PAM-OCT setup

PAM and OCT can be performed simultaneously or consecutively by using a sin- gle light source in a combined system. By using a SC source, a different spectral band can be used for each modality. The SC source used in section 5.1.1 is used here as well. The energies available for sPA presented in Fig. 5.3a are not sufficient to perform MPAM on biological samples. The imaging range would be limited to few micrometres and little signal will be detected. Therefore, only PAM and OCT are performed. Figure 5.7 is a sketch of the setup. Both modalities have been char- acterised, then the system has been used to image a mouse ear in-vivo. By using visible light for PAM, the blood vessels are exclusively imaged, near-infrared (NIR) light is used for OCT to image the structure of the ear. The results are presented in section 5.3.3.

FIGURE5.7: Sketch of the setup used for PAM-OCT at Northwestern University. AMP: electrical amplifier, C: 50/50 coupler, DC: dispersion compensation, DM1, DM2, DM3: dichroic mirrors, L: collimation lenses, M: mirrors, Obj: objective lens, PC: polarisation controller, PH: pinhole, T: telescope to adjust the focus on sample.

⋆ Triggered together.

Light source

As shown in Fig. 5.7, dichroic mirrors are used to separate the SC spectrum in a visible band (500-700 nm) and a NIR band (800-900 nm) for PAM and OCT, respec- tively. The visible band has a short wavelength edge of∼ 500 nm due to the blue edge of the SC source, the longer wavelength edge is determined by the dichroic mirror (DM1) cut off wavelength (775 nm). The NIR band consists of the band transmitted by DM1 and reflected by DM2 that presents a cutoff wavelength of 950 nm.

Setup

The NIR band is coupled into a 50/50 fibre coupler (FUSED-22-850, OZ optics) which splits the beam into a sample arm and a reference arm. The light scattered back from the sample interferes with the light reflected by the mirror in the ref- erence arm, a homebuilt spectrometer is detecting the signal. The spectrometer consists of a diffraction grating (1200 lines/mm @ 840 nm, Wasatch Photonics), a focusing lens (AC508-150-B, Thorlabs) and a line camera (spL2048-140km, Basler). The visible beam used for PA excitation and the NIR beam used for OCT are com- bined by a dichroic mirror (DM3, cutoff wavelength 785 nm) then, reflected by a pair of galvo-scanners (QS-7, Nutfield Technology) that allow transversal scanning and, focused on the sample by an objective lens of 30 mm focal length (AC254- 030-B, Thorlabs). Glass is introduced in the OCT reference arm to compensate for dispersion introduced in the sample arm. A telescope permits to adjust the focus in depth (vertical) of the visible beam on the sample to be superposed to the NIR fo- cal position. 10 mW of light is measured after filtering (after DM1 and DM2) in the visible band and 6 mW in the NIR band. However only 3 mW (120 nJ) is measured on sample for PAM (visible band) and 0.25 mW for OCT (NIR) due to losses in the setup. These values are below the MPE, according to section 2.2.5 (chapter 2). Fig- ure 5.8 represents the spectra of the visible and the NIR bands after filtering and of the visible band on sample. The sample is mounted on a three-dimensional trans- lational stage that allows alignment to choose the imaged area. A specific holder can be implemented to accommodate the mouse when the imaging of its ear is per- formed. The same transducer as that in section 5.1.1 is used here with the electrical amplifier. A drop of water is added on the surface of the sample to allow cou- pling of the ultrasound signals generated with the transducer. The transducer is

FIGURE5.8: Spectra of the visible and the IR bands after DM1 and DM2 respectively, and of the visible band on sample.

mounted on a three-dimensional translational stage to optimise its alignment. The system performs in reflection and not in transmission like in section 5.1.1.

Signal processing

The PA signal is digitised at 200 MHz and recorded by an acquisition card (CS1622, Cage). An analogue output board (PCI-6731, National Instruments) was used to synchronise the raster scanning, the PAM and OCT acquisitions with the trigger of the SC source. Because of the limited amount of analogue outputs, PAM and OCT could not be performed simultaneously. The source is operated with a PRF of 25 kHz and synchronised with the scanners that give an A-scan rate of 5 kHz. The exposure time of the spectrometer is fixed to 190 µs and synchronised with the scanning. At each position, the PAM A-scan is taken as the average of five consecu- tive A-lines. Each A-line is the absolute Hilbert function (envelope) of the temporal electrical signal (see details of signal processing in section 2.2.4, chapter 2). For OCT, the light from five pulses is integrated to obtain an A-scan. The signal re- construction for OCT is based on the use of a FFT (section 2.3.5, chapter 2). The algorithm was developed by the group of professor Hao F. Zhang at Northwestern University.

Characterisation

The theoretical axial and lateral resolutions of PAM and OCT are calculated by us- ing formula 2.2, 2.4, 2.17 and 2.19 (chapter 2), respectively. The numerical aperture is defined for each spectral band by:

N A = φ

2· f, (5.1)

where φ is the beam diameter before the objective lens of focal length f . Here, f = 30 mm. φ depends on the spectral band used, and is estimated to be∼ 2.5 mm for the visible band and ∼ 1.8 mm for the NIR band. In the visible, a telescope magnifies by a factor two the beam collimated at the output of the SC source that

Imaging modality PAM OCT

Theoretical lateral resolution [µm] 8 10.5 Experimental lateral resolution* [µm] 8.8 13.9 Theoretical axial resolution [µm] 42 4.25 Experimental axial resolution [µm] 51 4.6

TABLE 5.1: Theoretical and experimental resolutions. *: taken as twice the line width of the smallest element resolved on a USAF target.

(a) (b)

FIGURE 5.9: (a) OCT of a paper 1951 USAF resolution test chart with summed voxel projection and B-scan corresponding to the position indicated by the orange line and, (b) PAM of a positive 1951 USAF test target (R1DS1P, Thorlabs) with MAP and B-scan corresponding to the position indicated by the green line. Scale

bars: 150 µm.

presents a beam diameter of ∼ 1.25 mm at 650 nm (1 mm at 530 nm, 2 mm at 1100 nm, according to datasheet of SuperK COMPACT, Appendix B.2, the same collimator is used with same PCF core diameter). In the NIR, in the OCT channel, the beam diameter is calculated by considering that the collimator used presents a focal length of 7.5 mm (PAF-X-7-B, Thorlabs) and the fibre has a NA of 0.12. For the visible and the NIR bands, 650 nm and 850 nm are respectively taken as central wavelengths. A 1951 USAF resolution test chart is imaged to estimate the experi- mental lateral resolution of each imaging modality. Figure 5.9, adapted from Fig. 2 of paper [2] (Appendix A.2), represents the OCT summed voxel projection and the PAM maximum amplitude projection (MAP), with B-scans. With PAM the three lines could be resolved down to element 6 of group 6, and to element 2 of group 6 for OCT. The lateral resolution is taken as twice the width of the line that corre- sponds to the smallest element that can be resolved. Theoretical and experimental lateral resolutions are presented in Table 5.1. For the calculation of the theoretical PAM axial resolution, the speed of sound is taken as 1.5 mm/µs and the band- width of the detector is taken as 90 % of the central frequency: 31.5 MHz. The 1951 USAF resolution test chart coloured part (black) is highly absorbing and less than 5 µm thick, which is considered small in comparison to the PAM axial resolu- tion. Therefore, the PAM experimental axial resolution is taken as the full width at half maximum (FWHM) of the A-scan generated (expressed in s) multiplied by the speed of sound. The axial resolution of the PAM setup is presented in Table 5.1. For the calculation of the theoretical OCT axial resolution, 850 nm is taken as the cen- tral wavelength with a FWHM of 75 nm. The experimental OCT axial resolution is the FWHM of the A-scan generated by placing a mirror as the sample. The results are presented in Table 5.1, and very little deviation is observed between theoretical and experimental values. For OCT, a sensitivity of 77 dB is measured and a signal

attenuation of -3 dB at 0.55 mm, measured in air (roll-off of -10 dB/mm). Both OCT and PAM present characteristics that reach state-of-the-art results.