FPALM – Principle of Operation
Above images A-F:
An area containing photactivatable molecules (here, PA-GFP) is illuminated simultaneously with two frequencies of light, one for readout (here, an Ar+ ion laser, its spatial illumination profile shown in A), and a second one for activation (here, a 405-nm diode laser, its profile superimposed in B). Within the region illuminated by the activation beam, inactive PA-GFPs (small dark blue circles) are activated (C) (small green circles) and then localized (D). After some time, the active PA-GFP’s (E) photobleach (red Xs) and (F) become irreversibly dark (black circles). Additional molecules are then activated, localized and bleached until a sufficient number of molecules have been analyzed to construct an image.
Above schematic G:
The experimental geometry shows the 405-nm activation laser (X405), which is reflected by a dichroic mirror (DM1) to make it collinear with the Ar+ readout laser. A lens (L1) in the back port of an inverted fluorescence microscope is used to focus the lasers, which are reflected upward by a second dichroic mirror (DM2), onto the back aperture of the objective lens (OBJ). The sample, supported by a coverslip (CS), emits fluorescence which is collected by the objective, transmitted through DM2, filtered (F), and focused by the tube lens (TL) to form an image on a camera (charge-couple-device, CCD)
A: Fibroblast (tagged with PA-GFP) imaged by widefield fluorescence microscopy
B: Fibroblast imaged by FPALM showing greater resolution
C,D: Green box zoom of widefield fluorescence microscopy (C) and FPALM (D)
F,G: Further magnification shows dramatic increase in resolution power of FPALM (G) over widefield fluorescence microscopy (F)