Fluorescence PhotoActivation Localization Microscopy (FPALM)
A Better View of the Molecule
New method of microscopy using fluorescence photoactivation help address biological questions
Science has limits. Defined by theories, laws and formulaic equations, these limits define the boundaries within which scientific discoveries are made.
In most cases, having boundaries that are clearly defined by justifiable rules is a good thing. In science, however, the existing limits often interfere with the overall quest: the search for a cure, the advancement of technology, the depth of our understanding of ourselves and our universe. For scientists, the goal is often to break through those limits to reveal the discoveries on the other side. Breaking the diffraction barrier became our goal.
Our microscope system, dubbed FPALM (Fluorescence Photoactivation Localization Microscopy), combines existing technologies to build an image based on the florescence of individual molecules. The device’s magnification capabilities exceed those of the most powerful confocal light microscopes available.
A normal microscope looks at all of the molecules at once, which can make the individual molecules difficult to see, like drops in a stream of water. The separation between individual objects needs to be larger than the microscope’s resolution, otherwise the light coming back through the microscope is blurred and the objects are indistinct. This new technique allows us to find out where the molecules are and separate them as individual entities. The key is in the use of photoactive dyes.
FPALM uses lasers to excite dye molecules on the surface of the subject being observed. The laser causes a portion of the molecules to fluoresce, and the light given off creates an image that is captured digitally. The process is repeated as new sets of molecules are excited, and the individual images, each reminiscent of a starry sky at night, are layered with the help of a computer to create a composite image. The resolution of the new image is at least twenty times better than any traditional light microscope available today, easily creating images with as low as 10 to 20nm resolution.
Above image is a Fibroblast tagged with photoactivatable-GFP (PA-GFP) CamKII
A: Traditional widefield microscopy images all the molecules in a specimen simultaneously
Image turns into a blur beyond the resolution limit (200nm)
B: FPALM localizes individual molecules over a period of time
Which can be assembled to form an image of much higher resolution (10-20nm)