A different kind of microscope

I apologize for the light posting around here.  Unfortunately, it’s likely to continue until I finish these experiments.  I might find a little more time for blogging while I’m writing my thesis–who knows?

Lately, I’ve been spending my time trying to learn how to use a confocal microscope and bend it to my will.  The score so far is:  Confocal-5, Elisabeth-1.  But, I think I’m getting the hang of it.

A confocal looks much the same as a regular fluorescence microscope.  However, there are a few key differences in the way that the fluorescent image is acquired.   First, instead of using a mercury bulb to shine light on your sample to cause the molecules to fluoresce, a laser beam is used.  Second, the microscope is set up in such a way as to filter out much of the fluorescence not in the current plane of focus.  Third, instead of being able to look directly at the image using the eyepiece, the image is scanned with a special electronic scanning apparatus that is controlled via a computer.  The image is then displayed on the computer screen.

There are many advantages to this system, however, I am using it for a very simple reason.  I need to be able to collect an image from a sample in which my fluorescent molecule gives off infra-red light.  My regular microscope can’t do that whereas the confocal microscope can.  On the one hand, it’s good for me to learn how to use this important instrument.  On the other hand, I feel a little silly for using it for my stated purpose.  It feels a bit like using a supercomputer to write an article because it’s the only one that has the word processing program you like.  The confocal microscope has tremendous power but I’m only using it for it’s most mundane purpose.  In a way that’s good, I suppose, because I’m learning how to walk before learning how to run, but I still feel like I should be doing something a bit more profound on the scope than collecting an infrared image.

Trying to NOT falsify data

The vast majority of the data for my paper consists of photographic images. I have five figures and all but one of them are going to be mostly photographs. These images are of my cells and the images are supposed to answer the following question: where is Protein 1 when I do something severe to Protein 2?

Now, these data are exclusively qualitative. So, you would think that there wouldn’t be much of an issue with image processing. As long as I show images that look like what the cells actually do look like through the scope, then I’m fine, right?

Well, there exists in this world a program called Photoshop. With it, you can do any number of things to photographic images. For instance, I can take an image that looks like this:

And turn it into this:

And then, I can crop the image so that it looks like this:

But, should I? And why would I want to?

First of all, is the first image representative of what I see through the microscope? No. There is way too much green background (green that is not in spots) in the cells. That is most definitely not what they look like when I look at them through the scope. So, I can attempt to subtract out the green background. However, I can’t get rid of all of it because when I look through the scope there is a bit of green background fluorescence (cells naturally have some green background fluorescence). Besides which, when I try to get rid of all of it, I end up losing the greens spots which are my data.

Even more concerning, what if that green background is not actually background, but diffuse staining of my protein throughout the cell? That would mean that not all of my protein is found in spots. So, by getting rid of the background, I could be getting rid of data. If my purpose in showing this figure is to say that all of my protein is in these particular green spots and I manipulate the image to show that you only see green in spots, then I am misrepresenting my data.

Finally, by cropping the image, am I selecting a subset of images that look like what I want them to look like rather than what the majority of cells actually look like? I have no choice about cropping the image–I can’t possibly include the whole thing in the paper. The first image I showed you isn’t even the entirety of the original image. The original image is 18 x 11 inches in size, whereas my final image for the paper is probably going to be 1.5 inches in size. In order to be able to include more cells, I can resize the image to be 11 inches wide and I have done that with the actual image I’m going to use for my paper. Then, I can crop the image so that it’s only 1.5 inches wide–but only if the cells I include are representative of the majority of the cells I see when I look through the scope.

The Journal of Cell Biology has this to say about image manipulation:

No specific feature within an image may be enhanced, obscured, moved, removed, or introduced. The grouping of images from different parts of the same gel, or from different gels, fields, or exposures must be made explicit by the arrangement of the figure (i.e., using dividing lines) and in the text of the figure legend. If dividing lines are not included, they will be added by our production department, and this may result in production delays. Adjustments of brightness, contrast, or color balance are acceptable if they are applied to the whole image and as long as they do not obscure, eliminate, or misrepresent any information present in the original, including backgrounds. Without any background information, it is not possible to see exactly how much of the original gel is actually shown. Non-linear adjustments (e.g., changes to gamma settings) must be disclosed in the figure legend. All digital images in manuscripts accepted for publication will be scrutinized by our production department for any indication of improper manipulation. Questions raised by the production department will be referred to the Editors, who will request the original data from the authors for comparison to the prepared figures. If the original data cannot be produced, the acceptance of the manuscript may be revoked. Cases of deliberate misrepresentation of data will result in revocation of acceptance, and will be reported to the corresponding author’s home institution or funding agency. [emphasis added]

It’s somewhat tricky ground. I have to try to get rid of some of the green background in order to make the image true to what I see when I look through the scope, but if I get rid of too much of it then I’m massaging the data. Add to that the fact that I took these images a couple of weeks ago and am only now getting around to processing them which means that my memory of what things truly looked like when I looked in the scope is a little bit fuzzy. Do I really remember it having less green background or is that what I remember because ideally there would be less green background? Because of this, I’m going to repeat the experiment and process the images immediately after I collect them.

As you can see, it is relatively easy to manipulate your data to the point of falsification, even if you don’t mean to. The vast majority of scientists truly intend to present their data in a way that is truthful, but it is difficult to completely eliminate personal bias. I would love for all of my images to look like that last one, but that’s not reality and to present that image in a paper would be misleading. In some ways, I wish the image collection and processing were being done by someone other than me–someone who is not emotionally attached to the project in the way that I am. On the other hand, then I would have to trust that person to not manipulate the data in a way that is not truthful.

Immunofluorescence Step 2, Part 2

Previously, on Attack of the Clones….

Mr. Protein O’Interest was standing next to the bathroom in Millenium Park (or the boathouse in Central Park, etc) wearing a distinctive hat. An A. Body clone had recognized the hat and grabbed onto it. A group of ten Clone Grabber (CG) clones had recognized various bits of apparel of the A. Body clone and grabbed onto them. And, you are high overhead in a helicopter and it is night-time and all is dark. You’ve made your target slightly bigger, but you still can’t see it.

Time to shed some light on the situation.

This procedure is called immunofluorescence for a reason. And that is because the secondary antibodies (the CG clones above) have a fluorescent molecule attached to them so that, after they are put onto my sample, I now have a large glowing dot in my cell. In the park analogy, this is somewhat equivalent to having each of the CG clones wear a miner’s cap with a lantern on it. Now, from your helicopter, you can see a glowing spot on the ground and that spot tells you where the bathroom is!

Actual fluorescence is a bit more complicated than that. In order for the molecule to fluoresce, you first need to shine light of a particular wavelength on the molecule which will then emit light of a different wavelength (for reasons of physics*). In my case, I am shining green light on the sample and the secondary antibody then glows red.

There are some differences between the park analogy and real-life immunofluorescence (you know, besides the facts that I’m looking at a cell, not a park, and proteins, not clones). First, I’m not looking just at one molecule of my protein. If I was, I probably wouldn’t be able to see it even with the signal amplification I get from using a secondary antibody and the fluorophore. There are many, many, many molecules of my favorite protein in the cell (ie Mr. O’Interest is a clone himself and all one hundred of them are surrounding bathroom). Additionally, my cells have been “fixed.” Before I ever even put antibody on my sample, I have incubated my sample in formaldehyde which kills the cell and freezes it in time. This is because in order to get the antibody into the cell to begin with I have to do some fairly harsh things to the cell that would kill it anyway. In the case of my yeast, I have to destroy the cell wall and then make the remaining plasma membrane permeable to my antibody solution (both of these things allow the antibody into the cell so that it can recognize my protein). These things make the cell very unhappy. The advantage to killing the cell and fixing it with formaldehyde is that it kills the cell very fast and preserves the intracellular environment as it was just before I killed it (it’s a bit like the volcano erupting in Pompeii and gas and lava killing everything so quickly and freezing the scene so well that they were actually able to find people in the midst of whatever they were doing when the eruption started) (I love analogies, in case you haven’t noticed). The disadvantage is that the fixation process causes some artifacts that need to be dealt with (ie the people of Pompeii were probably not uniformly gray nor did they spend their days huddled in a corner with their faces in their hands). Also, you can’t view a process when everything is frozen in time, you can only infer what was going on based on the picture you now have.

And, for years, scientists lived with these caveats and we were mostly happy to do so because it allowed us to get data that we wouldn’t be able to obtain otherwise. But, still, if you wanted to view intracellular dynamics, you were pretty much SOL.

But one day, people thought, “You know, if we can attach an epitope tag to our protein, couldn’t we attach something else to our protein? Something that fluoresces all by itself without the need for chemical treatment?” And that’s exactly what they did.

Next Technique Series: Direct fluorescence——————————-

*Physics is a subject that I am not particularly good at, but which my husband is very good at (he’s an astrophysicist). Therefore, I try to avoid explaining physics if at all possible, leaving it for him to handle.

Immunofluorescence Step 2, Part 1

So, when last we left our Mr. Protein O’Interest and the A. Body clones, the A. Body clones had each attached itself to the hat of a different Mr. O’Interest in a different park. As I mentioned before, the fact that the A. Body clones have attached themselves to the Messr. Interest hardly helps you to find the bathroom, boathouse, ranger station, or campground since it is well nigh impossible to see people from high up in the air.

This is the situation I am in with my sample of cells after step 1. I have put antibodies on my sample that will recognize the epitope tag on my protein and this in turn will tell me where the ER is in the cell. However, proteins are incredibly small. So small that you cannot see an individual protein even if you magnified it 1 million times (and I assure you, the microscope I’m using does not have that sort of magnification capabilities). The fact that I’m now looking at a complex containing two proteins doesn’t help me very much.

So, the next step in the procedure is to 1) give me something to amplify the signal–so that I’m not just looking at a complex of two proteins–and 2) to give me something that I can actually see. This is, again, done with antibodies. However, these antibodies are a little more indiscriminate. They will recognize anything that came from a mouse (remember, my first antibody–called the primary antibody–originally came from a mouse). And, these secondary antibodies will recognize a variety of parts of the first antibody. This means that you will have many molecules of the secondary antibody attached to one molecule of the primary antibody.

Back to the park analogy. Okay, you’re high up in a helicopter and below, an A. Body clone has found the hat-bearing Mr. O’Interest and has grabbed onto that hat. Now, you release a second set of clones into the park–the Clone Grabber clones. The CG clones are a mixed group of clones. They have been trained to recognize the A. Body clones that are in the park, but they have been trained to recognize different parts of the A. Body clones. Some of the CG clones recognize the A. Body shoes, some recognize the A. Body right pantleg, some recognize the Star Wars insignia on the A. Body t-shirt, some recognize the A. Body hair, etc., etc. Therefore, when the CG clones find the A. Body clones, each of them will grab onto the part that they recognize–the shoe-recognizing clones to the shoes, the pantleg-recognizing clones to the pantleg–such that in the end you have quite a number of CG clones attached to the A. Body clone (who is, in turn, holding onto Mr. O’Interest’s hat). So now, instead of having a group of two people to detect from your helicopter, you have a group of 10 people to see. But, if you’ve ever been in an airplane flying high over a city, you will know that is not enough. Especially if you are flying over it at night. When you are flying over it at night, in fact, mostly all you see are lights….

To be continued…..

Immunofluorescence

A huge portion of the experiments I’m doing involves a technique called immunofluorescence (IF). Therefore, I thought I’d spend some time (several posts, actually) explaining what that is.

IF uses antibodies in order to identify proteins inside the cell that you are studying causing them to fluoresce under particular wavelengths of light. This allows you to see where in the cell a particular protein is found (we would say, where the protein is localized within the cell). In my case, I am looking at proteins that are always found on/in a certain place in the cell (the Endoplasmic Reticulum (ER), actually) and therefore can be used to mark (or “label”) that particular region of the cell.

Antibodies are a type of protein found in the immune systems of higher organisms. An antibody recognizes a specific protein (or part of a protein) and binds tightly to it. In your immune system, this occurs so that your body can identify that protein as foreign (because it’s attached to a virus or bacteria) and therefore target it for destruction. Immunofluorescence takes advantage of the specificity of antibodies for a particular protein in order to identify a protein in the crowded environment of the cell.

How does this all work? Let’s say you are in a helicopter, high above the ground, and need to locate where a particular structure (let’s say the bathroom) is in Millenium Park in Chicago (aka the ER) on the evening of July 4 when everyone and their brother is in the park. You can’t really identify the bathroom from in the helicopter because it is the same gray as the concrete surrounding it. However, you know that Mr. Protein O’Interest (aka my protein) hangs around the bathroom so if you can find Mr. O’Interest, you have found the bathroom. Now Millenium Park is huge and there are millions of people there, milling about (this is true in a cell, too; the volume of the cell is huge with respect to the size of a single protein and there are millions of proteins inside the cell). How do you locate Mr. O’Interest? Well, if you train a large number of A. Body clones (the antibody, of course) to recognize the face of Mr. O’Interest (which is presumably different enough from every other face in the park and therefore will specifically identify him), you can send the clones out into the crowd and when one of them finds Mr. O’Interest, it latches onto him.

This process is the basis of immunofluorescence. I want to find the ER in the cell. To do that, I need to locate a protein that is always at the ER. To find that protein, I dump some antibodies on my sample. The antibodies are specific for my protein and when they find it, they latch onto it (so to speak).

So, how do you get antibodies for your favorite proteins? Well, you expose an animal (often a rabbit, but in my case, the antibody comes from a mouse) to the protein by injecting purified protein into them. The animal’s immune system then creates antibodies to that protein (like how you trained the A. Body clones to recognize Mr. O’Interest) and the antibodies are collected and purified.

Generally, a researcher will make purified protein and then send it off to a company that specializes in making antibodies, who will then send the antibody back to you. This can be a very expensive process, though, and it can go wrong in any number of ways leaving you out a whole lotta money with nothing to show for it. So, what’s a researcher to do?

Fortunately, there are companies who mass produce antibodies for commonly studied proteins. However, these antibodies are usually for mammalian versions of the proteins and almost never recognize yeast versions. So, researchers have come up with a way around this problem.

Remember, I said that antibodies can recognize a particular region of the protein. That region can be really quite small. So, somebody figured out that if you took the antibody recognition region of a specific protein (let’s call it myc) that already had an antibody made for it, then stuck it on the end of your favorite protein.* The antibody would stick to myc which was in turn attached to your protein, effectively giving you an antibody to your protein. Which means that you don’t need a different antibody for different proteins. All you need to do is attach myc to your protein and use the myc antibody. These protein regions are called epitope tags and there are a few commonly used tags. Because so many people use these tags, companies have mass produced antibodies to those tags, thus saving the researcher from having to have a custom antibody made. Very nearly all of the IF I do uses antibodies for epitope tags.

Let’s put this into the context of the Millenium Park example. Let’s assume that you do not just want to find the bathroom in Millenium Park, but also the boathouse in Central Park, the ranger station in Yellowstone Park and the camping grounds in Yosemite Park. Each of these locations has a different Mr. Protein O’Interest hanging around. Now, you can train separate, customized groups of A. Body clones, each recognizing the face of a different Mr. O’Interest (very expensive and time consuming), or you can have each Mr. O’Interest wear the exact same kind of hat (the epitope tag)**–a hat that nobody else in the park would wear–and purchase a single group of hat-recognizing A. Body clones from a company. The company has put the time and energy into training the A. Body clones into recognizing the hat. All you have to do is buy the clones and set them loose on the various parks to locate the hat-wearing Mr. O’Interest and therefore, your structures (bathroom, boathouse, ranger station, campground) of interest.

Now, you are probably wondering how it helps to have an A. Body clone attached to Mr. O’Interest because all we’ve really done is replaced recognizing Mr. O’Interest with recognizing the A. Body clone. For that matter, how is it easier to see Mr. O’Interest in the first place? Isn’t the bathroom bigger than Mr. O’Interest? Wouldn’t a larger object (even if it blends into the surroundings) be easier to see from a helicopter than a much smaller one? This brings us to step 2 of the immunofluorescence process.

To be continued……

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*This of course raises the question of how one sticks a bit of one protein onto another protein. The answer is that you put the DNA coding for the tag (bit of protein) into the DNA of the protein you want to attach the tag to. Which raises the question, how do you put DNA from one protein into DNA from another protein? Well, it’s complicated. I’ll have to have another series of posts just for that.

**How Mr. Interest gets the hat will be dealt with another time (see note on about tagging a protein).