Cfi75 lwd 16x w

After habituation and training, mice were used for Ca²⁺ imaging. Multiphoton fluorescence microscope (Femtonics, Budapest, Hungary) with a 16× NA 0.8 water-immersion objective (CFI75 LWD 16X W, Nikon, Tokyo, Japan) or with a 20× NA 0.8 water-immersion objective (XLUMPLFLN20XW, Olympus, Tokyo, Japan) was used for Ca²⁺ imaging in vivo. Ti:Sapphire laser (Chameleon Ultra, Coherent, USA) with an excitation wavelength of 920 nm was used to excite the fluorescence of GCaMP6f. Fluorescence signal from a brain area of 600 × 600 or 175 × 175 µm² (512 × 512 pixels) was recorded in resonant scanning mode at 30 frames/s. The signal was filtered with a 520/60 nm bandpass filter (Semrock, Rochester, NY, USA) and then detected with a GaAsP photomultiplier (H11706P-40, Hamamatsu, Japan). Simultaneously, autofluorescence was filtered with a 650/100 nm bandpass filter and detected with a second identical photomultiplier. Several imaging sessions of 10 min were carried out for each mouse. In parallel with Ca²⁺ imaging, the animal movements on the platform were monitored with locomotion-tracking software (Neurotar, Helsinki, Finland). All sessions were video recorded in infrared light. The experimental session did not exceed 2 h per mouse.

Two-photon laser scanning microscopy (2PM) was employed to validate the imaging depth of RBC-aided WFLM. The imaging was performed with a custom-built 2PM system^{50 (link)}, equipped with a tunable femtosecond laser (Chameleon Discovery NX, Coherent Inc, USA). In this study, 940 nm excitation wavelength was selected for FITC excitation. A 16× water-immersion objective (CFI75 LWD 16X W, NA = 0.8, Nikon, Japan) was used for the validation experiment. 3D structural imaging was achieved by 2D lateral scanning with galvo mirror and axial scanning of the objective with a piezo motor-driven linear stage at 2 µm step size. Backscattered fluorescence was collected by the same objective, passing through a band-pass filter (535/50 nm, Semrock, USA), and then focused on the photomultiplier tube (H9305-03, Hamamatsu, Japan). Image acquisition was performed with ScanImage (r3.8.1, Janelia Research Campus)^{51 (link)}.

Four weeks after the surgery, mice were imaged under an Ultima IV two-photon microscope from Bruker/Prairie Technologies^{56 (link)}, with a Nikon 16 × 0.8 NA water-immersion objective (model CFI75 LWD 16XW). The fluorescence of GCaMP6f and jRGECO1a was excited at 999 nm with a Spectra-Physics InSight DeepSee laser. All optical filters mentioned in the following description of the two-photon microscope are by Chroma Technology Corporation: after the collected light is reflected towards the detection unit by the main dichroic filter (ZT473-488/594/NIRtpc), the signal light enters the detector house (four channels), passing a ZET473-488/594/NIRm filter, that is shielding the photomultiplier tubes from reflective light. Inside the detector house, the light is then split into two fractions separated at a wavelength of 560 nm by a dichroic filter (T560lpxr). The green light (GCaMP6f) is further guided by a secondary dichroic beam splitter at 495 nm (T495lpxr) and filtered by a ET525/50m-2p bandpass filter, whereas the red light (jRGECO1a) is similarly guided by a secondary beam splitter at 640 nm (T640lpxr) and subsequently filtered by a ET595/50m-2p bandpass filter. The emitted photons were detected with Peltier cooled photomultiplier tubes (model 7422PA-40 by Hamamatsu Photonics K.K.). Images (512 × 512 pixels) were acquired at 30 Hz in layer 2/3 of barrel cortex.