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After choosing the optical design and aligning the system, the final step is to characterize the performance of Version 1 of the LF-OCT system. We do this by measuring the axial and transverse resolution, the SNR, and the axial and transverse pixel calibration (to get imaging depth and FOV). First, to calibrate to microns/pixel, we imaged a plastic ruler with millimeter tick marks.

Figure 2-14 LF-OCT transverse calibration of Version 1.

Millimeter tick marks on a ruler are used to calibrate the transverse distance (arrows indicate the positions of the ticks, which attenuate the beam and create a shadowing effect).

Taking the average of the OCT signal amplitude of every column yields a transverse profile with dips corresponding to the black, millimeter tick marks (because the black lines on the ruler do not back scatter as much light as the clear plastic). Knowing the physical distance represented by each peak in the transverse profile, we find the average pixel calibration to be 8.58 µm per transverse pixel. Recalling from the optical design that the beam diameter in the X

dimension of the sensor is only 8 mm (compared to the sensor size of 17.4mm), we are filling only 46% of the transverse pixels. This corresponds to a transverse FOV of 4.04 mm at the sample, compared with a theoretical value of 4.01 mm.

To calibrate the axial pixels, we image a flat, silver mirror. Because silver mirrors are nearly perfect reflectors (RS ~ 1), we make use of a neutral density filter (NDF) to avoid

in known increments and imaged at each depth. We then plot the row number in the image corresponding to the depth profile peak vs. the relative position of the mirror, Figure 2-15. Taking the slope of this line yields the axial calibration of 0.994 µm per axial pixel in air. For 512 spectral pixels (half of the 1024 array due to spectrum redundancy caused by the negative frequencies given by the inverse Fourier transform of the modulated interference spectrum), this corresponds to an imaging depth of 509 µm. The theoretical maximum imaging depth in air for the system is 524 µm.

Figure 2-15 Axial calibration curve for LF-OCT System Version 1.

A flat silver mirror is lowered by known increments from coherence zero using a micrometer- driven translation stage. The sample position is plotted as a function of the center A-line in each

image to obtain an axial calibration of 0.994 µm per pixel in air. From the images of the mirror, we noticed that there is a smile artifact in the

reconstructed images: the image of a mirror appears to curve downwards at the transverse edges of the frame. We computed the difference in row number between the peak in the depth profile of the center A-line compared to the peak in the depth profile of the most extreme A-lines and

y = 0.994x - 21.851 R² = 1.000 0 100 200 300 400 500 0 100 200 300 400 500 S a m p le P osi ti on ( µ m )

Row Number of Peak position (pixels)

found that the difference in row number of the peak was 2 pixels at depths up to 200 µm increasing to 5 pixels at 500 µm. The effect of such a curvature is distortion in the axial calibration for the transverse edges of the B-mode.

From the images of the flat silver mirror at various depths within the image, we also compute the SNR as a function of depth (see Figure 2-16). The images were recorded with a framerate of 1000 frames per second with an exposure time of 3.33 µs, and the total sample power incident on the NDF in the sample arm was ~400 mW. With the detector’s quantum efficiency of 0.4, this gives a theoretical SNR of 104 dB. The sample power was attenuated with an NDF to ~8 mW. With a ref power of ~ 2 mW, the system was not set up in the shot noise limited regime. (We did not characterize the operating conditions needed for the shot-noise limited regime until after the demo NKT light source had been returned.) The experimental SNR of the system is taken to be 10*Log10 of the peak intensity of the center A-line (the mean-

squared OCT signal) divided by the standard deviation of noise (which is the mean-squared OCT signal evaluated above and below the peak position). Adding back the 34 dB of optical

attenuation, the maximum experimental SNR was 88 dB at a depth of 60 µm with a roll-off of - 12 dB over ~250 µm in depth. We measured the power loss from every optical element in the collection optics, and found that the second pass through the beam splitter incurred a loss of 3 dB (as expected from a 50:50 beam splitter), the diffraction grating a loss of 10 dB (from the other diffraction orders), and the other lenses and mirrors a loss of 1.7 dB. The total losses of 14.6 dB in the collection optics brings the theoretical SNR down from 107 dB to 92.4 dB, in good agreement with our experimental SNR of 88 dB.

Figure 2-16 SNR vs depth for LF-OCT System Version 1. There is a roll-off of ~10 dB over ~250 µm.

To measure the axial and transverse resolution, Δx, we imaged the same TiO2 point scatterers used in the digital dispersion compensation algorithm. To measure the PSF of an optical imaging system, it is important to use point scatterers, which are particles with a mean diameter much less than the theoretical resolution. Because the physical dimensions of the point scatterer are smaller than the smallest resolvable element of the system, the intensity profile of the scatterer- the convolution of the system’s point spread function (PSF) and a Gaussian profile with FWHM equal to the point scatterer’s diameter- provides us a measurement of the system’s PSF, both axially and laterally, as long as the point scatterer’s diameter is much less than the system’s PSF FWHM. 76 78 80 82 84 86 88 90 0 50 100 150 200 250 300 S N R ( d B )

Distance from Coherence Zero (µm)

Figure 2-17 Image of Point Scatterers for Resolution Measurement.

Image of TiO2 point scatterers embedded in PDMS. Twenty-five scatterers were selected and the FWHM of the axial and transverse profiles recorded.

Arbitrarily, we chose 25 scatterers, all with intensity greater than 700 arb. unit as

illustrated in Figure 2-17. For each scatterer, we found the axial and transverse resolution (mean ± standard deviation) to be 2.4 ± 0.6 µm and 24 ± 3 µm respectively. The theoretical axial and in-plane transverse resolutions are 1.01 µm and 17.0 µm respectively. This means that the experimental axial resolution was worse than was expected. This may be explained by poor focusing of the lenses in the spectral plane (so-called, as in the right side of Figure 2-5, because this is the plane in which the beam is focused onto the spectrometer). The in-plane transverse resolution, Δx, is also worse than the theoretical lower bound; however, it is very consistent with the theoretical value for the out-of-plane transverse resolution, Δy, which is determined by the Gaussian beam waist focused by L4 (estimated to be 25.5 µm). We may be able to achieve an

experimental Δx closer to the theoretical lower bound with improved alignment techniques. Additionally, to check the spatial oversampling, with a magnification of 2, the size of Δx on the sensor is 2*24µm = 48 µm. With a pixel size of 17 µm, each resolution element is sampled by 2.9 pixels, so we are above the Nyquist sampling criterion. The Rayleigh range is estimated using the experimental value of 24 µm for Δx in equation 2-18; it is close to the Rayleigh range expected in Y because the experimental value of Δx is so close to the theoretical value of Δy.

We also used an image stack of the TiO2 point scatterers in PDMS to measure the phase resolution of the system. We choose three of the brightest scatterers located near the top of the B- mode and compute the standard deviation of the optical phase in time, from a stack of 1000 frames recorded at 1 kHz. The average of those three standard deviations is 0.175 radians, and this is our estimate of the phase resolution. The theoretical value is computed using the measured SNR from the TiO2 point scatterers which was 60 dB (with an exposure time of 167 µs and a sample power of 7.66 mW). The theoretical value of the phase resolution given a measured SNR of 60 dB is 0.124 radians.

One major concern about line-field OCT is the potential for crosstalk causing resolution degradation. Crosstalk occurs when multiply scattered light is recombined with light from one scatterer and is mapped to the same camera pixel. Most point-scanning OCT systems couple the light back into a single mode fiber, which acts as a confocal gate to reject multiply scattered photons. Because LF-OCT systems are free-space optical systems, they lack the same confocal gating, so crosstalk is a concern. The experimental value of Δx being larger than the theoretical could be an indicator of crosstalk; however, because the measured value is very close to the upper bound given by Δy we would not say that resolution degradation is a huge problem for this system. Indeed, we found in later optical designs and alignments that the experimental value of

Δx was in excellent agreement with the theoretical value. Possibly, the SC source has lower spatial coherence than the lasers and SLDs typically employed in point-scanning OCT systems. These results are summarized in Table 2-5.