So, what does an expensive microscope look like?

A few months ago, I took a picture of the microscope I work on to show my family.  Here it is:

Some closer shots:

This scope allows me to look at my slides and take pictures of them.  It’s not a top of the line fluorescence microscope, and it’s a few years old besides, but it’s nice scope with good optics and it gets the job done.

Recently read books

I mentioned in a previous post that I was (some might say) obsessively reading fantasy books. Fantasy is the genre I indulge in when I need to relax or escape from whatever is going on in my life at the moment.

Right now, I’m reading books by Raymond E. Feist. I read the first few of his Midkemia books quite a number of years ago (10 or 11, I think) and haven’t read anything since then. I have just finished his two latest (in paperback), Flight of the Nighthawks and Into a Dark Realm, which are set in the same world as those first few books I read years ago. In fact, I seem to have jumped over a good 6 or 8 books in the series to get to these. Normally, I don’t like to do that (I prefer to read a series in the order in which it was written), but I was at the university bookstore and their fantasy selection is rather small so these were the only ones of the series available. Fortunately, the author writes these books in sets of trilogies so that I’ve essentially started at the beginning of a new storyline, so I’m not horribly confused (which would not be relaxing at all). The last book in this most recent trilogy has been released in hardcover, but I’m not sure I want to spend the money on a hardcover right now.

In general, I tend to buy books rather than borrow them from the library. First of all, because I love books and love owning books and love the feel and smell of books so I much prefer to own a book than borrow a book. Second, because I often read books over and over again and it’s easier to do that if I own it. Third (and possibly most importantly), I really suck at returning books to the library. Therefore, I rack up huge fines and in the end it is not much cheaper for me to borrow a book from the library than to buy the book (if it’s a paperback). When I was a kid, I ended up with so many fines the library wouldn’t allow me to check out books anymore (I had no way to pay the fines). So, I would read books in the library. This required me to hide whatever book I was currently reading so that nobody could check it out until I was done. I’m not sure if the librarians ever caught on. The books were always where I had stashed them, so if the librarians did know, they were kind enough to allow me to keep the book on hold, as it were.

At any rate, I’m now going back and reading the books in between the first books I read in the series and the books I just read starting with the trilogy just before this one. Of course, I know which characters make it to the next trilogy so the life-and-death passages are not so gripping, but I’m still enjoying them.

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.

Blog blah

Lately, I’ve been finding it difficult to write in my blogs.  I’m also way behind in reading some of the blogs I normally keep up with.  Some of this is just science overload, I think.  I was reading a popular science book, reading science blogs, writing a science blog and working in a lab all at the same time and I think I experienced some kind of upper limit and now, in backlash, I’ve read three fantasy genre books in the last five days.

I really wanted to keep this a strictly science blog and talk about my scientific interests but, ironically, I think that might be easier to do on a regular basis when I’m not actually doing research science.  For one thing, labwork takes up the bulk of my time.  Because of this, I have little time for leisure activities and less patience for studying and writing about science in what leisure time I do have (which seems to be about 15 minutes a day, but I suspect it may be slightly more than that).

So, my choices seem to be:

1.  Shut down this blog
2.  Keep this blog only about science and write in it very infrequently
3.  Write about things other than science when the mood strikes and write about science when I feel like it.

Of all of these, number 3 seems like the best option.   So, that’s what I’m going to do.

Banner Images

You may have noticed that the banner images have been changing every so often (or you may not have because you’ve never been to this blog before; I am currently on my 3rd (4th?) banner).  This isn’t because I’ve been unhappy with the banners that I have, but because I have these images that I take on the microscope, many of which will never be published, so I figure I may as well do something with them.*  The details of what is in the images can be found by clicking “Header Image Details” under pages on the left.

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*I’ve also considered printing a few and hanging them up in my living room, but I think that may be a little too geeky….

MIA

I’ve been having a hard time finding time to blog recently. Mostly, I get up in the morning, go through my morning hygiene regimen, eat breakfast, go to lab, come home, eat something small, take a shower and go to bed. That doesn’t leave much time for blogging. I was thinking about this the other day (albeit in the context of why other knitters are able to complete a sweater in a reasonable amount of time) wondering how anyone ever managed to even do laundry when I realized that most normal people have several hours between the time they get home and the time they go to bed. I vaguely remember when that was true for me, too, but I think it’s going to be awhile before I get back to that sort of sane schedule.

One disadvantage to being a scientist in the molecular biosciences is that it is virtually impossible to stop in the middle of an experiment due to the fragile nature of our samples. In some experiments, there are certain defined stopping points, but you really cannot stop until you get to one. It’s a bit like making cookies. You can freeze cookie batter, but it’s better if you finish adding all of the ingredients and mixing and so on before you freeze it. This is very comparable to my labwork because I am often freezing things (or putting them into the fridge) until the next day.

Anyhoo, the immunofluorescence experiments I’ve been doing have required that I grow my cells in special conditions for 8 hours, then start four hours of work. I can’t grow the cells for longer than 8 hours or I might have them grow overnight which would make things easier. No, I must grow them for 8 hours, then incubate them in fixative for 2 hours, then spend another hour preparing them for the antibody incubation and then I can stop until the next day. And, I always I think to myself, I’m going to go home for an hour or two in the afternoon before I start the fixation, or for dinner during the fixation, but I get busy doing other stuff and I never manage to take a break.

Anyway, the bottom line is, posting may be scarce until I get these particular experiments behind me.

Lab update 3-1-08

Mostly, I have been working on immunofluorescence experiments and taking pictures of same. I’ve actually gotten some pretty good images of my experiments and that makes me very happy. I now have good images for:

Figure 1A: Panels 2 and 3
Figure 2B: Panels 1, 2 and 3

There’s still a lot left for me to do, though. Unfortunately, I’m still having problems with the deconvolution software. Fortunately (?) I’m not the only one having problems. While this is definitely inconvenient, at least it means that it’s not just something I’m doing wrong or that my images are too sucky to deconvolve (actually, they look much better before I try to deconvolve them). Importantly, the other person who is having problems with the software said that it was working fine several weeks ago, but is now causing problems. Hopefully, it can be fixed because it’s pretty expensive software.

Anyhow, for now, I’m not doing the deconvolution and the images look fine so I’m going to set up the figures with the images that I have.