Fluorescence photobleaching of microplastics: A cautionary tale.

Fluorescence microscopy is an important step in visual identification of microplastics and is used to highlight white and transparent plastics that are otherwise easily missed or misidentified. Investigators using fluorescence must proceed with caution, however, as fluorescence photobleaching can significantly reduce the fluorescence output of samples within experimentally relevant time frames. We report on the photobleaching rate and subsequent lack of fluorescence recovery of five common plastics. Our results reveal statistically different photobleaching rates across plastic types. In the best-case scenario of low illumination intensity and a robust plastic, initial fluorescence intensity decayed by 10% in just 11(3) s and by 33% in 230(40) s. In all cases, fluorescence failed to recover more than 13(8)% in 3 h. These results indicate that significant bleaching can occur while searching a sample for plastics to identify and that the lack of recovery can compromise samples for further study.

Single- and two-photon fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules and can be applied both in vitro and in vivo for two- and three-dimensional systems. This introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

Multiphoton fluorescence recovery after photobleaching in bounded systems.

Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to measure diffusion coefficients of macromolecules in biological systems. The three-dimensional resolution and superior depth penetration within scattering samples offered by MP-FRAP make it an important tool for investigating both in vitro and in vivo systems. However, biological systems frequently confine diffusion within solid barriers, and to date the effect of such barriers on the measurement of absolute diffusion coefficients via MP-FRAP has not been studied. We have used Monte Carlo simulations of diffusion and MP-FRAP to understand the effect of barriers of varying geometries and positions relative to the two-photon focal volume. Furthermore, we supply ranges of barrier positions within which MP-FRAP can confidently be employed to measure accurate diffusion coefficients. Finally, we produce two new MP-FRAP models that can produce accurate diffusion coefficients in the presence of a single plane boundary or parallel infinite plane boundaries positioned parallel to the optical axis, up to the resolution limit of the multiphoton laser scanning microscope.

Measuring diffusion coefficients via two-photon fluorescence recovery after photobleaching.

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).

Improved model of fluorescence recovery expands the application of multiphoton fluorescence recovery after photobleaching in vivo.

Multiphoton fluorescence recovery after photobleaching is a well-established microscopy technique used to measure the diffusion of macromolecules in biological systems. We have developed an improved model of the fluorescence recovery that includes the effects of convective flows within a system. We demonstrate the validity of this two-component diffusion-convection model through in vitro experimentation in systems with known diffusion coefficients and known flow speeds, and show that the diffusion-convection model broadens the applicability of the multiphoton fluorescence recovery after photobleaching technique by enabling accurate determination of the diffusion coefficient, even when significant flows are present. Additionally, we find that this model allows for simultaneous measurement of the flow speed in certain regimes. Finally, we demonstrate the effectiveness of the diffusion-convection model in vivo by measuring the diffusion coefficient and flow speed within tumor vessels of 4T1 murine mammary adenocarcinomas implanted in the dorsal skinfold chamber.