U lh100l 3

For zona drilling, the embryos were moved to LifeGlobal Total^®w/HEPES. A laser (NaviLase, OCTAX Microscience GmbH) attached to a microscope
(U-LH100L-3, Olympus^®) was used. Three laser pulses of 1.3ms
were applied in the zona pellucida of the blastocyst, opposite to the inner cell
mass. After zona drilling, the blastocysts were placed in LifeGlobal
total^® media (LifeGlobal^®) for at least
8 hours, to facilitate the trophectoderm (TE) herniation for later biopsy.
Biopsy of two different TE fragments of each blastocyst were collected by
aspiration (between 4 and 16 cells each, depending on the characteristics of the
TE).

For the spectral
image analysis, an Olympus inverted microscope, equipped with a halogen
lamp (Olympus, U-LH100L-3) as a light source and coupled to a multispectral
camera (CRI Nuance EX) controlled using Nuance 3.0.2 software, was
used in the characterization of the silk films doped with DTE. Images
were acquired and unmixed in the different spectral components. The
absorbance magnitude was obtained by comparison between a reference
area (non-UV-irradiated silk) and the area under study (UV-irradiated
silk).
In the analysis of the silk biosensors, images were acquired
using a Canon EOS 250D in a photobox illuminated with a cool white
LED (6000 K; 18 W power) to avoid the interference of the environmental
light. Images were then analyzed using ImageJ. First, they were split
into the three primary color channels (i.e., red, green, and blue),
only considering the green one for the analysis since it presents
the highest sensitivity to the measurement. The rectangle tool was
then used for the selection of the colored areas to be analyzed, ignoring
the edges.

The polarizing image measurement was performed at ISSP, the University of Tokyo^{48 (link)}. The sample temperature was controlled in the range of 8–20 K. A 100 W halogen lamp (U-LH100L-3, Olympus) was used to obtain bright images. The flat (001) surfaces for imaging were prepared by cleavage in the atmosphere, and the sample was immediately installed to the vacuum chamber within 10 min. The polarizing microscope images were taken in crossed Nicols configuration with the optical principal axes along [110].

The microfluidic device was kept on the microscope stage throughout the experiment (7 days). One set of the side channels (top and bottom) was used for medium perfusion and nitrogen gradient generation, while the other set had ends plugged to prevent evaporation. A constant flow rate at 0.7 µL/min through the side channels was maintained by a syringe pump (KDS230, KD Scientific, Holliston, MA) and two 10 mL syringes (Exelint International Co., Redondo Beach, CA). For the nitrogen gradient experiment, the syringe connected to the top channel was filled with 10% TAP with 35.3 μM N, while the syringe connected to the bottom channel was filled with 10% TAP-N. For the lowest nitrogen (5.3 µM) condition, the source and sink channels were both perfused with 10% TAP-N, with habitats either under a light intensity gradient, or under dark (0 PAR).
The transmitted light path of an inverted microscope (Olympus IX81, Center Valley, CA) was modified to generate a light gradient as described previously^{51 (link)}. Briefly, a half-moon mask was inserted into the light path, perpendicular to the light beam from the bright field halogen lamp (Olympus U-LH100L-3), which resulted in a light gradient with a light–dark transition region near the middle of the sample plane. The microscope room temperature was controlled at 25 °C.

Exfoliated Gr and functionalized Gr dispersions (1 wt% Gr in DMF) were prepared, then diluted with a fluorescence dye solution (RhB, 10⁻⁶ M in ethanol) using a vortex mixer, and left to stand without any perturbation for 3 h at room temperature. The RhB-treated Gr sheets were acquired by centrifugating and drying, then diluted with an appropriate amount of ethanol again. The labeled Gr solution was dropped onto SiO₂/Si wafer and dried at 80 °C for FM imaging. The bare NCA particles (0.2 g) were subjected to vortex mixing in 0.2 g of Gr solutions (1 wt% Gr in DMF) containing 0–0.2 wt% DSPE-mPEG, then were diluted with 20 ml of RhB solution (10⁻⁴ M in ethanol), and left to stand without any perturbation for 12 h at room temperature. After centrifugation and drying at 80 °C, the RhB-treated NCA powder was spread onto a carbon adhesive tape and planarized with a slide glass for FM imaging. The FM images were obtained using a fluorescence microscope (Olympus corp., BX51M) equipped with green excitation filter (U-MWGS3) and halogen lamp (100 W, U-LH100L-3).

Photoreceptor outer segments (POSs) were isolated from fresh bovine eyes and labeled with fluorescein isothiocyanate (FITC), as described earlier.¹⁰ (link)^,11 (link) The RPE cells were co-cultured in conditioned medium containing 200 µg/mL FITC-labeled POSs for 4 hours at 37°C after exposure to the smokers’ serum for 7 days. Then, the excess FITC-labeled POSs were removed by rinsing with PBS, and cells were fixed with paraformaldehyde. The cells were observed and imaged using a fluorescence microscope (U-LH100L-3; Olympus Corporation, Tokyo, Japan). Phagocytosis was quantified by green fluorescence (representing the total POS phagocytosed by RPE cells) with Image J software. Four visual fields from four quadrants were randomly chosen from each group for evaluation. Total proteins were extracted from the RPE cells, which had been cultured in media containing POS, and then rhodopsin was detected by Western blot.