Posts

Aligning the IRAM system

admin

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.

 

Compare normal fitting with direct fitting

admin

These days work are mainly about comparing the performance of normal fitting and direct fitting.

A review for whole process. We have a parent Gauss or Normal distribution(parameter μ and σ). In math that should be continuous. Then we sample the distribution got a set of discrete data. Then we use the discrete population to map to a set of scattergram that generate by Mie scatter thoery in different particle diameter. After that we use cross-section to  weight the data. Now we get the raw simulation data we use in normal fitting.

In normal fitting we use log-normal population model + cross-section weighted model+Mie theory to fit to the scattergram. We want to report best estimate of I vs d.

For direct fitting, the raw simulation data generate is similar to the former one. We have a parent Gauss or Normal distribution(parameter μ and σ), then we sample the distribution get a set of discrete data. We weighted these data though cross-weighting model. Now we get the raw simulation data.

In direct fitting, we use a log-normal or gaussian population model to fit raw data directly(without Mie thoery, that is why we call it direct fit).

The purpose of normal fitting is we want to analyze cell’s angular scattergram we taking from our experiment devices. We would like to report best estimate of I vs d.

The purpose of comparing these two fitting process is we want to know several things: how well our normal fit compared to the direct fit; when the samples are very sparse, the Mie thoery model’s contribution could be neglected, and so on.

Followings are several simulation results for comparing direct fitting and normal fitting. Read More “Compare normal fitting with direct fitting”

Cell culturing protocol

admin

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 (SCC7)

  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

Passaging (HeLa)

  1. Aspirate (remove) media.
  2. Add 10 mL of HBSS or PBS.
  3. Aspirate.
  4. Add 2-4 mL trypsin.
  5. Incubate for 3 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

admin

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

Cell Media (HeLa)

  • 500 mL RPMI 1640
  • 10% FBS (50 mL)
  • 5 mL Hepes Buffer
  • 5 mL Pen Strep

*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

SUPERFASLIGAND (for 100 ng/10 uL concentration)

  • 5 ug of SFL
  • 500 uL of HeLa Media

9/9/15 IRAM Meeting

admin

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