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«Novel Biophotonic Imaging Techniques for Assessing Women’s Reproductive Health by Tyler Kaine Drake Department of Biomedical Engineering Duke ...»

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The index of refraction of the gel had previously been measured to be 1.34 using a refractometer, and the physical thickness of the gel can be related to OPL measurements by OPL = nt.

A linear correction coefficient can then be found for the gel measurements by dividing the slope by the refractive index of the gel, 1.3812/1.34 = 1.03, which is in agreement with previous results using the single channel instrument.26, 71 To demonstrate application to measuring gel atop tissue, a small amount of placebo gel is placed on top of a tissue phantom (optical properties described in Section 3.2.2), and a matrix of 6x6 points, with 1.0 mm spacing, is scanned with the mLCI system in order to measure local gel distribution.

The geometry of the scan pattern is shown below in Figure 3.7.

–  –  –

Typical A- scan data are shown below in Figure 3.8(a).

Peak A and peak B result from reflections from the front and back surface of the coverglass.

Peak C results from the gel and tissue phantom interface.

44

–  –  –

Figure 3.8:

(a) A- scan data from channel 1 of the phantom study and (b) mLCI scan of gel distribution on the tissue phantom.

Figure is from Drake et al.71 The optical path length between peaks A and B is determined to be 238 µ;m.

Since the coverglass has an index of refraction, n, of 1.5, the physical thickness, t, of the coverglass is found to be 159 µ;m by OPL = nt, in good agreement with the expected thickness of a #1 coverglass.

The OPL between peaks B and C, which corresponds to the path through the gel, is found to be 247 µ;m.

By using 1.34 as the index of refraction of the gel, the physical thickness of the gel is found to be 183 µ;m at this measurement point.

26 The complete 6x6 area scan is displayed in Figure 3.8(b).

The measurements have a mean value of 165.6 µ;m and a standard deviation of 14.2 µ;m, which shows good agreement with measurements with digital calipers of 160 ± 10 µ;m.

The benchtop mLCI system demonstrates the ability to measure gel thickness with high axial resolution without the need for an exogenous contrast agent.

The system responds in a linear fashion with increasing depth and demonstrates good OSNR.

The 45 axial resolution is found to be very close to the expected limit as governed by the coherence length of the SLD source.

Since all lateral A- scans of the measurements are recorded simultaneously, speed is improved as compared to a typical OCT or LCI system where each A- scan is recorded sequentially.

The Fourier domain approach also improves speed as the system is capable of capturing up to 1620 A- scans/s (270 Hz) compared to previous parallel time domain approach which operated at a speed of 112 A-  scans/s (14 Hz).49 The multichannel LCI system has a relatively sparse lateral sampling interval of approximately 1.0 mm, and a large lateral range of about 3.5 mm.

However, for measuring gel thickness distribution, the high axial resolution of the system along with the fact that there is no moving parts needed as a scanning mechanism, outweigh the sparse lateral sampling.

In the mLCI system, high data density is sacrificed for efficient sampling across a broad tissue area.

3.2.2 Clinical dual-modality optical imaging instrument With the knowledge gained from investigations using the benchtop mLCI system presented above, a clinical mLCI system was designed and constructed.

In order to translate the technology into the clinic, it was necessary to combine mLCI and fluorimetry into a single useable device to allow side by side comparison with an existing modality.

The device needed to have a footprint that was suitable for the typical OB/GYN examination room and needed to be easy to operate for a physician or technician with limited knowledge in the field of optics.

The instrument must also be 46 robust enough for repeated use without the need for optical maintenance and realignment.

The dual- modality optical imaging instrument features simultaneous imaging by fluorimetry and mLCI.

Figure 3.9 shows a basic diagram of the important optical components of the device.

Figure 3.9:

Diagram of the dual- modality optical imaging instrument, as taken from Drake et al.76 The mLCI system (left side) and fluorimetry system (right side) share the common components shown in the middle of (a).

Part (b) shows the endoscopic probe inside the polycarbonate tube;

PMT- photomultiplier tube.

Henderson et al.

has fully described the fluorimetry device and here the important operational features will be highlighted.27, 28 The fluorimetric system is built around a 4-  mm diameter medical- grade endoscope (Storz 27005CA;

Tuttlingen, Germany).

The endoscope both delivers the fluorescence excitation light and collects omitted light for measurement.

A 300- watt Xenon lamp (Wolf AUTO- LP 5131.001;

Vernon Hills, IL), 47 notch filtered to exclude the emission peak of fluorescein (500- 600 nm) provides excitation light.

The collected emission light is split into two signals.

One signal is routed to a video camera in order to provide visual context of the surface of the vaginal canal.





The second signal is routed via a fiber optic cable to a photomultiplier tube to provide quantitative information on fluorescence intensity.

The probe tip features a sealed well which is filled with distilled water to reduce reflections in the fluorimetry light path.

This feature also provides a thermal buffer between the vaginal tissue and the illumination probe tip.76 The clinical mLCI system is again based off of six parallel fiber optic Michelson interferometers.

A high- power, 15 mW SLD (λ0= 837.5 nm, Δλ = 54.2 nm, lc = 5.7 µ;m) is employed and the light is again split into eight channels, of which 6 are used, by a 1x8 tree coupler, as shown in Figure 3.10.

Six 50/50 fiber couplers are used to create separate reference and sample arms for each interferometer.

In the reference arms, light is collimated by fiber optic collimators and reflected of reference mirrors as in the benchtop system above.

Each reference arm also now includes a fiber optic shutter to enable gating of the reference signal for background subtraction.

48

–  –  –

In the sample arms, the fibers for each of the six channels are routed into a single protective PEEK (polyether ether ketone) sheathing and inserted into the base of the fluorimetric system’s endoscopic probe.

The sample arm fibers are again epoxied to a silicon v- groove chip (Figure 3.2), and this chip is now secured into a custom imaging module that is adapted to the distal end of the fluorimetric probe, as described in Figure 3.11.

49

–  –  –

Lens L1, collimates the light exiting the fibers and a 3 mm prism creates a 90- degree bend in the light path to create a side- firing probe.

Lens L2, then focuses the light through a 3.174 mm thick polycarbonate tube and onto the sample.

The average optical power measured at the sample for each channel is 0.624 mW ± 0.094 mW.

Zemax (Radiant Zemax, LLC;

Redmond, WA), an optical design software package, simulations of the geometry are shown in Figure 3.12.

50

–  –  –

The signal beam returned from the sample is recombined with the reference field for each channel at the corresponding 50/50 couplers and detected by the multichannel spectrometer as described in Section 3.2.3.

The same custom collimator mount from the previous system is also used for OPL matching.

The system operates at a speed of 48 scans/s in the clinical application, but the spectrometer is capable of reaching speeds of 1620 scans/s with optimization.71 The configuration in Figure 3.11 offsets the mLCI field of view by 180° azimuthally and 20 mm axially from that of the fluorimetry system, and this offset is compensated for in data processing.

The entire rigid endoscope is contained in a 27 mm diameter, 150 mm long, polycarbonate tube as shown in Figure 3.11(c).

The internal endoscope rotates and translates freely inside the outer tube, allowing measurements that span the length and circumference of the tube to be made.

This design allows for 51 broad tissue coverage without the use of electromechanical scanners typical of many OCT systems.

The completed mLCI system is housed on a 12”x12” optical breadboard, covered with an aluminum housing, and mounted to a stainless steel utility cart as, shown in Figure 3.13.

The clinical cart contains the

following:

PC, mLCI instrument, spectrometer, SLD driver, uninterruptible power supply, and positional encoder interface which is used to capture the positioning coordinates from the fluorimetric system for co- registration during data processing.77

–  –  –

A similar algorithm to that described in Section 3.2.3 is used for data processing with a few minor changes.

First, IR in Eqn.

(3.2) is captured before the endoscope is placed into the polycarbonate tube.

The endoscope is then placed into the tube and ID is 52 obtained.

Then, at each measurement point, computer controlled mechanical fiber shutters are closed and IS is obtained immediately prior to each data acquisition to minimize changes in the subtracted signal from Eqn.

(3.2).

Also, dispersion compensation is found with the polycarbonate tube in place to correct for chromatic dispersion due to the tube itself.

OSNR is again characterized for the clinical mLCI system using a mirror as a sample and utilizing Eqn.

(3.3).

OSNR values show improvement over the benchtop system, ranging from 87- 113 dB, and are similar to the published value of 106 dB achieved by Choma et al.

for a similar Fourier- domain OCT system.70 The OSNR values are summarized in Table 3.2.

Improvement in OSNR most likely come from better fusion splices in the system that resulted in decreased back reflections and lowered noise.

–  –  –

Theoretical axial resolution is found with Eqn.

(3.1) to be 5.7 µ;m for the new SLD, and the actual resolution values are shown in Table 3.2.

The increased resolution compared to theoretical expectation can be attributed to incomplete correction of chromatic dispersion form variations in the polycarbonate tube and spherical aberration 53 arising from off- axis focusing of the light in the lenses of the imaging module (Figure 3.10(a,b)).

Cross- talk is again investigated by leaving one channel connected to the source and measuring power in the adjacent channels.

The results from Eqn.

(3.6) are determined to be insignificant as the values were all less than

- 53 dB.77 Lateral sampling and imaging field of view are characterized for the clinical mLCI device.

The mLCI device’s imaging field was captured using a CCD and analyzed with ImageJ software (NIH).

Figure 3.14, below, shows the six measurement spots as imaged onto the CCD, as well as a Zemax simulation of the field from the geometry in Figure 3.

12.

–  –  –

Figure 3.14:

Spot size at mLCI focus.

(a) Measurement from clinical mLCI device captured with a CCD and (b) modeled field with Zemax.

The average spacing between the measured points is found to be 0.633 mm and the six spots cover 3.185 mm laterally, measured from the center of channel 1 to the center of channel 6.

The measured spots sizes for each channel in Figure 3.14(a) range from 41 µ;m in the center, to 100 µ;m for the outer channels.

These match fairly well to the simulation as shown in Figure 3.14(b), where the spot sizes range from 25.2 µ;m in the 54 center to 97.5 µ;m on the outside of the field of view.

The Zemax simulations also reveal that coma and spherical aberrations cause the optical distortions.

This could be partially compensated for by using custom designed aspherical lenses, as the device was built with off the shelf optics.

The custom machined calibration socket is used to test the accuracy and linearity of the clinical mLCI instrument.

This same test socket (Figure 3.15(a)) is used for calibration of the fluorimetry instrument, making it a suitable target to test both modalities against a single standard27, 28 A test gel, Replens, (LDS Consumer Products) is placed into the socket and measurements are taken in each of the five grooves with the mLCI instrument.

Measurements from the six channels are averaged and plotted against those from the digital calipers in Figure 3.15(b).

The slope is found to be 1.0082, and the correlation coefficient, R = 0.9947, indicates excellent agreement between the two measurements.

Again, the y- axis error bars are given by the axial resolution values in Table 3.2, and the x- axis error bars are given from the uncertainty of the digital caliper measurement.

These results are consistent with previous generation systems.26, 71 55

–  –  –

The calibration socket is translated axially and an A- scan is captured every 3.5 mm using a single channel (channel 3) to create a B- scan, or cross- sectional image, as shown in Figure 3.15(c).

The three deepest of the five wells are seen in the image, and Figure 3.15(d) shows a single A-  scan from the tomographic image captured at the blue line.

The peak at approximately 310 µ;m reveals the depth of the well.

3.2.3 Clinical application Once the mLCI imaging optics were adapted to the fluorimetric endoscope, simultaneous, dual- modality measurements of gel distributions in vivo are now possible.

A feasibility study was conducted using in vivo measurements of a test gel, Replens (n = 56 1.34) in a single human subject.

Replens is considered an accurate biophysical model of typical microbicide gel products that are currently being developed.18, 27, 28 In clinical application of the mLCI instrument, a physician inserts the endoscopic probe, which is lubricated with a thin layer of non- labeled Replens gel, into the subject’s vagina for a background scan.



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