Question: How does the lasers work to help you see the paths the drugs take?

Natalie Garrett answered on 12 Nov 2015:

OK prepare for a very long explanation!

Imagine you have a slice of a brain on a thin glass slide, and you’re shining laser light into it from underneath using a focussing lens. If you could see the shape of the laser beam as it is focussed inside the brain, you’d see that it has a kind of hour-glass shape, that the focal “spot” isn’t really a spot at all, but kind of spread over a small region.

If you put a detector (like a camera or a photomultiplier tube) above the sample, and steer the laser beam over the sample whilst collecting the scattered laser light, you can generate a picture of the sample itself. The imaging system I use raster scans the laser focus across the sample. This is like white light microscopy, which most people have a chance to try out at school at some point. The downside to this kind of imaging is that the sample is usually pretty blurry if it’s thick (because the focus is an hour glass shape, your picture is from a relatively thick piece of the sample). So this works best for thin things, like single cells in a dish. If you want to make a 3D image, this method won’t work because your resolution in the z-direction is just not good enough.

This picture of three different samples (which have used fluorescent dyes to make pretty colours) illustrates this well: the thicker the sample, the blurrier the picture becomes.

So how do you get around that? One method which has been used a lot is called confocal microscopy – this method uses a clever setup with pinholes that effectively block out light that doesn’t originate from the thin region you want to look at.

So with confocal microscopy, I no longer have to worry about blurred images, and now I can take one x-y picture (x and y refer to the axes of the sample, so it’s like a flat plane), move my lens up a bit, take another x-y picture, and so on, and then stitch these pictures together afterwards to make a three dimensional picture. Woot!

However, I don’t wanna use fluorescent labels. They change the chemistry of the sample. I am interested in looking at small molecules, and the fluorescent dyes are sometimes larger than the things I want to pin-point – clearly I’ll end up distorting the behaviour I want to look at if I use fluorescent dyes.

So, wat do? Well, I use a different technique that is based on a phenomenon known as Raman scattering. Simply put, with Raman scattering when laser light is shone at a sample a small proportion that bounces back off it will have a slightly different colour than it started out with. This is because the light either gains or loses energy to the sample in the process of scattering off it. The energy change is related to the particular chemical bond that was involved in the interaction: carbon-carbon bonds will result in a different energy shift than carbon-hydrogen, for instance. I like to compare this with a very oversimplified example: imagine throwing a ball at a weak friend who throws it back to you. The ball will likely be returned to you with less energy than you threw it with. However, if you do the same thing with a strong friend, the ball in this case will be returned with more energy than you threw it with. The energy difference is related to the “bond” (i.e. arm) strength, and you could be blindfolded but still be able to work out who threw the ball back to you based on how much energy the ball had when you caught it.

This process (with the light, not with the balls) is called spontaneous Raman scattering, named after the amazing guy who discovered it and who received a Nobel prize for his efforts: .

Raman scattering is awesome: you can plot the energy shift against light intensity and generate chemical fingerprints of your sample, which can be used to work out the composition of your sample. This technique is used to identify whether paintings are modern forgeries, as well as to determine which parts of a biopsy are diseased and which are healthy. There’s tons of applications. Unfortunately, it’s a very very weak process (something like only one in every ten million interactions between light and matter result in a Raman scattering event).

So wat do? You could just increase the laser power to get more signal, right? Sure. However, for biological samples, this is not good. You will literally fry your sample. You could instead sit there for a long time to take a reading, but this is no good for things that are alive and moving about – you will get a blurry picture. So how can you get around this?

Well, it turns out if you take not one but TWO laser beams and adjust their colours such that the energy difference between them matches the particular Raman bond energy you’re interested in (in most biological samples, this tends to be the carbon-hydrogen stretch, since CH is abundant in fats, and fats are all over the shop in bio stuff) you can produce a third colour of light. This is called an anti-Stokes shifted signal.

This third colour is bluer than the input light, so you can just stick some optical filters in front of your detector to block the input light, and therefore only see this signal. This technique gives much stronger signals because it’s a coherent process, using ultra fast pulses of laser light (most of the time the laser isn’t actually on, but in the teeny instances of time that it IS on, the laser beam is super duper intense) to generate tons of signal without destroying the sample.

This technique is called coherent anti-Stokes Raman scattering (or CARS for short). It’s a coherent process because all the bonds of interest in the sample are made to vibrate together (since we’re basically forcing them to wobble at their preferred frequency) and hence all the blue light that they produce in this interaction is also coherent. This means it all adds up, you don’t get destructive interference.

The bonus to this method is that for CARS since it’s a multiphoton process, the focus not an hourglass shape, it’s actually a teeny tiny spot. This picture gives you a comparison between a multiphoton focus and a normal single-photon focus with the traditional hourglass shape: So I can take label-free chemically specific x-y pictures, then move the lens up a bit, and take another, and so on, building up a three-dimensional picture without needing to add fluorescent dyes, and without needing to use a confocal setup. I can use this method to specifically look at drug chemical signatures against the background of the biological tissue. This means I can track and trace where the drugs are going!

This is a picture I took of a liver using CARS and another multiphoton process called two photon fluorescence: from this paper: http://onlinelibrary.wiley.com/doi/10.1002/jbio.201200006/epdf