Quantitative assessment of visual microscopy as a tool for microplastic research: Recommendations for improving methods and reporting.

Microscopy is often the first step in microplastic analysis and is generally followed by spectroscopy to confirm material type. The value of microscopy lies in its ability to provide count, size, color, and morphological information to inform toxicity and source apportionment. To assess the accuracy and precision of microscopy, we conducted a method evaluation study. Twenty-two laboratories from six countries were provided three blind spiked clean water samples and asked to follow a standard operating procedure. The samples contained a known number of microplastics with different morphologies (fiber, fragment, sphere), colors (clear, white, green, blue, red, and orange), polymer types (PE, PS, PVC, and PET), and sizes (ranging from roughly 3-2000 µm), and natural materials (natural hair, fibers, and shells; 100-7000 µm) that could be mistaken for microplastics (i.e., false positives). Particle recovery was poor for the smallest size fraction (3-20 µm). Average recovery (±StDev) for all reported particles >50 µm was 94.5 ± 56.3%. After quality checks, recovery for >50 µm spiked particles was 51.3 ± 21.7%. Recovery varied based on morphology and color, with poorest recovery for fibers and the largest deviations for clear and white particles. Experience mattered; less experienced laboratories tended to report higher concentration and had a higher variance among replicates. Participants identified opportunity for increased accuracy and precision through training, improved color and morphology keys, and method alterations relevant to size fractionation. The resulting data informs future work, constraining and highlighting the value of microscopy for microplastics.

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.