We discounted the hypothesis that speckles were generated by uorescent aggregates by showing that labeled tubulin dimers sediment similarly to unlabeled puried dimers in an analytical ultracentrifugation assay.Speckle formation in actin lament networks.Phalloidin image showing the organization of actin laments in amorphous lament networks and bundles.In the single FSM image much of the structural information is lost, but timelapse FSM series contain dynamic information of lament transport and turnover not accessible with higherlevel Oleic acid labeling of the cytoskeleton.Colorized speckle signal overlaid onto a quickfreeze deep etch image of the samesized region of the actin cytoskeleton in the leading edge of a broblast with hypothetical uorophore distribution that could give rise to such speckle pattern.This indicates the scale of FSM compared with ultrastructure of the polymer network and illustrates that a small proportion of the total actin uoresces and that uorophores from different laments contribute to the same speckle.Thus, the most plausible explanation for speckle formation in microtubules was that variations existed in the number of uorescent tubulin subunits in each resolutionlimited image region along the microtubule.To understand how speckles originated, we considered how the images of uorescent microtubules were formed by the microscope. Microtubules assemble from tubulin dimers into the nmdiameter cylindrical wall such that there are dimers per micron. The nal image results from a convolution of the uorophore distribution along the microtubule with the pointspread function of the microscope.A given fraction of uorescent dimers, f, produces a mean number of uorescent dimersn fper PSF.The speckle pattern along the microtubule is produced by variations in the number of uorescent dimers per PSF relative to this mean.Thus, the contrast of the speckle pattern can be approximated by the ratio of the standard deviationnelements:c. For a microtubule, or more speckles arise from a single uorophore whenfis less than. Technologically, the noise level and sensitivity of the imaging system, instability of the microscope, and the dynamics of the observed process may all deteriorate feasibility of single uorophore detection.In contrast to speckle formation in isolated microtubules, labeled actin subunits associate with a highly crosslinked threedimensional network of actin laments. The same concept of speckle formation has been exploited to visualize molecules making up focal adhesions. As with actin networks, speckles represent randomly distributed uorescent FA proteins that are temporarily clustered in the FA Dicumarol complex within the threedimensional volume of one PSF.A speckle is thus dened as a diffractionlimited image region that is signicantly higher in uorophore concentration than its neighboring diffractionlimited image regions.The duration of association must be equal to or longer than the camera exposure of one frame in a timelapse image sequence.Conversely, the disappearance of a speckle would mark the local dissociation of subunits.In other words, FSM allows, in principle, the direct kinetic measurement of subunit turnover in space and time via speckle lifetime analysis.In addition, once a speckle is formed, it may undergo motion that indicates the coordinated movement of labeled subunits on the platform andor the movement of the platform itself.The graph displays the mean speckle density from ve simulations of a network that starts with no uorophores, assembles for s, and disassembles for s at equal rates.

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