As mentioned above, in the first step of the experiment a collimated laser beam was used to illuminate the ear of a rabbit and record the transmitted light in a hologram. In step two, the phase conjugate of the transmitted light was read out from the hologram and back-propagated through the ear. The transmitted signal was recorded in time and analysis of the signal decay yielded the time scale of the in vivo tissue perturbation. We repeated the measurement 0.5, 1, 2, 3, and 24 hours after the ear was excised, which revealed several perturbation mechanisms of different time scales.
shows the top view of the experimental setup. The collimated output of a solid state CW laser at 532nm (Spectral Physics, Excelsior) was split into three vertically polarized beams: a signal beam, a writing beam, and a reading beam. In the first step, shutter 1 and 2 were open and shutter 3 was closed to pass the signal beam and the writing beam. The signal beam illuminated the sample and the transmitted scattered light interfered with the writing beam inside the 45° cut, iron doped LiNbO3 photorefractive crystal to form a 3D holographic grating along the c-axis of the crystal. In the next step, shutter 1 and 2 were closed and shutter 3 was opened to pass only the reading beam, which counter-propagated through the crystal with respect to the writing beam and generated the phase conjugate of the recorded scattered wavefront. The transmission of the phase conjugate light through the sample was focused onto a CCD camera. The powers of the signal, writing and reading beams were 48mW, 48mW and 4.8mW, respectively. A 5 second recording time was used throughout the experiments. The reading process in photorefractive materials can erase the stored hologram. Using a power ratio of 1:10 between the reading and writing beams ensured a hologram life time of ~6 min.
Fig. 1 (a) Top view of the experimental setup of in vivo TSOPC. The light source is a solid state CW laser at 532 nm (Spectral Physics, Excelsior). M = mirrors, WP = half wave plate for 532nm, BS = beam splitter, S1,2,3 = shutters, SH = sample holder, L = lens, (more ...)
The ear of a New Zealand rabbit under deep anesthesia was gently held between two glass slides and mounted onto the sample holder. shows the reconstructed TSOPC images through the ear of the rabbit (histology shown in ) when the rabbit was alive (i, ii), 30 mins after euthanasia (iii), and through a tissue phantom of comparable scattering properties (iv). The images reconstructed through the ear of the euthanized rabbit (iii) and the tissue phantom (iv) were similar round spots ~68 micron in diameter as expected from the 1.5 mm input beam diameter and the 150mm lens in front of the camera. The images (i, ii) reconstructed through the live rabbit’s ear however deviated from the expected round spot, indicating that the scattering structures in the tissue varied during the recording process (5 sec) and distorted the hologram.
We were interested in measuring the sensitivity of the reconstructed TSOPC signal to tissue variation. To investigate the minimum length scale on which sample perturbations affected the TSOPC measurement, we mounted a 1.6 mm thick tissue phantom composed of polystyrene microspheres (1 micron in diameter, weight concentration 1.77%) suspended in a polyacrylamide hydrogel on a translational stage driven by a piezo actuator. The product of the scattering coefficient of the phantom and the sample thickness was µsL = 130. A laboratory-built laser fringe tracking system was employed to monitor the stage position with better than 30 nm accuracy. After the holographic recording (experimental step one), we displaced the sample and monitored the TSOPC signal variation.
shows the experimentally measured TSOPC signal decay as a function of sample displacement during TSOPC playback (experimental step two). Gaussian fitting (red line) yields a FWHM of 523 nm which is comparable to the wavelength (532 nm) of the laser used in the experiment. Based on this study, we expect that the vibration caused by heart beat should significantly affect the reconstructed TSOPC signal.
TSOPC signal vs. sample displacement during playback. The data are fitted with a Gaussian function (red line).
show the TSOPC signal decay curve measured through the living ear, as well as 0.5, 1, 2, 3, and 24 hours after the ear was excised. An exponential function a
exp(-t/τ) was used to fit the decay and yield the decay constant τ. As we predicted from (i, ii), the sample perturbation in a living rabbit ear ( τ = 1.5 sec) was indeed faster than the holographic recording time (5 sec). After the excision, the decay time quickly increased and then gradually reached a plateau (τ = 0.5 min). To separate the decay caused by the tissue variation from other experimental factors including the hologram decay and the laser and mechanical instabilities, we performed the TSOPC experiment without a scattering medium present, from which we identified a hologram lifetime of 6 min. We then measured the TSOPC signal decay as a function of time for a polyacrylamide tissue phantom with µs
L comparable to the ear of the rabbit. The measured decay time (τ = 2 min) is due to the laser and mechanical instabilities of our setup.
(a-f) TSOPC signal decay measured when the rabbit is alive and 0.5, 1, 2, 3, 24 hours after the ear is excised. The data are fitted with an exponential function (red line).
After excision of the ear, the decay rate of the TSOPC signal initially drops quickly. We attribute this immediate change to the fact that the heart beat no longer affects the ear of the animal. The heart beat (i.e. pulse) appears to be responsible for tissue vibrations and bulk motion that move the tissue to a much greater length scale than the optical wavelength. An additional source of motion in the tissue is the microscale motion caused by cells undergoing active processes, varying their shape, size, and location over time. The ear tissue is alive upon excision, but gradually ceases activities as the tissue dies. After ~2 hours, the decay rate reaches a plateau. This plateau, however, is still much faster than the decay rate of the tissue phantom. We attribute this finding to the fluidic environment inside the tissues and associated Brownian motion that exists even when the tissue is not longer living. Unlike the tissue phantoms, the scattering structures of the tissue are not held in place by a hydrogel. Each of these factors can significantly perturb the time reversal process, and each of them has its own time scale.