Fv10 ver4

Individual hair fibers treated with the KP-Cryst-FITC conjugates were embedded in epoxy resin and transversal cuts (20 μm) were prepared using a microtome (Microtome Leitz, Oberkochen, Germany). Transversal cuts of Asian and Caucasian hairs, with and without the KP-Cryst-FITC conjugates, were analyzed by Confocal Scanning Laser Microscope (Olympus BX61, Model FluoView 1000). Acquisition for all the hair samples was done using the same settings (filter, exposure time and brightness). Images were acquired with the program FV10-Ver4.1.1.5 (Olympus) integrated with Line Series Analysis. The detection was obtained with a laser excitation line at 488 nm and emissions filters BA 505–605. Images were acquired with the program FV10-Ver4.1.1.5 (Olympus) integrated with Line Series Analysis.

Microscopy images were taken using CSLM (Olympus BX61, Model FluoView 1000, Shinjuku City, Japan) to characterize the samples prior to digestion, as well as to assess the aggregation of Lf–curcumin nanoparticles during the in vitro digestion process, using a 60× oil-immersed objective lens. The samples of native Lf, denatured Lf, and Lf–curcumin nanoparticles, as well as samples from stomach emptying 4 and 8 (i.e., digestion times of 12.68 and 25.38 min, respectively) were stained according to Liang et al. [15 (link)] Briefly, a volume of 5 µL of FITC (10 mg·mL⁻¹ in dimethyl sulfoxide) was added to 200 µL of sample. The stained samples were then vortexed for 5 min and 7 µL were transferred to the microscope slide. The samples were then analysed using a green laser (laser with the reference laser488 BA: 505–540) since the emission and excitation wavelengths of FITC are 488 and 545 nm, respectively. All images were acquired and processed with the software FV10-Ver4.1.1.5 (Olympus).

CLSM was performed as described before [56 (link)] with some modifications. Briefly, the 13 mm in diameter Thermanox^® Plastic Coverslip (Rochester, New York, USA) were placed in 24-well plates, and mono and dual-biofilms were formed as mentioned before. Coverslips were further washed twice with saline solution, and treatments were applied. After the treatment, the suspension was aspirated, and the wells were washed twice with 0.9% saline solution. The fluorescence probes, EPA1_TFP with mCherry (laser excitation line 635nm and emissions filters BA 655–755, red channel) and LM12_AMI-SH3 with GFP (laser excitation line 488 nm and emissions filters BA 505–605, green channel) were used for detection of cells in biofilms. The coupons were stained with 15 µL of probes in a final concentration of 20 mM for 15 min. After, the images were acquired in a Confocal Scanning Laser Microscope (Olympus B × 61, Model FluoView 1000) with the program FV10-Ver4.1.1.5 (Olympus). For each condition, three independent biofilms were used.