PDMS

The final goal of our diffraction grating lab was to make something we could actually use as a grating. Our silicon wafer isn't transparent though, so we can't shine light through it! We need to make something transparent, with the same pattern as wheat was on the wafter. Here is out finished wafer. You can see the printed pattern in it:

PDMS stands for Poly-dimethyl-siloxane, which is a polymer made out of silicon, oxygen, carbon, and hydrogen. If you have ever seen silicone (what's in breast implants, cookware, some bike seats, and a myriad of different uses) this is a very similar substance, and sets as a clear gel. So we're basically putting sili-CONE on sili-CON. When I hammer on how important using the right words are, this is what I mean. One is a soft squishy polymer, the other is a shiny brittle crystaline substance. Here we are coating the silicon wafer in the PDMS:

Once the PDMS sets, it should mold to the same pattern as is on the silicon, except in reverse, just like any other mold:

The final part is de-bubbling and baking the PDMS so it sets. Inside this oven is a vacuum, so any air bubbles inside the PDMS suddenly have a much higher pressure than what is in the oven. They will expand and bubble out to the surface. Remember gas laws! The bubbles are not sucked out,t hey push themselves because there is no longer any equal pressure holding them down! (imagine pushing against someone, and then they move out of the way!) Here is the PDMS bubbling out all the air in the vacuum oven:

They need to stay in there for close to two hours, so we left them in the capable hands of engineer who was walking us through the process.

Next: Playing with lasers using our new diffraction gratings!

project video

Here's a video explaining the setup and purpose of our project. Now you can really see what we are working on!

Cell cultures: take two!

We started a new cell culture a few days ago, this time using culture bottles. These bottles have a air-permeable membrane on them so that the culture can "breathe" and have enough of the gases it needs to divide and grow. It is on its side because we want a very large amount of culture medium, but we also want only a thin depth so the cells can still get enough air from the surface:

Thus far they seem to be doing well, and there is no sight of mold. When we checked on it today, it had not grown enough that we needed to split the culture into new growing media. We were able to check and see whether we needed to split the culture by counting the cells and doing some math to find the number of cells per milliliter. To do this, we used a hemocytometer. This is a little mirrored glass slide that has a grid on it, so we can count the number of cells in an area. Here is an example of cells in a hemocytometer when looked at though a microscope:

The volume of that area is known, so we can just scale up to find the cells per milliliter, and compare this to a minimum that tell us it's time to split the culture. This is just like calculating a concentration using chemicals, it's all unit conversions! For our cell counting, we use a blue dye to help us see the good cells, since it only stains dead cells. It also makes the solution a little darker, so we can see our cells a bit easier as they stand out more. This is our hemocytometer on the microscope in the culture lab:

Our cell count was unfortunately too low for the culture to be ready to split, but hopefully tomorrow we can check again and continue to multiply our cells!

cell culturing

One of the idea things we would like to do with our machine is run real cells through it to test it out and see what kind of signals we get from the detector. In order to do this, we need to grow the cells ourselves, because it is much more practical than ordering a whole bunch of them and trying to keep them alive.

Growing cells, or "culturing" as it is usually called, is a very simple but specific process. You need to give the cells a place to be, and medium to grow in. Most importantly, you need to make sure that only your cells are growing, otherwise your culture won't be what you wanted it to, and this can really mess up an experiment. One of the PhD students in Dr. Bigio's lab showed us how to grow these cells. First, we needed to get a starter culture to begin with. These are kept frozen in a giant freezer at -80 degrees. We have to warm up these cells, so that they come out of their suspended animation and can grow and multiply in the medium. We can just do that in a water bath.

It is very important that we don't introduce any other types of cells into the medium, so everything has to be extremely clean, we carried all of our stuff to the cell culture room in its original packaging, sow e knew it was sterile. Here is the bucket with our pipettes (used to suck up liquid like the growth media) and our cell plates, all still in their sterilized containers.

We then just mix the cells with the media so there is a bout 10mL total, and let them grow! When they grow too much, they will sue up all the nutrients in the media, and need to be "split". This means that a portion of the culture is put into a new plate with more media that it can grow into. This is just like dilution of a chemical solution: we know that each plate is 10mL, if we have a full plate of cells and want to make 1:10 dilutions, how many plates would we need, and how much of the cell-solution and media would we need to use in each? Think about it for a little while, and you could be culturing your own cells!

Update

So, remember how important it was to keep everything vclean and to not contaminate your cultures with any other type of cell? When we checked on our cultures to see if we needed to split them so they could grow more, we found this:

That, is mold. White fluffy mold growing in what is supposed to be filled with invisible little cells. When this happens, we need to dump it out and start all over with a fresh batch of uncontaminated cells. The plates need to be bleached before we can dump them though, and the reaction between the indicator and the pH change caused by the bleach can lead to some pretty interesting colors:

Clean room training

On Wednesday, we were trained in how to dress and work in a clean room. Clean rooms are areas with very little dust or debris in them, and are often used for hi-tech fabrication of small or delicate components that would be ruined by any contamination. Many parts of you computer are made in a clean room. Some chemical analysis labs are also in clean rooms, so that external contamination doesn’t ruin your results. An example of this might be testing for arsenic in water, when arsenic is commonly found in soils. If you just walked into a room doing this kind of testing, microscopic dirt on your shoes could skew your results! You would have to at least wear some kind of bootie over your own shoes to prevent this.

Our covering for the first clean room required a “bunny suit” (a giant coverall, like little kid pjs), slip-on booties to cover our feet, goggles, a hair net, and nitrile gloves. However, this was a class 1000 clean room. There was a class 100 as well, which was even “cleaner”, meaning the controls on dust and other particles were even stricter. To our 1000-level outfit, we added higher booties that snapped shut at the knee, a hood, and a mask if we were worried about contaminating things with the saliva and other particles we exhaled. Here is a picture of me completely gowned in my full bunny suit and 100-level gear:

The clean rooms we were in are primarily used to create diffraction gratings and other optical gratings. These gratings change laser light into new and interesting patterns, and can even make holograms. To make a grating, we must first make a “mask”, or a basic template for the grating. This is basically like a mold for the grating, and s

erves as our template. A laser is programmed by a computer to expose a chemical on a piece of chromium and photo-resist coated glass in a specific pattern. This chemicals can be washed off when it is exposed, leaving behind a pattern based on where the laser was. Here is another mask made by someone in the lab, you can see the chromium has been etched away, leaving clear glass in some places.

A lot of different chemicals are used in this process, such as sulfuric acid. Using these chemicals can be very dangerous, so sometimes more protection than the normal gloves and goggles is needed...

Our pattern was a series of equally spaced dots, about 50 micrometers across. (Can anyone convert this to meters? How about nanometers?) Next week we will take our mask and make the next part of our grating by using light to etch a pattern based on our mask.

End of the 1st week

(This post was originally made on 7/5/2010, but was reposted to a new blog for technical reasons)

It feels like this first week of BU's RET program has gone by so fast, we've gotten so little done! Here I will try and summarize the first week of working in the lab.

I am working with Ms. Sewell from Swampscott high school in the Biomedical Optics Laboratory. We were introduced to the lab space by the lab's head faculty member, Doctor Bigio, a former researcher at Los Alamos laboratories. Both Ms. Sewell and I have done research before, but in labs much more oriented towards chemistry, so seeing what is is like in an optics lab was quite a new experience! The benches were set up very differently, and most of the familiar glassware wasn't present. Instead, there are lasers and mirrors, designed to allow the researchers to use directed light in their experiments. Optics is a branch of physics and engineering concerned with the use of light. Many sensors that you are commonly familiar with, such as the scanner in a grocery store, use lasers and optics. Biophotonics uses these principles combined with biology to make new and interesting devices, such as a probe that can tell if the tissue it is touching is cancerous based entirely on how it reflects light.

Our project involves using the properties of light to determine mechanical properties of a single cell. Since the cells have a different refractive index than water (remember physics anyone?) any light shone through them will be bent. We can then detect that light and tell something about how dense the cell is.

This project takes it one step further. By hitting the cells with a burst of ultrasound, we can cause them to wiggle! This wiggle is seen by the light level at the place where we are detecting rising and falling. Theoretically, we can use this wiggle to determine how rigid the cells are.

Why is this important?

Healthy cells have a cytoskeleton, a framework holding them in shape. This makes them somewhat rigid, but with enough flexibility to function wherever they need to be in a body. It also helps the cell move. Cancer cells show a drastically different structure in the cytoskeleton, and we should be able to detect the resulting change in rigidity. Also, cells that are dying begin to lose their structure. They would get softer and oscillate (wiggle) differently.

Right now, the machine we are using doesn't quite work, but our job is to "optimize" it. This means get it to work, and to hopefully work very well. The first week was mostly about seeing the lab and getting to know where everything is. this next week we should really get down to finding out what will make this project take off!