Cfi plan fluor

The perovskite crystals of the thin film were mechanically exfoliated using the Scotch tape method (Nitto SPV 224). The exfoliation guarantees a freshly cleaved and atomically flat surface area for inspection, which is crucial to avoid emission from edge states and guarantee direct contact with the glass substrate. After several exfoliation steps, the crystals were transferred on a glass slide and were subsequently studied through the glass slide with a ×100 oil immersion objective (Nikon CFI Plan Fluor, NA = 1.3). A big advantage of this technique is that the perovskites are encapsulated through the glass slide from one side and by the bulk of the crystal from the other side. It is important to use thick crystals to guarantee good self-encapsulation and prevent premature degradation of the perovskite flakes to affect the measurement^{43 (link)}.

Before recording, oocytes were subjected to fluorescence labeling using methanethiosulfonate-carboxytetramethylrhodamine (MTS-TAMRA, Santa Cruz Biotechnology) at 10 μmol/L in a depolarizing solution (mM: 110 KCl, 1.5 MgCl2, 0.8 CaCl2, 0.2 EDTA and 10 HEPES, pH 7.1) for 30 min on ice. Fluorescence emission and ionic current were recorded simultaneously on a custom rig that combines cut-open voltage clamp and an epifluorescence upright microscope (FN1, Nikon) via a 40X water-immersion objective with 0.8 NA (CFI Plan Fluor, Nikon). A green, high-powered LED (Luminus, PT-121) provided the excitation source and was controlled through a driver (Lumina Power, LDPC-20-6-24VDC) by Clampex software. The emitted fluorescence signal was detected by a photodiode (PIN-040A, United Detector Technology) which then was amplified by a patch clamp amplifier (Axopatch-200A, Molecular Devices). The recording was repeated about 7–10 times for each cell to average the fluorescence traces recorded. The internal solution was (mM): 105 NMG-Mes, 10 Na-Mes, 20 HEPES, and 2 EGTA, pH 7.4, and the external solution contained (mM): 25 NMG-Mes, 90 Na-Mes, 20 HEPES, and 2 Ca-Mes₂, pH7.4.

The optoacoustic excitation was performed using an Nd:YAG pulsed laser operating at the wavelength of 532 nm (Optogama, “WAVEGUARD”) with a pulse width of 1 ns, a repetition rate of 1 kHz, and pulse energy of $80\mu \mathrm{J}$ [532 nm laser, Fig. 2(b)]. The laser beam was attenuated to reduce the energy [A; Fig. 2(b)] then guided through two mirrors [M; Fig. 2(b)] and coupled using an aspheric lens [L; Fig. 2(b)] to single mode fiber with a core size of $3.2\mu \mathrm{m}$ (P1-460B-FC-1, Thorlabs). The average pulse energy at the fiber output was $\sim 400\mathrm{nJ}$ . After collimation, the light was focused using a collimator comprising plano-convex and aspheric condenser lenses, followed by an objective lens with an NA of 0.3 (CFI Plan Fluor, Nikon), resulting in a focal width of $1.3\mu \mathrm{m}$ [Fig. 2(c)]. The scanning of the optical beam was performed by mechanically translating the imaging head using X-Y-Z mechanical stages (XY: M-126.2S1, Z: M-112.12S, PI). For each position of the optical beam, the resulting acoustic signals were detected by SPADE, which was static during the measurement. Imaging was performed in both epi- and trans-illumination configurations, as shown in Figs. 2(d) and 2(e), respectively.

Successful fertilization was assessed using an inverted microscope (TE2000U, Nikon USA, Melville, NY, USA) equipped with 2 × , 4 × , and 10 × objectives (Nikon CFI Apo), and 20 × and 40 × objectives (Nikon Polarized optics CFI Plan Fluor) [36 (link)]. In preparation for embryo transfer, patients received 50 mg of intramuscular progesterone supplement daily, starting from the day after ovulation. The overwhelming majority of patients (n = 26) underwent single embryo transfers of blastocysts on day 5, while the remaining (n = 4) transferred 2 embryos at the cleavage stage, on day 3. None of the couples included in this study opted for PGT-A testing. Serum βhCG levels were measured between 10 and 14 days post-embryo transfer. Clinical pregnancy was defined as fetal heart activity (+ FHB) detected on ultrasound at 7 weeks of gestation.

Cell samples were prepared in the same way as for high-speed video tracking. After dilution, cells were located in Chambered #1.0 Borosilicate Coverglass System (Lab-Tek). Phase images of bacteria that attached to the glass surface were recorded with 100x oil immersion objective (Nikon CFI Plan Fluor) at 50 frames per second.

Built on a research quality, vibrationally isolated 4′ × 8′ optical table, the system is constructed around a Nikon Eclipse Ti-U inverted microscope base using a × 60/1.4NA CFI Plan Fluor oil immersion objective. The microscope base is outfitted with a precision, 2-axis stepper motor sample stage (Optiscan II; Prior) and a custom-designed confocal optical bench with three, independent detection channels. Each detection channel is configured with an optimized band-pass filter set for wavelength selection and a low-noise, single photon counting APD unit (SPCM-AQRH-15; Excelitas). Photon pulses are collected and time stamped with either a multichannel hardware correlator (correlator.com) or high speed TTL counting board (NI9402; National Instruments). Sample excitation is provided by either one or a combination of three lasers: two diode lasers (488 and 642 nm; Omicron) and one diode-pumped solid state laser (561 nm; Lasos). The free-space beams of each laser are each coupled to a three-channel fibre combiner (PSK-000843; Gould Technologies) and the combined output is directed into the sample objective with a custom, triple-window dichroic filter (Chroma). Each laser is addressable from the integrated control and data acquisition software, custom developed using LabView (National Instruments).

All voltage-clamp fluorometry (VCF) experiments were carried out on fluorophore-labeled cysteine mutants. Oocytes were labeled in the dark with 5 μM CF488- (Biotium) or AlexaFluor488 C-5 maleimide (Invitrogen) for 15 min at room temperature. VCF experiments were done in a RC-26Z recording chamber (Warner Instruments). The VCF setup was equipped with an Intensilight mercury lamp (C-HGFI; Nikon). A 40× Nikon oil-immersion objective (CFI Plan Fluor; Nikon) was used to detect the fluorescence signal emitted by the labeled oocytes. The optical signal was measured by a photodiode (S1336-18BQ; Hamamatsu Photonics) coupled to the headstage of an amplifier (List EPC-7; HEKA). An offset device was used to adjust and measure the offset of the signal, allowing the measurement of the total fluorescence intensity. Changes in fluorescence intensity (ΔF) were normalized to the total fluorescence signal (F).