Mai tai hp

For both 10-day- and 6-week-old (P42) mice, two-photon imaging was acquired from the left S1 using a laser scanning system (LSM 7 MP system; Carl Zeiss, Oberkochen, Germany) with two types of water-immersion objective lenses (10 ×, numerical aperture (N.A.) 0.5; 20 ×, N.A. 1.0; Carl Zeiss, Germany) and a Ti:sapphire laser (Mai Tai HP; Spectra-Physics, Santa Clara, CA) operating at 950-nm wavelength. Fluorescence was collected using GaAsP photomultiplier tubes (Hamamatsu Photonics, Shizuoka, Japan)^{73 (link)}.

Fluorescence and time-resolved images were obtained using an LSM 880 (Carl Zeiss, Jena, Germany) equipped with a short-pulse femtosecond Ti: Sa laser Mai Tai HP with a pulse repetition rate of 80 MHz, and a duration of 140 ± 20 fs (Spectra-Physics, Milpitas, CA, USA) and a FLIM system for time-resolved microscopy (Becker&Hickle GmbH, Berlin, Germany). Fluorescent images of NAD(P)H were obtained with two-photon excitation of fluorescence at a wavelength of 750 nm, fluorescence was received in the range of 455–500 nm. Fluorescence images of FAD were obtained from pixels of at least 5000; all studies were carried out under constant conditions (37 °C and 5% CO₂). Next, we calculated the optical redox ratio (ORR = IFAD/INAD(P)H) using ImageJ 1.52p software (NIH, Bethesda, MD, USA). Using SPCImage software (Becker&Hickle GmbH, Berlin, Germany) we analyzed FLIM images and registered the following parameters: the fluorescence lifetimes (τ1, τ2 (ps)) and the lifetimes’ contributions (α1, α2 (%)).

We used a custom-built two-photon microscope controlled by HelioScan^{57 (link)}, equipped with a Ti:sapphire laser system (~100-fs laser pulses; Mai Tai HP; Newport Spectra Physics), a water-immersion objective (16 × LWDPF, 0.8 NA; Nikon), galvanometric scan mirrors (model 6210; Cambridge Technology), and a Pockel’s Cell (Conoptics) for laser intensity modulation. For calcium imaging, we chose the ratio-metric GECI YC-Nano140 as it allows for the correction of movement artefacts in the Z plane typically observed in awake recordings, which can otherwise give rise to the detection of false positive active cells from out of focus planes. Furthermore, YC-Nano140 has a near linear relationship between the amplitude of the calcium transient and the number of action potentials, as verified by single-cell electrophysiology^{27 (link)}, unlike the GCaMPs. YC-Nano140 was excited at 840 nm and fluorescence collected with blue (480/60 nm) and yellow (542/50 nm) emission filters for CFP and YFP fluorescence detection, respectively. Images were acquired at 15.6 Hz with 128 × 64-pixel resolution. This gave rise to a relatively small field of view, but acquired with a high frame rate to enhance the reliability of detecting neuronal firing. Single trials of 9 to 10-s duration were recorded at a time with 1-s breaks in between trials to allow for hard disk storage during inter-trial interval periods.

Multiphoton experiments employed a Leica SP5 MP scanning confocal/multiphoton system using a Leica DM6000 inverted microscope and HCX IRAPO L x25.0 NA0.95 WATER objective lens with Spectra Physics MaiTai HP tunable IR-pulsed and 561-nm CW lasers as light sources. N13 microglia cells within the chambered slide were maintained under optimal conditions (37 °C and 5% CO₂ in a semi-closed, humidified microscope incubator) throughout the experiment.
Experiments were performed using a sequential time-lapse mode at a single focal plane. Samples were alternately measured for fluorescence using the multiphoton laser tuned to 720 nm (1% power with 3 accumulations) and a 413–532-nm detection window, and then activated by scanning the sample a total of 50 times using the 561-nm laser with the transmission set to between 0% and 8% maximum power. Each complete cycle of detection and activation was completed in 27 s and immediately followed by the next cycle. Conditions were chosen to allow photooxidized molecules to be detected while minimizing the photobleaching effect of the multiphoton laser and maximizing the ¹O₂ generation. All photographs were processed and analyzed using the Fiji J software (Adobe Systems, San Jose, CA, USA) [28 (link),29 (link)].