same spot in the near field (close to the pellicule).
  4. Adjust reference mirror 2 so that the two spots overlap in the far field (farther away from the pellicule).
    1. When the spots are very close to overlapping, it may be beneficial to stop down both beams so that the spot sizes are small. Just make sure the iris’s used are centered!
  5. Put the ND filters back in, and make final adjustments to mirror 1 so that the two beams overlap perfectly.
    1. If this part of the alignment is correct, you should have circular fringes with good contrast.
  6. Put a sample of fixed beads into the system (larger beads make alignment easier).
  7. Place a bead on the edge of the elastic excitation beam, diagonal from the center. Adjust BS1 and reference mirror 1 so that the tilt fringes are nulled in the interferogram.
  8. Move the bead to the center of the excitation beam. Adjust the BS and mirror until the frequency of the tilt fringes approximately doubles in both x and y directions.
  9. Put in two pinholes along the reference beam path to make future alignments easier.

Aligning the IRAM system

Raman alignment (all in epi-illumination mode):

1) With the air objective and no condenser lens, and a glass coverslip in the sample plane, use the 1st Raman mirror to make the two reflections from the coverslip overlap on the hitachi CCD.

2) With no objective or condenser lens, and a mirror facing downwards in the sample plane, use the 2nd Raman mirror to make the retro-reflection exiting the microscope hit the same spot as the illumination beam. (This can be done by using the IR viewer and a piece of lens paper to see both beams.)

3) Repeat steps 1-2 until they converge.

4) Put in the oil objective and a glass coverslip in the sample plane. Adjust the 2nd Raman mirror so that as the objective goes in and out of focus, the beam of the hitachi CCD has equal power in each quadrant of the spot.

5) Put in two pinholes after the periscope. These pinholes designate the path of a vertical beam.

Elastic alignment (in trans-illumination mode):

6) Use the two Elastic mirrors to direct the beam through the pinholes before the periscope, using the 1st mirror to align to the first pinhole and the 2nd mirror to align the second pinhole.

7) To fine-tune the beam so that it is going through the center of the objective with no condenser lens: Adjust the 2nd Elastic mirror so that the beam is hitting the center of the objective (will need IR viewer to see this). Then adjust the 1st Elastic mirror so that the beam is still going through the pinholes before the periscope. Repeat until the two converge.

8) Put in the condenser lens, and adjust its focus and position.

Quick Alignment

1) In epi-mode: Adjust 1st and 2nd Raman mirrors so that the beam is going through the 1st and 2nd pinholes, respectively, in the Raman excitation path.

2) In epi-mode: Without the condenser or objective, adjust the periscope mirrors so that the beam is going through the two pinholes after the periscope.

3) In trans-mode: Adjust the 1st and 2nd Elastic mirrors so that the beam is going through the two pinholes before the periscope.

4) Put in the objective and condenser. Adjust the focus and position of the condensor.

 

Cell culturing protocol

Thawing Frozen Cells

  1. Cells arrive deep-frozen.
  2. Add cells to 10 mL of growth media with 20% FBS.
  3. Incubate overnight or until confluent.
  4. Passage to 2 dishes with 10 mL of growth media with 10% FBS
  5. Continue to passage about twice a week.

Passaging

  1. Aspirate (remove) media.
  2. Add 1 mL of trypsin, coating the entire bottom of the dish.
  3. Swirl for 20 seconds, then aspirate.
  4. Add 1 mL trypsin.
  5. Incubate for 4 minutes.
  6. Use the pipette to “pressure wash” cells off of one dish with trypsin.
  7. Add the trypsin with the lifted cells to the second dish.
  8. Use pipette to wash cells off of the second dish with trypsin.
  9. Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
    1. (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence

Cell Media/Solutions

Cell Media (EMT6)

  • 500 mL BME (Basal Medium Eagle. We have always gotten this from the Foster lab)
  • 2.5 mL Pen Strep
  • 2 mL Fungizone
  • 1 mL plasmocin
  • 1 mL primocin
  • 10% FBS – 55.5 mL (Fetal Bovine Serum)

*Note: 20% FBS media is also needed for when cells are first thawed.

*For SCC7 cell line, components are all the same except instead of BME base media, use RPMI media

*All Nada solutions are based on 500mL total final solution

Nada Solution #1 (basic cell measurement solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008  g D-glucose
  • 6 mL Hepes
  • 0.0832 g CaCl2
  • 0.0571 g MgCl2

Nada Solution #2 (permeabilizing solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008 g D-glucose
  • 6 mL Hepes
  • 0.476 g MgCl2
  • 20 μM Ionomycin

Nada Solution #3 (calcium shock solution)

  • 3.9447g NaCl
  • 0.2199g KCl
  • 0.9008g D-glucose
  • 6mL Hepes
  • 0.0888g CaCl2

9/9/15 IRAM Meeting

During the weekly angular scattering group meeting we discussed:

  • Incubator: Cells are never growing to confluence in the incubator. Yesterday frozen SCCVII cells were brought over to our lab from the Foster lab, thawed, and put into media. Today all of the cells were dead. How can we confirm that the incubator is the problem? If it is, then the most likely issue is the % humidity. There is usually condensation on the incubator door, and the water bath is evaporating quickly.
    • We will contact Phoenix Equipment about possible sanity checks/repairs or purchasing a new incubator.
  • Cell measurements will be on hold for about a week while the Foster lab gets CO2 for their incubator and gets new cells up and running.
  • Reinstating the holographic arm of the elastic system: Determining how much more stable size estimates we can get by going to lower angles and (a) doing no processing, (b) intensity smoothing, (c) coherent smoothing of speckle. Eventually move to a common path interferometer? ROBERT
    • We need to buy another BK7 flat or wedge so that we can put in a double bounce periscope that preserves polarization, like Zach’s system had.
  • Reinstating the Raman arm (probably not in the near future)
  • Measure the scattering from multiple (>5) small beads to see the effect of speckle on the fits when we know what the right answer should be. JANET
  • Possibility of larger field of view when doing interferometric measurements
  • Possible uses for SLM
  • Update on Xing’s sparse sampling simulation study. Xing is creating a sparse distribution of scatterers (mitochondria) and converting the sparse probability density function (versus diameter) into a cumulative distribution function. This CDF is then being fit, and parameters like the maximum diameter (dmax) and diameter that equally divides the area under the PDF (d50) are reported. We are looking for robust parameters. This will tell us how much the sparseness of our sample alone is limiting the stabilty of our fits.
    • The next step is to repeat this process ~100 times. XING