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The weak phalloidin signal could include sparse or very short residual actin filaments, together with nonspecific background; it is not a direct image of G-actin. phalloidin preferentially binds filamentous actin, so genuinely specific residual signal would indicate some polymeric actin. Long exposure also amplifies autofluorescence, out-of-focus light and nonspecific staining. Vehicle controls, unstained biological cells, matched acquisition settings and background subtraction are needed before assigning a small residual signal to actin filaments.

For wavelength $\lambda$ and numerical aperture $\mathrm{NA}$, the Rayleigh criterion gives a conventional lateral resolution of roughly $d\approx 0.61\lambda /\mathrm{NA}$, typically a few hundred nanometres under these conditions, far larger than the roughly 7-nm actin filament diameter. It cannot resolve every short actin filament, distinguish all closely spaced actin filaments, or infer polymer mass directly from the displayed intensity. The tenfold exposure change is not itself a calibrated tenfold decrease in F-actin. Fixation, dye accessibility and detector saturation introduce further uncertainty.

Super-resolution fluorescence microscopy could localize remaining labelled structures more precisely, while electron microscopy or cryo-electron tomography could test ultrastructure, subject to preservation and sampling limits. Biochemical separation of polymeric and soluble actin, with the mass-balance and aggregation controls already discussed, supplies an independent population measurement. A live actin reporter can test dynamics but must be checked for perturbation of assembly. Agreement between these approaches would be stronger than concluding complete actin filament absence from weak conventional staining alone.