- 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.
- 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).
- 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.
- Aspirate formaldehyde and wash twice with PBS. Aspirate PBS.
- For measurements, fill with HBSS at 37C as is typical with live cell measurements.
Raman Spectra of 1um beads taken at 20s exposure times. Spectra were taken at different axial positions of trapped bead(s?) and a group of beads that were adhered to the cover slip. These positions ranged from about 5 (microns?) above and below the cover slip (full range is shown for individual beads while select points are shown for the ensemble). The “glass edge” on the right is dominant in all of the adhered ensemble spectra while it disappears as the focus moved farther into the sample for the trapped bead.
Some sources of uncertainty are: 1)finding a place where a bead could be trapped and wouldn’t get knocked out by other beads coming into the trap or leave on its own (I had to start 5 microns above the coverslip to get a stable trap). 2)I used the back reflection of the glass on the microscope’s ccd as a reference point. When I was finishing measurements, I noticed the position of the back reflection was not where I originally set my “0” point (This is not the first time this has been noticed, but I wanted to make a note of it here) 3)I’m pretty sure that 1 increment on the fine tuning knob (which is what I was using to move the objective) corresponds to 1 micron, but I haven’t been able to 100% verify this. 4)Looking at published spectra, the pixel to wavenumber calibration may be slightly off (peaks are shifted to the right a bit). 5)This spectra was taken after looking for and reducing sources of power loss in the Raman path. Part of the process involved adjusting a filter that cleans up the laser light and that may explain the floor around 200 cm^-1
Raman spectra of a plastic block taken at different exposure times ranging from 1 second to 2 minutes (range in images shown is 1 to 30 seconds). Increasing exposure time reduced noise but also increased background. Spectra was collected with the oil objective and the block in direct contact with the objective, so changing depth was not an option. I also looked at how the spectra looked using the full CCD (fvb) and a subset corresponding to an individual fiber from the bundle (mt).
- We plan to use 10-20 pig (ideally human) corneas and take Raman Spectra. Then Professor Buckle’s lab can perform mechanical tests (modulus identification) on the same cornea samples to compare with Raman signal. We hope to see some correlation between the mechanical properties and Ramen signal, which may show up in collagen concentrations or in hydration.
- We also can take the spectra of several corneas at different levels of swelling, which may provide an alternative way to compare Raman signal to mechanical properties.
- Crosslinking of corneal collagen is of some interest as well. We will need to investigate if the Raman spectrometer is sensitive to changes in the of corneal collagen.
- In the end Professor Buckley is interested in finding a way to diagnose Keratoconus in human cornea before the onset of corneal thinning. Our goal is to develop a way of analyzing the mechanical properties of the cornea via Raman spectroscopy.
- Future: In vivo Raman imaging of cornea is also of some interest, however we are not sure if this can be/has been done before. Air pump test – correlation between pressures and mechanical properties?
Here is the link to the Judy Mourant paper that we’ll be discussing at this week’s group meeting. Should be another good discussion, especially for us on the angular scattering project!
These are Raman spectra of ethanol taken using the IRAM system. Images were taken at multiples of 5 microns into the sample, starting from the coverslip/ethanol interface to 25 microns into the sample. As the focus goes deeper in to the sample, the effects of the coverslip (ex broad peak at the high end) become less prominent.
Exposure time was 10 seconds. Spectra was taken from a section of the image that was 10 pixels tall, corresponding to light going into a single fiber of the fiber bundle.
Following is the paper we are going to talk about on Friday.
And if you have some notes or comments, you could comment in our blog, under this post!
Amide III (protein): 1225-1275 cm-1
Amide I (protein): 1640-1675 cm-1
Phenylalanine (amino acid): 1003 cm-1
Tryptophan (amino acid): 760 cm-1 and 881 cm-1
Tyrosine (amino acid): 646 cm-1
CH2-CH3 bending band: 1275-1500 cm-1
Most peaks are accurately represented in our data, however some peaks seem to be shifted. Such as our Phenylalanine peak at 1010 cm-1 when it should be at 1003 cm-1. We believe this is an issue with the calibration from pixel to wavelength.
1. How does the quality of our data set compared to what we expect to see out of Ramen signal from Cornea?
a. Set up two different data sets: one with raw data, and another one with smooth data (in matrix).
b. Set up a mean spectrum and calculate the trend of different peaks relative to the mean spectrum.
2. What biological information can we extract from the data set?
a. Hydration? Collagens? Mechanical Property? Chemical Property? Ratio of multiple peak strengths?
3. How do certain peaks change as we image different spatial positions or depth positions of sample?
a. Using the depth scanning / spatial actuator to quantify the peak strength.
4. Find publications regarding the area of interest in mechanical property of cornea.
For today’s group meeting, only the IRAM members (Xing, Robert, and Ashley) were in attendance. We primarily discussed the big picture scope of our project and the upcoming Photonics West presentations. Continue reading