The organization of actin filaments into higher-order structures depends on two classes of interaction. First, enthalpic interactions between actin filaments and crosslinks provide a chemical means of controlling the filament organization . Biological crosslinks are protein molecules containing two or more actin-binding domains . There are 13 distinct actinbinding proteins, known as crosslinks . Examples include fascin , α-actinin , scruin , and ARP2/3 complex . Furthermore, actin binds a variety of other actin-binding proteins that do not function as passive crosslinks, such as gelsolin , profilin , cofilin , and myosin . Actin-binding proteins bind actin via specific interactions using a variety of actinbinding domains, such as DNase I loop, nucleotide binding cleft, calponin-homology domain, and β -trefoil domain . Second, a number of physical interactions affect the filament organization. Counterion condensation and depletion interactions can also induce bundling. Similarly, steric confinement induces bundling and affects the filament organization . Shear forces can induce nematic patterns and introduce hysteresis . Van der Waals attraction between filaments promotes adhesive interfilament interactions that lead to gradual relaxation of prestress . Polyethylene glycol (PEG) is known to induce actin bundle formation owing to crowding and depletion interactions between filamentous actin and the polymer . Morphological changes in the F-actin depend on the molecular weight and concentration of the polymer in the solution . For instance, 0.1% w/v (or 1 mg/mL) PEG with a molecular weight of 20 kDa can result in strong bundling, whereas PEG with 6 kDa at the same concentration produces weakly bundled networks . Although the bundling degree of actin filaments under morphology control has been established and studied , the effects of bundling degree on actin molecular Actin morphology and biochemical properties are closely related. Tension-induced structural changes in filaments affect their binding to certain effector proteins . The actin structure is sensitive to the solvent environment, including water and cations, which affects the (de)polymerization and binding capability of actin filaments . Therefore, understanding physical properties, such as the structure and dynamics around the backbone, is critical for understanding the biochemical properties of the actin network. Furthermore, studying the impact of morphological control, such as a higher or lower bundling degree, on the physical and biochemical properties of the actin network can inform the development of soft materials using biopolymer assemblies. To this end, ultrafast infrared spectroscopy is utilized to probe the structural and dynamic changes in the actin secondary structure or actin backbone in crowded environments, that is, polymer-crowded aqueous solutions. Ultrafast infrared spectroscopy has a higher spectral resolution and better sensitivity than traditional IR techniques such as FT IR. Additionally, it can perform real-time measurements of non-crystallized actin samples to study transient motions along the protein backbone, which cannot be performed using microscopic techniques.