Gvsm002

In vivo imaging was performed on a custom-built two-photon setup based on a Zeiss upright microscope (AxioExaminer Z1) equipped with a 25 × water immersion objective (NA 1.05, WD 2 mm, Olympus XLPLN25XWMP2). Femtosecond pulses from an ultrafast Ti:Sapphire laser (Newport, Tsunami) whose intensity was modulated using a half-wave plate (Thorlabs, AHWP05M) and a polarizer (Thorlabs, GL10-B) is used as light source. The excitation beam is raster scanned using a galvo scanning mirror (Thorlabs, GVSM002) before entering the microscope body and is focussed on the imaging plane using the objective lens. The fluorescence that is collected by the objective lens in an epi-illumination geometry is then separated using a dichroic before being detected by photomultiplier module (H7422, Hamamatsu Corporation, Japan).
A low noise current preamplifier (Stanford Research Systems, SR570) was used to amplify the photomultiplier tube photocurrent, which was further digitized using a data acquisition board (National Instruments, PCI-6110). ScanImage (r 3.8.1) software was used to interface instrument control and generation of galvometric scan command. Image acquisition was accomplished using a custom Matlab script interfaced with z-drive of the microscope. The digitized signal was analysed using Matlab, Origin and ImageJ for further analysis.

Synchronized pump and Stokes beams are provided by a picoEmerald S system from APE (Applied Physics & Electronic, Inc.) The fundamental IR fiber laser at 1031.2 nm with 2-ps pulse width and 80-MHz repetition rate is served as the Stokes beam. The pump beam is provided by incorporation part of fundamental laser to OPO which is tunable from 700 to 990 nm. Spatially overlapped pump and Stokes beams were expanded and coupled into an Olympus IX71 microscope to overfill the back-aperture of the objective by passing through a dichroic mirror (FF825-SDi01, Semrock). A 60× water immersion objective (1.2 N.A., Olympus UPLSAPO) was used for all imaging experiments. All laser power mentioned were measured after objective. Laser scanning was accomplished by a 2D galvo scanning system (GVSM002; Thorlabs). For SREF detection, fluorescence signal was detected on the backward with a high-efficient single photon counting module (SPCM) (SPCM-NIR-14-FC; Excelitas). The 100-μm active area diameter of avalanche photodetectors (APD) forms a loose confocal configuration. Two high-OD bandpass filters (FF01-729/167-25; Semrock) were used to block the reflected excitation laser beams and another two high-OD bandpass filter (FF01-735/28-15; Semrock) were utilized to block the CARS background. A home-written LabVIEW program was used to control the galvo scanning and data acquisition.

The coherent fibre bundle (core diameter: 12 μm, lumen diameter: 2.70 mm, outer diameter: 3.15 mm, minimum bend radius: 450 mm; Fig-50, Fujikura, Japan) achieved a one-to-one mapping between the spatial distribution of excitation light at the proximal end and ultrasound transmissions at the distal end. Via a pair of orthogonal galvo-mirrors (GVSM002, Thorlabs, Germany), collimated excitation light (beam diameter: 2.6 mm) was focussed (focal length: 50 mm; AC254-050-A, Thorlabs, Germany) onto the proximal end of the fibre bundle. Using this scanning configuration, a light spot was projected on the optically absorbing coating, which could be arbitrarily positioned across the distal end face to scan a finite acoustical source aperture.