A series of experiments have been conducted to characterize the designed PPOCT system. These experiments were carried out on a tissue phantom constructed by embedding a black human hair (diameter ~110 µm) in chicken breast tissue. The hair was embedded at an angle relative to the tissue surface to enable continuous variation in the hair tissue depth. Black hair in particular contains eumelanin. Eumelanin is also found in the eye and skin.
is a series of OCT and PPOCT cross-sections recorded with the hair at various depths in the chicken breast. The pump was modulated at 6 kHz. The average power of the pump and probe was 2.2 and 2.5 mW, respectively, with the pump leading the probe by 280+/− 40 ps. The line rate was 12.2 kHz with a 50 µs integration time. The label above each column corresponds to the tissue depth of the
Fig. 3 PPOCT and OCT images of a human hair embedded in chicken breast at various depths in the tissue. The depth corresponds to the center of the hair. The scale bar is 100 μm. For the 315 mm depth both pump on and pump off images are shown while only (more ...)
center of the hair. The 319 µm image was generated using 500 OCT lines per PPOCT A-line. All others used 1000 lines. The second image in the PPOCT row was recorded with the pump beam blocked. In order to facilitate direct comparison of the PPOCT images in , each image was placed on an signal (S) to noise (σN) dB scale (SNR = 20log(S/σN). The standard deviation of the noise was calculated by finding the standard deviation of the signal above the tissue surface, which should be a good approximation to the noise floor.
The PPOCT images show strong signal from the embedded hair. Only the signal from the hair is pump dependent, as evidenced by the pump blocked image. The residual signal when the pump is blocked is due to intensity noise in the laser source at the pump modulation frequency.
The hair was visible in the PPOCT image at the largest depth measured, 725 µm center depth and 780 µm maximum depth. The PPOCT signal should be more strongly attenuated as a function of depth than the OCT signal because of the much stronger attenuation of 415 nm light in tissue than 830 nm light. The images in appear to be in qualitative agreement with the expected trend. Ultimately the choice of pump wavelength is a trade-off between absorption cross-section of the target chromophore and tissue scattering.
is a plot of OCT, PPOCT, and PPOCT background (pump off) A-lines corresponding to the center of the hair in the 319 μm images. All three A-lines have been placed on an absolute SNR dB scale in order to facilitate a quantitative comparison of signal strengths. At the tissue surface indicated by the left most arrow the OCT, PPOCT, and PPOCT background SNR was 38 dB, −4 dB and 12 dB, respectively. While there is a fairly strong OCT signal, the PPOCT and PPOCT background signals are very near the noise floor. At the peak signal in the hair indicated by the right most arrow the OCT, PPOCT, and PPOCT background SNR was 63 dB, 49 dB, and 29 dB, respectively. If we subtract the PPOCT background from the PPOCT signal we find that there is 20 dB of background free PPOCT signal. The OCT signal is 43 dB stronger than the background free PPOCT signal.
OCT (blue), PPOCT (green) and PPOCT background (red) A-lines on an SNR dB scale. The A-lines were taken from the 319 μm B-scans. The PPOCT background corresponds to the pump off image.
The series of images in
were generated using a single data set (319 µm depth, ). Images in each column utilize a different number of OCT lines/PPOCT A-line thereby changing the effective PPOCT A-line rate. The columns are labeled with the effective line rate, 24.4 Hz, 244 Hz, 1220 Hz, and 2440 Hz which correspond to 500, 50, 25, 10, and 5 OCT lines/PPOCT A-line. At 50 A-lines per image in , the total acquisition time varies from 2 s to 20 ms. While we clearly loose signal to noise at high line rates we are able, with the current system, to measure PPOCT images at line rates exceeding 1 kHz. This represents a more than 1000 fold improvement over the previous time-domain system.
Fig. 5 OCT (grayscale) and PPOCT B-scans of a human hair embedded in chicken breast, recorded at different PPOCT line rates. In all cases the OCT line rate was 12.2 kHz, hence 24.4 Hz corresponds to 500 OCT lines/ PPOCT line. The scale bar is 100 μm. (more ...)
We have also investigated the pump power and modulation frequency dependence of the signal in the range of 0.1-2.7 mW average power and 1-6 kHz, respectively, using a black human hair as a sample. Each point in
represents the integrated signal in a PPOCT B-scan of a black human hair divided by the integrated signal of the OCT B-scan. This algorithm was developed to improve the repeatability of the measurements (estimated at +/− 5%). The power dependence in panel A remains linear up to ~700 μW. The curvature after 700 μW indicates that we are approaching saturation of the state(s) responsible for generating the PPOCT signal. Average pump powers beyond ~3 mW will not substantially improve the SNR. Since the saturation intensity is independent of chromophore concentration these measurements may be used as a guide for imaging other eumelanin containing tissues with this system, e.g. the retinal pigment epithelium and iris.
A) Pump power dependence of the PPOCT signal measured with a black (eumelanin) human hair sample. B) Pump amplitude modulation frequency dependence of the PPOCT signal from a similar sample. The estimated error is ± 5%
The signal strength in visibly begins to drop off (10%) starting at 2 kHz. It continues its downward trend up to the highest frequency measured, 6 kHz, where the signal is down by ~70% from its maximum. The pump induced signal clearly has a long lived component that persists on the order of several hundred microseconds. If the signal was entirely due to transient absorption from allowed transitions (scheme 2) the chromophore should completely relax between pump pulse trains and therefore not be a function of the pump modulation frequency. This does not preclude the existence of short lived states which could also contribute to the pump induced signal. If the long lived component proves to be the dominant source of pump-probe signal, it may be possible to replace the 5 MHz Ti:Sapph laser with a continuous wave source. In general, continuous wave sources are less expensive, smaller, and more robust than the femtosecond laser used in this study; all properties that would be helpful in any effort to transfer this technology to a clinical environment.
Given the complexity and uncertainty of the melanin excited state manifold, it is not obvious a priori what specific mechanism or mechanisms are responsible for the pump-probe signal, i.e. scheme 1, scheme 2, scheme 3, and/or local heating. The phase of the PPOCT signal relative to the pump modulation can help narrow the options. We have measured the pump modulation while acquiring an OCT M-scan by directing the weak pump beam reflection from the dichroic mirror (DM in ) into a photodiode. The camera trigger was used to synchronize the digitization of the photodiode signal. An M-scan of the hair sample was collected in this manner. The strongest reflection in the M-scan was filtered (in time) around the modulation frequency with a digital bandpass filter (600 Hz bandwidth). The photodiode signal was processed with the same filter. A comparison of the unfiltered and filtered photodiode signal indicated that there was no inherent phase shift due to the filter itself. The filtered line in the M-scan and photodiode signal from a representative data set is shown in
over a 4 ms time interval. The average phase difference from eight data sets was 1.1π radians with a mean standard deviation of 0.1π radians.
Modulated pump signal (blue) and peak of the OCT M-scan (red), filtered around the pump modulation frequency.
The OCT signal amplitude is nearly perfectly out of phase with the pump, i.e. when the pump is on, the OCT signal is more strongly attenuated than when the pump is off. The π phase shift is consistent with schemes 2 and 3 and excludes scheme 1 as a significant contributor. It does not rule out the possibility that at least a portion of the signal is due to thermal effects which should have a similar phase relationship.
Signal due to thermal effects, either phase washout from the pressure (acoustic) wave or localized refractive index changes from heating, should persist over relatively long pump-probe delays because of the slow acoustic wave velocity (~1500 m/s) and slow heat dissipation. In 1 ns a pressure (acoustic) wave would travel ~1.5 μm, far less than one coherence length of the source, hence we would expect little change in signal originating from pressure wave phase washout. Similarly, a time scale of 1 ns is too short for dissipation of localized heating. Any reduction of PPOCT signal at delays of ~1 ns could then be reasonably attributed to transient absorption (schemes 2 and 3).
Since our optical delay line was not readily scanable we inserted two indexed mirrors which could easily be removed and replaced with minimal alignment in order to allow us to quickly measure signals with fixed pump-probe delays of 100 ps and 1.2 ns. A PPOCT A-line of a black human hair was recorded at both delays under similar conditions. The PPOCT signal was integrated and normalized to the integrated OCT signal. A comparison of the two delays indicated a decrease in the normalized signal of 11% at the longer delay, based upon the average of three measurements. This result is consistent with at least 11% of the signal arising from transient absorption. Furthermore, the mechanism is more likely akin to scheme 2 because the long ground state recovery times expected in scheme 3 due to the forbidden transition.
A consequence of the integrative nature of absorption is that reflections distal to the chromophore deposit in the tissue will also report on the attenuation due to the chromophore. The result should be PPOCT signal at all depths distal to the chromophore deposit. Such behavior is not evident in the images of , i.e. there is no apparent PPOCT signal below the depth of the hair. One explanation is the strong shadowing from the chromophore absorption masks this effect. There is clearly a shadow in the OCT image. This masking was also noted in previous work [3
]. An alternative explanation arises if the PPOCT signal can be attributed to thermal effects. Since over relatively short time scales the heating should be localized to the area of the tissue containing the chromophore, PPOCT signal due to thermal effects should be localized to that tissue. Unfortunately, we do not have enough information to distinguish the precise contribution of the several physical phenomena that may contribute to the PPOCT signal. Further experimental investigation is needed.
As an initial demonstration of PPOCT in a biological sample we have chosen the porcine iris which contains eumelanin. A fresh porcine eye from a healthy domestic pig (Sus scrofa domesticus) was obtained from the Department of Animal Science’s, E. M. Rosenthal Meat Science and Technology Center at Texas A&M University. In order to avoid the need to compensate for the refractive power of the cornea, the cornea and the aqueous humour in the anterior chamber were removed to expose the surfaces of the iris and the lens. A B-scan was recorded over a 2 mm span which included the junction of the iris and lens. Memory limitations with the custom LabVIEW ® software written to acquire PPOCT images necessitated the acquisition of the B-scan in five 400 μm sections which were merged in post processing. The data acquisition time for each segment was ~4 s. There are several vertical artifacts visible in the images of
due to the segmented acquisition. The incident average pump and probe power were ~2.5 mW and ~1.9 mW, respectively with a pump modulation frequency of 4.2 kHz. The integration time for the camera was set at 50 μs with a line rate of 12.5 kHz. The lateral sampling was 4 μm with 500 A-lines per M-Scan used to extract the PPOCT A-line. The pump-probe delay was set to ~120 +/− 40 ps. shows the OCT and corresponding PPOCT images.
Fig. 8 A) OCT image of the porcine lens and iris. The scale bar is 200 μm. B) PPOCT image mapping the melanin in the iris. The color bar is in dB SNR. C) PPOCT background image recorded without the pump radiation. The color bar is in dB SNR. D) Background (more ...)
The OCT B-scan image, , clearly depicts the iris and lens. The corresponding PPOCT image, , shows a strong signal from the iris, a melanin rich region, and weak (background) signal from the lens. A B-scan recorded without the pump, , showed similar signal strength from the lens. We may generate a background subtracted image by subtracting 8C from 8B. Since the speckle is not correlated between and (there was a short delay between acquiring the two images), before making the subtraction we had to reduce the speckle by spatially averaging (16 μm x 33 μm) both images. The subtracted image is in . The maximum SNR went up even though we are subtracting signal because of the spatial averaging. The background subtraction is not perfect, however in many areas where there should not be a PPOCT signal (around the lens) the background is completely removed. The maximum SNR of the lens signal stayed approximately constant between 8C and 8D, but the SNR of the iris went up by approximately 5 dB.
The OCT image is essentially a measure of the depth dependent reflectivity. The PPOCT image bears the same reflectivity information except as a product with the molecular dependent part of the signal (See Eqs. (1)
), and (3
)). We can therefore create a molecular image that is independent of the tissue reflectivity by taking the ratio PPOCT/OCT. The ratio image in was calculated via the following algorithm: The OCT image was spatial averaged to match the subtracted PPOCT image and normalized. The background subtracted PPOCT image (8D) was normalized and then thresholded at 4.0% of the maximum to avoid taking the ratio of noise. The resulting image is displayed on a false color scale where black is zero (thresholded) and above zero to 1 varies from blue to red. The ratio image is displayed on a linear intensity scale, in contrast to the other images which are displayed on a log intensity scale. It is interesting to note that the strong iridial surface signal in the PPOCT image is no longer the dominant signal in the ratio image. The signal in the ratio image is a much weaker function of depth.
A potential application of this technology is imaging melanoma in the iris and retinal pigment epithelium. Melanoma is the most common form of adult ocular cancer which originates in the melanin producing melanocytes. In order to image melanin in the eye we must adhere to the ANSI limits for ocular exposure. OCT is typically performed at or below 600 μW (@ 800 nm) which is safe for continuous viewing. The pump laser need only illuminate the eye after an initial OCT scan to choose the area of interest, hence the dwell time for the pump laser may be relatively short. If we assume we are able to acquire a suitable number of images for diagnosis in 1-10 s the maximum permissible exposure at the pump wavelength (415 nm) is 692 μW for a 1 s exposure and 390 μW for a 10 s exposure. Based on we would expect a PPOCT signal reduction of 50-80% based on the pump power reduction. This reduction could likely be compensated by system improvements such as a faster spectrometer. The imaging speed would also need to be improved or a retinal tracking system incorporated in order to avoid the motion artifact endemic to in vivo
imaging of the eye. The blue edge of the visible spectrum has poor ocular transmission [18
], hence the pump would also likely need to be moved to longer wavelengths in order to make it feasible to image the living iris and retina. We have moved the pump to 450 nm (same ANSI limits) with no significant drop in the PPOCT signal. Longer wavelengths were not possible at equivalent power with our current laser system. We believe that with system improvements and a longer pump wavelength it would be possible to image the living iris and retina.