*4.1.1 Doping networks with pre-formed labeled filaments*

This method is ideal for measuring filament length distributions and resolving single-filament fluctuations and mobility (**Figure 3**).

#### **Figure 3.**

*Two-color laser scanning confocal imaging of fluorescent-labeled equimolar actin-microtubule composite ([T-P] = 11.6 μM). (A) The actin (green) channel, (B) microtubule (red) channel and (C) both channels show actin filaments and microtubules within composites form networks that overlap with each other forming a homogeneous network with no phase separation or clustering. The scale bar is 50 μm and applies to all images. The 512 512 image is taken on a Nikon A1R laser scanning confocal microscope with a 60 objective and QImaging CCD camera.*

#### *4.1.1.1 Labeled actin filaments for actin networks (Sections 2.2 and 2.3)*

Prepare 10 μL of a 5 μM solution of 1:1 [*5-A*]:[*A*] to polymerize prior to adding to actin network:

7.75 μL G-buffer

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks DOI: http://dx.doi.org/10.5772/intechopen.84401*

0.72 μL 5-A 0.54 μL A 1 μL 10 F-buffer

Interestingly, the scaling exponents for the long-time relaxation exhibits a nonmonotonic dependence on *ϕT*, reaching a maximum for equimolar composites (*ϕ<sup>T</sup>* = 0.5), which suggests that filament diffusion (i.e., reptation) is fastest at *ϕ<sup>T</sup>* = 0.5. This non-monotonic trend likely arises from a competition between increasing mesh size as *ϕ<sup>T</sup>* increases, which increases filament mobility, versus increasing filament rigidity (replacing actin with microtubules), which suppresses

**4. Fluorescence imaging and characterization of network transport,**

results and parameters that can be obtained with the described methods.

**4.1 Fluorescence labeling of proteins for varied measurement methods**

discrete filament segments for particle-tracking [19, 28].

*4.1.1 Doping networks with pre-formed labeled filaments*

single-filament fluctuations and mobility (**Figure 3**).

*4.1.1.1 Labeled actin filaments for actin networks (Sections 2.2 and 2.3)*

actin network:

**204**

*QImaging CCD camera.*

**Figure 3.**

7.75 μL G-buffer

Prepare 10 μL of a 5 μM solution of 1:1 [*5-A*]:[*A*] to polymerize prior to adding to

*Two-color laser scanning confocal imaging of fluorescent-labeled equimolar actin-microtubule composite ([T-P] = 11.6 μM). (A) The actin (green) channel, (B) microtubule (red) channel and (C) both channels show actin filaments and microtubules within composites form networks that overlap with each other forming a homogeneous network with no phase separation or clustering. The scale bar is 50 μm and applies to all images. The 512 512 image is taken on a Nikon A1R laser scanning confocal microscope with a 60 objective and*

A key question regarding the cytoskeleton is how the mechanical force response couples to both network structure as well as the mobility and deformations of the comprising filaments. To address this problem, a range of fluorescence labeling schemes can be incorporated into in vitro networks, and various microscopy methods can be employed to image networks and quantify mobility and structure. This section describes different in vitro labeling and imaging methods as well as key

Below are protocols for three different labeling schemes optimized for different network characterizations and imaging methods: (1) doping networks with preformed labeled filaments [18, 27], (2) in situ network labeling [23], and (3) labeling

This method is ideal for measuring filament length distributions and resolving

filament mobility. See Section 4.2 for more discussion of this result.

**mobility and structure**

*Parasitology and Microbiology Research*

Incubate for 60 min at RT.

Prepare a 1:2 dilution in PEM-100 or F-buffer (depending on desired final buffer). Add 1 μL of dilution to final sample chamber solution from Section 2.2 or 2.3, replacing the equivalent volume of PEM-100 or G-buffer (depending on network).

*4.1.1.2 Labeled filaments for actin-microtubule composites (Section 2.4)*
