Lucplanfln

The laser module consists of an Nd:YVO₄ laser source (Cougar, Time-Bandwidth Products, Switzerland). It produces 1064 nm at the source, and by frequency-doubling, we achieve the excitation wavelength of 532 nm. The laser is pulsed at a 20-MHz repetition rate, and the pulse width is 12 ps. The PicoHarp 300 (PicoQuant, Germany) device was used for TCSPC measurements. The excitation beam enters the microscope (Olympus IX71 inverted microscope, Germany) through a dichroic mirror (ZT532SPRDC, short pass 532 nm, AHF Analysentechnik, Germany) and illuminates the chip through the objective (60 × , UPLANSApo water immersion, numerical aperture (NA) = 1.2, WD = 280 μm, Olympus or 40 × LUCPlanFLN, NA = 0.6, WD = 3.00–4.20 mm, Olympus, Germany). The fluorescent emission passes the same lens and is filtered by a bandpass filter of 295–385 nm (DUG 11, Schott, Germany) right after the dichroic mirror. A photomultiplier tube (PMA 165 nm, PicoQuant, Germany) detects the emission. The droplet sorting event was captured by a customized upright microscope, as illustrated elsewhere [39 (link)].

Ependymal cilia were prepared as previously described [56 (link)]. The motility of ependymal cilia was observed under a 60x objective (LUCPlan FL N, Olympus) on a differential interference contrast microscope (BX53, Olympus) and recorded at 200 fps through a HAS-220 high-speed camera (DITECT, Tokyo, Japan).

To form microcolonies, 1 μL of bacterial culture was deposited onto soft TSA plates (0.7% agarose, wt/vol) and incubated at 37°C for 3 h. The thickness of soft TSA plates was controlled to be ~1 mm by adding 2 mL of growth media into a 60-mm petri dish. Bacterial microcolonies on agar plates were observed directly in phase contrast mode using an Olympus X71 inverted microscope with a 60×/0.7 Ph2 Air objective (Olympus LUCPlan FL N). The white light source was provided by an Olympus TH4-100 lamp. Digital images (672 by 512 pixels with a pixel size of 107.5 nm) were acquired by an ORCA-ER digital camera (Hamamatsu, Japan).

Single cells were identified in the bright field for imaging of selected areas by confocal Raman microscopy (WITec alpha 300 R, grating monochromator with f = 30 cm focal length, EM‐CCD grating). A 40× air objective with cover glass correction and a NA of 0.6 (Olympus LUC Plan FLN) was used. The 632.8 nm radiation from a He‐Ne laser with a power of 1.2 mW at the sample was employed for exciting Raman scattering. The integration time per pixel was 100 ms. For the localization of PD‐L1 on single cells, the cells were at first identified in the bright‐field. Next an area was selected for the SERS mapping and investigated with an integration time of 100 ms and a laser power of 1.2 mW at the sample. After performing the mapping experiments the recorded SERS spectra were processed. Raw spectra were smoothed via the Savitzky‐Golay algorithm (7th order polynomial, 31 points). Then a baseline correction (Whittaker‐Henderson procedure 29) was performed. Mean spectra with error bars were calculated using a MATLAB algorithm (shadedErrorBars). For each pixel the maximum intensity of the Raman peak centered at ca. 1590 cm⁻¹ was determined by using a Lorentzian line profile and a nonlinear least square algorithm in order to generate the corresponding SERS false‐color images.

The fixed Ciona larvae on a glass-based dish were observed by fluorescence microscopy with a 3CCD camera. A long-term Ca²⁺ transient was captured with pSP-CiVAChT:GCaMP6s or pSP-CiVAChT-H2B:GCaMP6s transduced Ciona embryos (N = 10, Figure 2; Figure 3., Supplementary Table S1). The same results can be obtained with either pSP-CiVAChT:GCaMP6s or pSP-CiVAChT-H2B:GCaMP6s (data not shown). An inverted microscope (Nikon Eclipse, IX71) with a 10×, 20×, 60× oil immersion objective lens (LUCPlanFLN) was used for fluorescence imaging with a U-MWBV2 mirror unit (Olympus, Shinjuku, Japan). SOLA LED light (Lumencor, Beaverton, OR) was used as a light source; fluorescence images were acquired with a 3CCD camera (C7800-20, Hamamatsu Photonics, Hamamatsu, Japan) and processed by the AQUACOSMOS software (Hamamatsu Photonics). Room temperature was maintained at 20°C. As a result, Ca²⁺ transients for both MN2L and MN2R, located at the Ciona motor ganglion region (Figure 1C), were continuously imaged from St.22 (mid tailbud II) to St.34 (late tail absorption) (N = 10, Supplementary Table S1). Ciona developmental staging (Hotta et al., 2007 (link)) was used to estimate the developmental stage from morphology and time after fertilization.