Actin filaments, fluorescence images

FIGURE 8.8 A section of a single HeLa (human cervical cancer) cell stained with Phalloidin 350 (actin filaments) and MitoTracker488 (mitochondria) acquired using selective two-photon excitation, without fluorescence color filters. The image size is about 25 (xm. (From Dantus, M., Lozovoy, V. V., and Pastirk, I. Laser Focus World, 43(5) 101-104. 2007. Used with permission.)... 

Figure 10.4 (A) A schematic of globular actin monomer forming a protein filament, called F-actin. This filament is one of the important components of muscle cells, as well as the cytoskeleton of other cells. (B) Oriented actin filaments inside a fibroblast cell, called stress fibers, seen through a fluorescence microscope. Image obtained from Nguyen et al. [148] and reprinted with permission.

The three-dimensional network of actin stress fiber, which is an association of actin filaments, provides mechanical support for the cell, determines the cell shape, and enables cell movement. Thus the shape change in the cell due to the laser tsunami can be examined by observing the laser-induced dynamics of fibers. The actin stress fiber was visualized by binding it with enhanced green fluorescence protein (EGFP), and monitored by total internal reflection fluorescence (TIRF) imaging [34]. 

Figure 11. Fluorescence images of actin filaments labeled with various TMR-dye contents. N represents the average number of TMR-stained actin units per micron of filament. The filaments were in F-buffer (0.1 M KC1,2 mM MOPS, 1 mM MgCl2, 1.5 mM NaN3, 0.1 mM ATP, pH = 7.0) and bound to polylysin-coatcd quartz coverslips. The excitation intensity was 0.14 (a), 0.33 (b). 2.8 (c), and 11 (d) mW, respectively, for an illuminated area of about 35-/mi diameter. All images were obtained by integration of 16 video frames, that is, 0.53 s, and are reproduced with the same brightness level. (Adopted from [64].)...

Unlike Alexa Fluor 594, rhodamine phalloidin increases its fluorescence upon binding to actin filaments. This makes it preferable to Alexa Fluor 594. However, for imaging in the green channel, we would use Alexa Fluor 488 or similar fluor-ophore over the rapidly bleaching FITC. 

Fig. 3 Actin cytoskeleton and focal adhesion. F-actins were visualized using rhodamine phalloidin (red), and vinculins found in focal adhesions were stained using fluorescently conjugated antibodies. (A) Immunofluorescent images of a thin filamentous actin meshwork and vinculins showed that osteoblasts contain fewer and smaller focal adhesions. (B) In contrast, hMSCs showed thick actin stress fibers, and multiple and large adhesion contacts.

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