*2.4.3 Crosslinked network for any given R, ϕ<sup>T</sup> and total protein concentration, [T-P]*

VPEM-100 = VF VTubulin VActin VCL VGTP VATP VTween VTaxol VOS\* VTubulin = (ϕT[T-P]VF)/([T] 2R[T-P]) VActin = ((1-ϕT)[T-P]VF)/([A] 2R[T-P]) VCL = VF/X VGTP = 0.1VF 10 mM GTP VATP = 0.1VF 10 mM ATP VTween = 0.025VF 1% Tween VTaxol = 0.025VF 200 μM Taxol (in DMSO) VOS = 0.05VF oxygen scavenging system\* \**If not imaging networks replace VOS with PEM-100*.

## **3. Optical tweezers microrheology measurements**

Given the importance of cytoskeleton mechanics to cell function, coupled with the complexity of mechanical properties that cells exhibit, understanding the response of cytoskeleton networks to stress and strain remains an important topic of research. Using standard bulk rheology techniques to measure the mechanical properties of cytoskeleton networks has been problematic due to the difficulty and expense in producing mL sample volumes often needed for these measurements.

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

Further, these measurements probe the macroscopic mechanical properties of the networks but are unable to probe mechanics at the molecular and cellular scales (μm). Finally, these methods are ill-equipped to measure spatial heterogeneities in network response, and can irreversibly disrupt or damage the network. Microrheology offers a complementary approach to characterizing the microscale mechanical and viscoelastic properties of cytoskeleton networks. While passive microrheology tracks freely diffusing microspheres embedded in networks to extract viscoelastic moduli, active microrheology uses optical tweezers to actively force embedded microspheres through networks and measure the force exerted to resist this strain. Active microrheology enables one to probe both molecular and mesoscopic scales and perturb networks far from equilibrium to access the nonlinear regime. Specifically, optical tweezers can be used to drag microspheres over distances that are large (5–30 μm) relative to the mesh size of the network (<μm) at speeds much faster than the molecular relaxation rates. The force exerted on the bead to resist the strain, as well as the subsequent relaxation of force following strain, is measured.

Reference [25] provides a thorough overview of the underlying principles and execution of optical tweezers microrheology to characterize the mechanics of biopolymer networks. Here, the focus is on the key results obtained using the in vitro cytoskeleton networks described in Section 2 [18, 19, 26–28].

#### **3.1 Entangled actin networks**

VATP = 0.1VF 10 mM ATP VTween = 0.025VF 1% Tween

*Parasitology and Microbiology Research*

Incubate at 37°C for 60 min.

*a volume* VFC

**Actin**: [B-P] = [B-A] **Microtubule**: [B-P] = [B-T]

sample chamber.

VCL = VF/X

**200**

VGTP = 0.1VF 10 mM GTP VATP = 0.1VF 10 mM ATP VTween = 0.025VF 1% Tween

VTaxol = 0.025VF 200 μM Taxol (in DMSO) VOS = 0.05VF oxygen scavenging system\*

depends on the type of crosslinking as follows:

Sonicate complex solution for 90 min at 4°C.

Add volume *VCL* to solution below.

VTubulin = (ϕT[T-P]VF)/([T] 2R[T-P]) VActin = ((1-ϕT)[T-P]VF)/([A] 2R[T-P])

VTaxol = 0.025VF 200 μM Taxol (in DMSO) VOS = 0.05VF oxygen scavenging system\*

\**If not imaging networks replace VOS with PEM-100*.

**3. Optical tweezers microrheology measurements**

\**If not imaging networks replace VOS with PEM-100*.

**Co-linked**: [B-P] = [B-A] + [B-T]; [B-A] = [B-T] = ½[B-P]

*2.4.2 Recipe for preparing crosslinker complexes that are concentrated by a factor X in*

Prepared complexes are viable for 24 h on ice. Biotinylated protein [*B-P*] used

**Both**: Prepare Actin and Microtubule solutions. Add equal parts of each to final

Concentration factor, X Number ranging from 2 to 20 4 μL PEM-100 volume VPEM-100 = VFC VNA VB-P VB 5.28μL NeutrAvidin volume VNA = X(VFCR[T-P]/[NA]) 2.79μL Biotinylated protein volume VBP = X(VFC2R[T-P]/[B-P]) 1.02μL Biotin volume VB = X(VFC2R[T-P]/[B]) 0.91μL

*2.4.3 Crosslinked network for any given R, ϕ<sup>T</sup> and total protein concentration, [T-P]*

VPEM-100 = VF VTubulin VActin VCL VGTP VATP VTween VTaxol VOS\*

Given the importance of cytoskeleton mechanics to cell function, coupled with

the complexity of mechanical properties that cells exhibit, understanding the response of cytoskeleton networks to stress and strain remains an important topic of research. Using standard bulk rheology techniques to measure the mechanical properties of cytoskeleton networks has been problematic due to the difficulty and expense in producing mL sample volumes often needed for these measurements.

**Equations for a given R Ex. R = 0.02**

Active microrheology experiments have been carried out on entangled actin networks (Section 2.1) to characterize the dependence of the viscoelastic response and stress relaxation on the rate of the applied microbead strain *γ*\_ and actin concentration *c* (1 mg/mL = 23.2 μM) [26, 27]. The results are largely described within the framework of the tube model for entangled polymers, pioneered by de Gennes and Doi and Edwards [29, 30]. Comparisons to new theories and extensions of the tube model are also highlighted [31–33].

#### *3.1.1 Strain rate dependence*

Entangled actin networks (*c* = 0.5 mg/mL; mesh size *ξ* = 0.42 μm) subject to strain rates of *γ*\_ = 1.4–9.4 s<sup>1</sup> (corresponding to speeds of *v* = 1.5–10 m/s) display a unique crossover to appreciable nonlinearity at a strain rate *γ*\_<sup>c</sup> comparable to the theoretical rate of relaxation of individual entanglement segments *τ*ent*<sup>1</sup>* . Above *γ*\_c, networks exhibit stress-stiffening, which, importantly, is not apparent at the macroscopic scale. This stiffening behavior occurs over very short time scales, comparable to the predicted timescale over which mesh size deformations relax *τξ*, and has been shown to arise from suppressed filament bending. At times longer than *τξ*, deformed entanglement segments are able to bend to release stress, and stress softening ensues until the network ultimately yields to an effectively viscous regime, over a timescale comparable to *τent*. This terminal viscous regime exhibits shear thinning due to release of entanglements, with scaling *η γ*\_ 0.34, which is notably less pronounced than the thinning exhibited by flexible entangled polymers (*η γ*\_ *1* ). Surprisingly, the force relaxation following strain proceeds more quickly for increasing strain rates; and for rates greater than *γ*\_c, the relaxation displays a complex power-law dependence on time, as opposed to the expected exponential decay. This power-law relaxation is indicative of dynamic strain-induced entanglement tube dilation and healing, which corroborates recent theoretical predictions for rigid rods [31, 34].

#### *3.1.2 Concentration dependence*

These studies were extended to entangled actin networks of varying concentrations (*c* = 0.2–1.4 mg/mL) to reveal a previously unpredicted and unreported critical concentration *cc* = 0.4 mg/mL for nonlinear response features to emerge. Beyond *cc*, entangled actin stiffens for times below *τξ*, with the degree of stiffening *S* and stiffening time scale *tstiff* scaling inversely with the theoretical entanglement tube diameter *dt*, i.e., *S* � *dt* �*<sup>1</sup>* � *<sup>c</sup> 3/5*. At longer times, the network yields to a viscous regime with the distance *dy* and corresponding force *fy* at which yielding occurs scaling inversely with the length between entanglements *lent* along each filament: *fy* � *dy* � *lent*�*<sup>1</sup>* � *<sup>c</sup> 2/5*. Stiffening and yielding dynamics are consistent with recent predictions of nonlinear strain-induced breakdown of the cohesive entanglement force, which predicts the onset of yielding to occur when the induced force balances the cohesive elastic force provided by the entanglements [27, 32]. Following strain, the force relaxation displays distinct behaviors for *c* > *cc* versus *c* < *cc*. For *c* < *cc*, relaxation follows a single exponential decay with a decay time that scales according to tube model predictions for the disengagement time *τ<sup>D</sup>* � *c 6/5*. For *c* > *cc* relaxation proceeds via two distinct mechanisms: slow reptation out of dilated tubes with *τD*<sup>0</sup> � *c 1/5* coupled with �10� faster lateral hopping. Tube dilation and the commensurate reduction in reptation time *τD*0*/τ<sup>D</sup>* scales as *c* �*1* , in agreement with recent predictions for entangled rigid rods [34, 35]. This model also predicts faster lateral hopping out of constraining tubes due to temporary fluctuation-induced yielding. The coupled emergence of lateral hopping with concentration-dependent dilation indicates that hopping only plays a significant role when entanglement tubes are sufficiently dilated to allow for fluctuation-induced transient yielding of tube constraints.

further discussed in Section 4.3, this long-lived post-strain stress is likely a result of the network distributing stress to a small fraction of highly strained connected filaments that span the network, allowing the rest of the network to relax [28]. These intriguing results, along with the corresponding actin filament deformations and stress propagation dynamics that lead to the force response, are further

As described in Section 2.4, techniques have recently been developed to create randomly oriented, co-entangled networks of actin and microtubules by simultaneously co-polymerizing varying ratios of actin and tubulin in situ. The relative concentrations of actin and microtubules, quantified by the molar fraction of tubulin *ϕT*, as well as the overall protein concentration *[T-P]*, can be systematically varied over a wide range of values while maintaining composite integrity and stability. Different crosslinking interactions and motifs can also be methodically

Seminal microrheology studies on these composites have been carried out for *ϕ<sup>T</sup>* values of 0 to 1 with *[T-P]* held fixed at 11.6 μM [18, 23]. These studies show that composites comprised of mostly actin (*ϕ<sup>T</sup>* < 0.5) initially exert a 100 higher resistive force in response to strain, compared to networks comprised of mostly microtubules (*ϕ<sup>T</sup>* > 0.5). However, the rise in force with strain distance is steeper for *ϕ<sup>T</sup>* > 0.5 networks such that at 5 μm, the force became larger for *ϕ<sup>T</sup>* > 0.5 composites compared to *ϕ<sup>T</sup>* < 0.5. Actin-rich composites are also initially relatively stiff but quickly softened, whereas microtubule-rich composites display an initially soft/viscous response followed quickly by stiffening such that at the end of the strain the stiffness for *ϕ<sup>T</sup>* > 0.5 networks was 10 higher than their actin-rich counterparts. The initial force response can be understood in terms of poroelastic models, which consider the dynamics of the mesh as well as the pervading fluid [14, 36]. In these models, the faster the timescale for water to drain from the deformed mesh (*τp*), the faster the system can relax, such that it will exert a concomitantly smaller initial force on the bead. The poroelastic timescale, which depends both on the elastic modulus and mesh size of the network, is 40 longer for actin networks than for microtubule networks [18], resulting in a comparably higher initial force and stiffness for actin-rich composites versus microtubule-rich composites. The subsequent sharp transition from softening to stiffening when *ϕ<sup>T</sup>* exceeds 0.5, arises from microtubules suppressing actin bending fluctuations. The presence of a large fraction of microtubules (*ϕ<sup>T</sup>* > 0.7) result in large heterogeneities in force response as well as increased average resistive force. Heterogeneities arise from the increasing mesh size of the composite as *ϕ<sup>T</sup>* increases, as well as more frequent microtubule buckling events. As *ϕ<sup>T</sup>* increases the mesh size of the composite increases from *ξ<sup>A</sup>* 0.42 μm for *ϕ<sup>T</sup>* = 0 to *ξ<sup>M</sup>* 0.89 μm for *ϕ<sup>T</sup>* = 1. Thus, at the microscale, the system becomes increasingly more heterogeneous as *ϕ<sup>T</sup>* increases. Further, for a composite with equal molar fractions of actin and microtubules (*ϕ<sup>T</sup>* = 0.5), the mesh size of the microtubule network is 2 that of the actin network (*ξ<sup>A</sup> 2ξM*), and the actin mesh remains smaller than the microtubule

mesh until *ϕ<sup>T</sup>* > 0.7. Thus, actin network characteristics dominate the force response until relatively large fractions of microtubules are incorporated. This effect, combined with force-induced buckling of microtubules to alleviate stress,

first decay arising from actin bending modes while the long-time relaxation is indicative of filaments reptating out of deformed entanglement constraints.

Force relaxation following strain exhibits two-phase power-law decay with the

leads to a nonlinear increase in resistive force as *ϕ<sup>T</sup>* increases.

**3.3 Co-entangled composite networks of actin and microtubules**

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks*

*DOI: http://dx.doi.org/10.5772/intechopen.84401*

explored in Section 4.3.

introduced and tuned.

**203**

#### **3.2 Crosslinked actin networks**

As detailed in Section 2.2, methods have been developed to produce highly stable and reproducible networks of randomly-oriented crosslinked actin filaments. With these methods, the crosslinker density can be systematically tuned while fixing the actin concentration and structural network properties (i.e., isotropic filament orientation, no bundling).

Nonlinear microrheological characterization of these networks have been carried out for crosslinking ratios of *R* = 0–0.07 (*c* = 0.5 mg/mL) [19]. For all *R* values, networks exhibit initial stiffening due to entropic stretching of filaments along the strain path, followed by stress softening and yielding to a steady-state regime. The maximum stiffness achieved *Kmax* as well as the time to yield to the terminal regime scale exponentially with *R*. The critical decay constant associated with this scaling, *R\** � 0.014, corresponds to a crosslinker length *lc* equal to the theoretical entanglement length *le*. Networks with higher *R* values also exhibit more sustained elastic resistance in the terminal regime such that the terminal stiffness *Kt* scales exponentially with *R* with a similar critical ratio *R\** � 0.018. These stress response characteristics suggest that softening and yielding arise from force-induced disentanglement and crosslinker unbinding while crosslinker rebinding events allow for the observed sustained terminal elasticity.

Following strain, all networks exhibit exponential force decay with two distinct timescales. Similar to the stress response characteristics, both fast and slow relaxation times scale exponentially with *R* with comparable *R\** values of � 0.008, which likewise corresponds to *lc* � *le*. For *R > R\** networks are able to maintain high levels of elastic stress following the strain, which is quantified by the terminal force value *Ft* at the end of the 30 s relaxation phase. Once again, *Ft* � *e R/R\** with *R\** � 0.007. As

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

further discussed in Section 4.3, this long-lived post-strain stress is likely a result of the network distributing stress to a small fraction of highly strained connected filaments that span the network, allowing the rest of the network to relax [28].

These intriguing results, along with the corresponding actin filament deformations and stress propagation dynamics that lead to the force response, are further explored in Section 4.3.

### **3.3 Co-entangled composite networks of actin and microtubules**

As described in Section 2.4, techniques have recently been developed to create randomly oriented, co-entangled networks of actin and microtubules by simultaneously co-polymerizing varying ratios of actin and tubulin in situ. The relative concentrations of actin and microtubules, quantified by the molar fraction of tubulin *ϕT*, as well as the overall protein concentration *[T-P]*, can be systematically varied over a wide range of values while maintaining composite integrity and stability. Different crosslinking interactions and motifs can also be methodically introduced and tuned.

Seminal microrheology studies on these composites have been carried out for *ϕ<sup>T</sup>* values of 0 to 1 with *[T-P]* held fixed at 11.6 μM [18, 23]. These studies show that composites comprised of mostly actin (*ϕ<sup>T</sup>* < 0.5) initially exert a 100 higher resistive force in response to strain, compared to networks comprised of mostly microtubules (*ϕ<sup>T</sup>* > 0.5). However, the rise in force with strain distance is steeper for *ϕ<sup>T</sup>* > 0.5 networks such that at 5 μm, the force became larger for *ϕ<sup>T</sup>* > 0.5 composites compared to *ϕ<sup>T</sup>* < 0.5. Actin-rich composites are also initially relatively stiff but quickly softened, whereas microtubule-rich composites display an initially soft/viscous response followed quickly by stiffening such that at the end of the strain the stiffness for *ϕ<sup>T</sup>* > 0.5 networks was 10 higher than their actin-rich counterparts. The initial force response can be understood in terms of poroelastic models, which consider the dynamics of the mesh as well as the pervading fluid [14, 36]. In these models, the faster the timescale for water to drain from the deformed mesh (*τp*), the faster the system can relax, such that it will exert a concomitantly smaller initial force on the bead. The poroelastic timescale, which depends both on the elastic modulus and mesh size of the network, is 40 longer for actin networks than for microtubule networks [18], resulting in a comparably higher initial force and stiffness for actin-rich composites versus microtubule-rich composites. The subsequent sharp transition from softening to stiffening when *ϕ<sup>T</sup>* exceeds 0.5, arises from microtubules suppressing actin bending fluctuations.

The presence of a large fraction of microtubules (*ϕ<sup>T</sup>* > 0.7) result in large heterogeneities in force response as well as increased average resistive force. Heterogeneities arise from the increasing mesh size of the composite as *ϕ<sup>T</sup>* increases, as well as more frequent microtubule buckling events. As *ϕ<sup>T</sup>* increases the mesh size of the composite increases from *ξ<sup>A</sup>* 0.42 μm for *ϕ<sup>T</sup>* = 0 to *ξ<sup>M</sup>* 0.89 μm for *ϕ<sup>T</sup>* = 1. Thus, at the microscale, the system becomes increasingly more heterogeneous as *ϕ<sup>T</sup>* increases. Further, for a composite with equal molar fractions of actin and microtubules (*ϕ<sup>T</sup>* = 0.5), the mesh size of the microtubule network is 2 that of the actin network (*ξ<sup>A</sup> 2ξM*), and the actin mesh remains smaller than the microtubule mesh until *ϕ<sup>T</sup>* > 0.7. Thus, actin network characteristics dominate the force response until relatively large fractions of microtubules are incorporated. This effect, combined with force-induced buckling of microtubules to alleviate stress, leads to a nonlinear increase in resistive force as *ϕ<sup>T</sup>* increases.

Force relaxation following strain exhibits two-phase power-law decay with the first decay arising from actin bending modes while the long-time relaxation is indicative of filaments reptating out of deformed entanglement constraints.

*3.1.2 Concentration dependence*

*Parasitology and Microbiology Research*

tube diameter *dt*, i.e., *S* � *dt*

*fy* � *dy* � *lent*�*<sup>1</sup>* � *<sup>c</sup>*

*τD*<sup>0</sup> � *c*

constraints.

**202**

**3.2 Crosslinked actin networks**

entation, no bundling).

These studies were extended to entangled actin networks of varying concentra-

*3/5*. At longer times, the network yields to a viscous

�*1*

*6/5*. For *c* > *cc* relaxation

, in agreement with recent

*R/R\** with *R\** � 0.007. As

*2/5*. Stiffening and yielding dynamics are consistent with recent

tions (*c* = 0.2–1.4 mg/mL) to reveal a previously unpredicted and unreported critical concentration *cc* = 0.4 mg/mL for nonlinear response features to emerge. Beyond *cc*, entangled actin stiffens for times below *τξ*, with the degree of stiffening *S* and stiffening time scale *tstiff* scaling inversely with the theoretical entanglement

regime with the distance *dy* and corresponding force *fy* at which yielding occurs scaling inversely with the length between entanglements *lent* along each filament:

predictions of nonlinear strain-induced breakdown of the cohesive entanglement force, which predicts the onset of yielding to occur when the induced force balances the cohesive elastic force provided by the entanglements [27, 32]. Following strain, the force relaxation displays distinct behaviors for *c* > *cc* versus *c* < *cc*. For *c* < *cc*, relaxation follows a single exponential decay with a decay time that scales according

proceeds via two distinct mechanisms: slow reptation out of dilated tubes with

*1/5* coupled with �10� faster lateral hopping. Tube dilation and the com-

As detailed in Section 2.2, methods have been developed to produce highly stable and reproducible networks of randomly-oriented crosslinked actin filaments. With these methods, the crosslinker density can be systematically tuned while fixing the actin concentration and structural network properties (i.e., isotropic filament ori-

Nonlinear microrheological characterization of these networks have been carried out for crosslinking ratios of *R* = 0–0.07 (*c* = 0.5 mg/mL) [19]. For all *R* values, networks exhibit initial stiffening due to entropic stretching of filaments along the strain path, followed by stress softening and yielding to a steady-state regime. The maximum stiffness achieved *Kmax* as well as the time to yield to the terminal regime scale exponentially with *R*. The critical decay constant associated with this scaling, *R\** � 0.014, corresponds to a crosslinker length *lc* equal to the theoretical entanglement length *le*. Networks with higher *R* values also exhibit more sustained elastic resistance in the terminal regime such that the terminal stiffness *Kt* scales exponentially with *R* with a similar critical ratio *R\** � 0.018. These stress response charac-

teristics suggest that softening and yielding arise from force-induced

allow for the observed sustained terminal elasticity.

*Ft* at the end of the 30 s relaxation phase. Once again, *Ft* � *e*

disentanglement and crosslinker unbinding while crosslinker rebinding events

Following strain, all networks exhibit exponential force decay with two distinct timescales. Similar to the stress response characteristics, both fast and slow relaxation times scale exponentially with *R* with comparable *R\** values of � 0.008, which likewise corresponds to *lc* � *le*. For *R > R\** networks are able to maintain high levels of elastic stress following the strain, which is quantified by the terminal force value

predictions for entangled rigid rods [34, 35]. This model also predicts faster lateral hopping out of constraining tubes due to temporary fluctuation-induced yielding. The coupled emergence of lateral hopping with concentration-dependent dilation indicates that hopping only plays a significant role when entanglement tubes are sufficiently dilated to allow for fluctuation-induced transient yielding of tube

�*<sup>1</sup>* � *<sup>c</sup>*

to tube model predictions for the disengagement time *τ<sup>D</sup>* � *c*

mensurate reduction in reptation time *τD*0*/τ<sup>D</sup>* scales as *c*

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

0.72 μL 5-A 0.54 μL A

composite:

1 μL 10 F-buffer

6.74 μL PEM-100 0.72 μL 4-A 0.54 μL A

2.00 μL 10 mM ATP

composite as follows:

Incubate for 60 min at RT.

equivalent volume of PEM-100.

Thaw R-T aliquot in hand. Add 0.55 μL 10 mM GTP. Incubate at 37°C for 30 min. Add 0.6 μL of 200 μM Taxol. Incubate at 37°C for 30 min.

equivalent volume of PEM-100.

*4.1.2 In situ network labeling*

6.3 μL PEM-100

**205**

entangled actin and actin-microtubule composites.

*4.1.2.1 Example of in situ labeled actin network*

*c* = 1 mg/mL, [*5-A*]:[*A*] = 1:9.6, *VF* = 20 μL

**Rhodamine-labeled microtubules**

Incubate for 60 min at RT.

*DOI: http://dx.doi.org/10.5772/intechopen.84401*

**Alexa-488-actin filaments**

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).

Prepare 10 μL of a 5 μM solution of 1:1 [*4-A*]:[*A*] to polymerize prior to adding to

Immediately prior to imaging prepare a 1:2 dilution in PEM-100 + 2 mM ATP. Add 1 μL to final sample chamber solution from Section 2.4, replacing the

Prepare 5 μL of a 37 μM solution of R-T to polymerize prior to adding to

Immediately prior to imaging prepare a 1:10 dilution in PEM-Taxol. Add 1 μL to final sample chamber solution from Section 2.4, replacing the

Labeled filaments can be stored at RT for up to 1 week. After day 1, shear microtubules with a sterile hamilton syringe before adding to the sample chamber.

In this method labeled monomers are added to solution prior to in situ network formation, rather than adding pre-formed filaments [23]. This method, demonstrated in **Figures 1** and **4** provides the most accurate depiction of network architecture and enables evaluation of network formation during polymerization. The drawback is that rarely are discrete single filaments visible, preventing filament length measurements. The ratio of labeled (*4-A*, *5-A* or *R-T*) to unlabeled (*A* or *T*) monomers can range from 1:50 to 1:5 depending on the overall protein concentration and type of fluorescent dye used. Below are recipes for optimized samples of

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

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks*
