Alex Palazzo is an Assistant Professor in the Department of Biochemistry at The University of Toronto.
Flipping through Nature, I stumbled onto this commentary: The good, the bad and the ugly. Here's a taste:
To correctly capture images using a modern microscope, researchers must have a good grasp of optics, an awareness of the microscope's complexity and an obsession for detail. Such skills can take months or even years to master, and yet, owing to inexperience or the rush to publish, are all too often squeezed into hours or days. Popular methods such as fluorescence microscopy are particularly fraught with dangers.
The problem? Here's more:
It is now a routine part of many studies to investigate, using microscopy, where in the cell a fluorescently labelled protein is concentrated and where it goes. This type of microscopy has hooked cell biologists because it allows them to gaze inside living tissues and monitor molecules in their native environment. But although most biologists graduate with some training in chopping and splicing DNA, few will have laid their hands on a pricey fluorescent microscope. "Your average molecular biologist can make all these fantastic fluorescent tools," says Kurt Anderson of the Beatson Institute for Cancer Research in Glasgow, "but then imaging is just a little bit tacked on the end."
Yes, exactly!
Anyone who has hung out with me, knows that I have gone apoplectic when it comes to microscopy. Most of the microscopy data in papers I read is just poor AND IT DRIVES ME CRAZY. And it also drives my microscopist friends crazy too. I have resisted writing too much on this topic (here is a rare exception), but now that I've read this article I'll have to rant a bit ...
OK let's start:
1) As far as I'm concerned, most qualitative microscopy lies at the bottom of the hierarchy of data. Poorly controlled and poorly presented microscopy is far too common. And the data is often overinterpreted. It drives me nuts. 95% of the endoplasmic reticulum staining in papers look like amorphous blobs. How can you derive any useful information from that? And then people adjust the brightness/contrast levels so that the images are what we call overcontrasted. And then there are the journals ... why are your images so freakin' small????? Microscopy is only valuable if can derive information, if your images are uninterpretable and do not add anything to your argument, why bother?
From the article (that quotes Jennifer who is right downstairs):
... one expert contacted by Nature estimated that as many as half of all experiments that report two proteins in one spot have not been performed properly. Another estimated that 5-10% of images don't match what is reported in the text. "It's easy to pick up any journal -- even Nature -- and see poor microscopy data," says Jennifer Waters, who directs the Nikon Imaging Center at Harvard Medical School. "I don't know how often the results are blatantly wrong, but I do worry about the accuracy."
2) Colocalization. From the article:
One of the most common uses of fluorescence microscopy -- and therefore the source of many problems -- involves looking for two proteins labelled with different coloured tags in order to determine whether they sit in the same place in a tissue or cell. Each fluorescent protein is excited by a particular range of wavelengths and emits at different wavelengths that are collected through microscope filters. If a researcher uses GFP in combination with a tag that emits red light then, in places where the two proteins are close together, combining digital images of these two tags will create a yellowish signal.
I hate it. Just because you flash some "yellow" at me, it does not follow that the green and red proteins colocalize!!!!! Please perform controls. Make sure that each protein/immunostain does not fluoresce in multiple channels. Bleedthrough is a horrible artifact. And here is another request show the distribution of each protein separately and please show the distributions of each protein with black and white images. This brings me to #3 of things that bug me.
3) Color. From the article:
.. journals and reviewers are too often impressed by pretty images. North tells the story of one postdoc who slaved to capture images of her small cells only to have the paper turned down because the reviewer said the images were not good enough. "I've seen how long she spent getting the highest quality images she could possibly achieve," says North. "I think it's a big problem when the reviewers are more concerned with how aesthetically pleasing an image is than whether the scientific content is clear."
Yes colour pictures are pretty, but they are awful when it comes to presenting data. If you are presenting the distribution of a single protein there is no need for color. This practice is wrong. Your eyes can distinguish more shades of grey then shades of any color (especially dark blue on a black background). I hate color. I hate color. Data is more important than esthetics.
4) FRET. ARRGGHHHH! For those that know me, you'll laugh because I've been ranting about this forever. This passage made me happy:
Microscopists save most of their expletives for more sophisticated techniques such as FRET (fluorescence resonance energy transfer), one of several four-letter acronyms for methods that are both popular and treacherous.
The idea is that you activate fluorophore A it will donate its energy to a nearby fluorophore B. So if A and B are close you should be able to flash A and see B. Sounds nice right? The problem is that this FRET signal is usually very small. We're talking about fluorescent changes of 5-10% in the best case scenario. And to calculate the amount of FRET you need to know how much A and B you have at any one spot on the coverslip. Add on background and bleaching effects and you can see why many microscopists call FRET the "photoshop effect". The only FRET that I can truly believe is cuvet FRET where the entire spectrum of both fluorophores can be analyzed. That is the only way to ensure that the spectral properties of each fluoropore have been altered.
The answer to all these problems? From the article:
... many biological questions simply cannot be answered without a working knowledge of microscopy. "In competitive biology, you don't necessarily need to be a mechanic but you need to be able to operate the machine," Davidson says. "If you don't know how it works you'll get creamed in the race."
Translation: get educated, get trained, be careful, and don't overinterpret your data. And please follow some basic principles like performing controls.
You are now warned.
Ref:
Helen Pearson
The good, the bad and the ugly
Nature (07) 447:138-140 doi:10.1038/447138a
Comments
I am by no means a microscopy expert, and I dislike FRET for other reasons (the big one being the angular orientation between fluorophores is unknown, and everyone just uses an average value), but I would imagine the FRET signal to noise is much better in, say, a total-internal reflection setup?
Posted by: PhilipJ | May 17, 2007 7:21 PM
Right with you, Alex.
There's a couple of papers in my (corner of) field with some appalling fluorescence microscopy; even in J Cell Biol, and I'm rather frustrated because I can't march across to the road and say "Your experiments are shite".
I should talk to $BOSS I guess; after all, I got this particular position because I could do cell biology (it's a structural lab), but there's a pile of politics going on here.
Posted by: BK | May 17, 2007 8:52 PM
I did a fair amount of microscopy in my postdoc, and the issues you bring up here really hit home with me. I wasn't trained to do fluorescent/confocal microscopy at all (other than a 1-2 hour this-is-how-to-use-the-scope session), and was left to figure it out myself. I realized quickly that it's very easy to turn an artifact into something real if you don't know what you're doing and don't do the right controls. Unfortunately, I think many people doing it aren't taking the time to learn how to do it right.
It's sad but true that pretty pictures will take you far. You'd think scientists would be more sophisticated than a child who gets distracted by all the pretty colors. And if you've got a movie with live-action fluorescent microscopy? Jackpot!
I've got to say, I really don't miss sitting in a dark room for hours on end working on the scope.
Posted by: Betsy | May 17, 2007 9:13 PM
Posted by: sparc | May 17, 2007 10:53 PM
I agree. Having a specialist helps tremendously for researchers not coming from a lab where microscopy is taught. If institutions want to increase the level of science they should seriously consider it. Marc Kirschner pushed for our department to have a central imaging facility (the HMS Nikon Imaging Center), and it is of great help to those with no experience.
Posted by: apalazzo | May 18, 2007 7:03 PM
yeah the color kills me too... especially when someone shows something in blue (on a black background), just so they have a red a green and a blue panel. And all those poor colorblind folks!
Posted by: Dave Bridges | May 20, 2007 1:21 PM
I'm glad you enjoyed the article Alex! (Karl told me about your blog). I dream of a day when journals will require public access to raw microscopy images/data as a prerequisite to publication...
Posted by: Jennifer Waters | May 21, 2007 2:16 PM
Yes yes yes!!! All of what you said. (Although I am trying to do FRET, with the Rho family GTPase probes, I find that most people who use them are very ignorant about how this even works!)
Posted by: Joolya | May 24, 2007 9:15 PM
Does anyone want to talk about BRET?
http://www.pnas.org/cgi/content/short/0701987104v1
Posted by: BK | June 5, 2007 10:12 PM
I had heard about this when I was a grad student, but as the abstract states, the signal is so low that it was hard to detect. Perhaps camera and image acquisition technology has advance sufficiently to allow for this type of imaging, we'll have to see how many new publications use it.
There was also this split GFP technology (called Biomolecular Fluorescence Complementation) that was used in a couple of papers. The idea was that you stuck half of the GFP on molecule A and half on molecule B. Only if molecule A and B were in close proximity you could detect fluorescence. Kinda neat but very tricky.
Here's the link to a post on this:
http://scienceblogs.com/transcript/2006/04/bimolecular_fluorescence_compl.php
Posted by: apalazzo | June 6, 2007 8:55 PM