Precision-tracking of individual particles By Fluorescence Photo activation Localization Microscopy(FPALM) Presented by Aung K.

Transcription

1 Precision-tracking of individual particles By Fluorescence Photo activation Localization Microscopy(FPALM) Presented by Aung K. Soe

2 This FPALM research was done by Assistant Professor Sam Hess, physics Dept. of Physics and Astronomy, University of Maine, Orono, ME Tel.:

3 What is FPALM? A new microscope system, called 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, allowing us to find out where the molecules are and separate them as individual entities. The key is in the use of photoactive dyes. Generally, the separation between individual objects needs to be larger than the microscope's resolution, if not then, the image is blurred and the objects are indistinct. 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. 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 is at least twenty times better than any traditional light microscope available today, easily creating images with as low as 10 to 20nm resolution.

4 The experimental geometry The 405-nm activation laser(x405), which is reflected by a dichroic (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 (CCD).

5 An area containing PA-GFP is illuminated simultaneously with two frequencies of light: (A) An Ar+ ion laser for readout, in its spatial illumination profile. (B) A second one for activation, a 405-nm diode laser, its profile superimposed. (C) Within the region illuminated by the activation beam, inactive PA-GFPs (small dark blue circles) are activated. (D) Activated PA-GFPs (here, small green circles) and then localized. (E) After some time, the active PA-GFPs photobleach (red Xs). (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.

6 Why frequencies of lasers matter?

7 k A = the activation excitation rate Φ A = the activation quantum yield K 0 = the spontaneous activation rate K BC = the spontaneous and light-dependent inactivation rate K X = the fluorescence excitation rate = the photo bleaching quantum yield Φ B where ρ is a ratio of photo-activation to (reversible and irreversible) photobleaching. ρ <<1 Typically, to control (limit) the number of active molecules at a given time, the rate of photo bleaching and spontaneous inactivation should be equal to or larger than the rate of activation.

8 What is PA-GFP and its property? A photoactivatable version of green fluorescent protein (known as PA-GFP) enables photo conversion of the excitation peak from ultraviolet to blue by illumination with light in the 400-nanometer range. Unconverted PA-GFP has an excitation peak at approximately 395 to 400 nanometers. After photo conversion, the excitation peak at 488 nanometers increases approximately 100-fold. This event evokes very high contrast differences between the unconverted and converted pools of PA-GFP and is useful for tracking the dynamics of molecular subpopulations within a cell.

9 Fluorescence emission before and after photo activation of PA-GFP molecules immobilized on a glass coverslip under continuous illumination at 488nm. (A)blue curve a represents the fluorescence intensity which increased significantly during illumination with 405 nm laser (intermittently). (B and C) Before activation. (D) During activation. (E-K) after activation, the downward black arrow indicates that the emission intensity was decreasing with time.

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13 Time dependence of positions of localized HA molecules within an HA cluster in a live fibroblast at room temperature. Hess S T et al. PNAS 2007;104: by National Academy of Sciences

14 Nanoscale visualization of intracellular proteins by FPALM. Hess S T et al. PNAS 2007;104: by National Academy of Sciences

15 Above image is a Fibroblast tagged with photoactivatable-gfp (PA-GFP) CamKII A: Traditional wide-field 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)

16 Did we break the diffraction barrier? The End