3.1.

Pouch Design

The heart of the system is a flexible polymer pouch filled with a mixture of a silicon-based elastomer and small silica particles (Fig. 2 ). The pouch consists of four distinct polymer layers (UltraFlex Packaging Corp, Brooklyn, New York) [Fig. 2a]. The top layer (1) is made of optically transparent polyester, while the bottom layer (4) is made of a metallized polyester, which reflects light back into the medium. Layers 2 and 3 are adhesive layers of linear low-density polyethylene (LLDPE) that were used to create a seal between layers 1 and 4 when heated. The scattering medium is located between layers 2 and 3. The bare end of the fiber is inserted into the medium in the pouch, where light emanates from the bare end of the fiber cable, is then scattered throughout the pouch medium, and eventually exits through the top transparent layers of the pouch.

Fig. 2

Pouch detail. (Top) View of pouch from above. Fiber-optic light supply enters pouch from the left. (Bottom) Side view of pouch showing multilayer structure and scattering media.

The pouch is fabricated by layering the polymer sheets in their appropriate order over a rubber mat, after which the edges are sealed using a hot stamp heated to a temperature of $275\text{to}300°\mathrm{C}$ and moderate hand pressure for $5\mathrm{s}$ . A thin sheet of Teflon between the stamp and the polymer layer prevents adhesion to the stamp. Two pouch sizes were fabricated; the first pouch was $20\times 20\mathrm{mm}$ by $3\mathrm{mm}$ thick and was used for the optical characterization. A second smaller pouch $10\times 10\mathrm{mm}$ by $2\mathrm{mm}$ thick was fabricated and used for the animal studies.

3.2.

Scattering Medium

The scattering medium is a silicate powder of mostly silica composition prepared via the solvent evaporation method using an acid solution [ethanol, hydrochloric acid, and (high-performance liquid chromatography HPLC)—grade water] with tetraethylorthosilicate (TEOS) as the silica source. The solution was heated on a hot plate on medium heat to evaporate all liquid, leaving a solid layer of silicate; this process took roughly $1\mathrm{h}$ . The silicate was removed and crushed using a mortar and pestle for $10\min$ to produce a fine powder. The silicate particle morphology was imaged by sputter-coating powder samples with $\sim 12\mathrm{nm}$ of gold in an Edwards S150b coating unit (Wilmington, Massachusetts) and placing in an LEO 1550 scanning electron microscope (SEM; Thornwood, New York) operating at $20\mathrm{kV}$ with a Robinson backscatter detector and a working distance of $9\mathrm{mm}$ .

The scattering medium was mixed into the PDMS silicone elastomer (Sylgard 184, Dow Corning, Midland, Michigan) precure (10:1 base:curing mass% mixing ratio) with concentrations of 20, 30, 40, 50, and 60 mass% silicate particles. The pouch was filled with the liquid mixture immediately after mixing using a syringe, after which it was placed in a vacuum desiccator for $4\mathrm{h}$ at $65°\mathrm{C}$ to remove air bubbles and cure the elastomer. Last, the bare end of the optical fiber was inserted into the pouch and secured with double-sided tape wrapped around the fiber.

3.3.

Specimen Preparation and Microscopy

The hamster cheek pouch tissue was chosen to evaluate this light source for two reasons. First, it is similar in thickness to the hamster gracilis muscle $\left(400\text{to}800\mu \mathrm{m}\right)$ , which is a tissue for which this light pouch is particularly well suited. The total transmittance of light through the cheek pouch is similar to that through the gracilis. Second, the cheek pouch tissue is readily exteriorized without compromising the vasculature. Thus, a comparison of the same vascular location is possible using standard bright-field transmitted light and the fabricated light pouch.

All animal studies were performed with the approval of the Animal Care and Use Committee for Stony Brook University. Adult male hamsters ( $124\pm 12\text{grams}$ , $N=4$ ) were anesthetized (pentobarbital sodium, $70\mathrm{mg}∕\mathrm{kg}$ ) and tracheostomized, and the right cheek pouch was prepared for intravital microscopy.¹¹ Briefly, the tissue was gently everted from the lateral side of the mouth and pinned over a quartz pillar; typically, the bright-field light passes through the quartz pillar and tissue for observation. The cheek pouch tissue was observed using a modified Nikon upright microscopy with a $25\times \text{objective}$ (Leica, $\mathrm{NA}=0.65$ ), using both a collimated bright-field light source (halogen lamp) with a long working distance condenser and the light pouch device. The light pouch $(10\mathrm{mm}\times 10\mathrm{mm}\times 2\mathrm{mm})$ was slid under the cheek pouch tissue between the tissue and the quartz pillar. Images were captured using an intensified charge coupled device (ICCD) camera (Solamere Technology Group, Inc.) and videotaped with an SVHS Panasonic AG 7350 recorder. Analog images were digitized offline (EPIX, XCap, Inc.), and blood vessel diameters were measured using ImageJ (freeware, NIH). Comparisons of diameters for the same vascular location with the different light sources were made using a paired $t$ -test with $\alpha =0.05$ .

4.1.

Scanning Electron Microscope Analysis of Silicate Morphology

Scanning electron microscope (SEM) images of the silicate powder at different magnifications are shown in Fig. 3 . The powder consists of small granular particles of varying size. The actual size distribution depends on the crushing operation. For this particular sample, the larger particles have a mean edge length of about $150\mu \mathrm{m}$ , with a standard deviation of $24\mu \mathrm{m}$ , as estimated from Adobe Photoshop. For the higher magnification figures [Figs. 3c and 3d], it can be seen that the larger particles have a highly angular morphology that corresponds to rhombohedral cleavage, characteristic of quartz. The smaller particles are also angular; however, they are more equi-axed than the larger particles [Figs. 3c and 3d]. The mean edge length for the smaller particles was $6.5\mu \mathrm{m}$ , with a standard deviation of $0.99\mu \mathrm{m}$ .

Fig. 3

SEM images of the silicate particles that were used to scatter the incoming light in the pouch. Magnifications of (a) $125\times$ , (b) $\mathrm{1,000}\times$ , (c) $\mathrm{3,000}\times$ , and (d) $\mathrm{30,000}\times$ .

4.2.

Characterizing Illumination Uniformity

Silicate particle morphology, size, and concentration play a strong role in the light scattering process. As some of the particles are on the order of microns in size, Mie scattering theory holds,¹² but it is complicated by the nonspherical nature of the silicate particles.¹³ In this work, the optimal (most uniform) light distribution was determined empirically by fabricating devices with different silica concentrations, supplying light, and visually determining the optimal concentration.

Figure 4 shows a top view of the illuminated pouch for particle concentrations of 20%, 30%, and 40%. The fiber enters the pouch from the left side. For the 20% silicate concentration (Fig. 4, left), much of the light emitted from the fiber end strikes the pouch edges, where it is either reflected, transmitted, or absorbed, as seen on the left of the top row in the figure for the 20% concentration. The highest silica concentration of 40% (Fig. 4, right), on the other hand, results in a high luminosity near the fiber end with a rapid decrease in intensity away from the end, due to strong scattering by the media. A reasonably uniform brightness was achieved with a silicate weight percentage of 30%, as shown in the center of Fig. 4. Negatives of the top images, shown in the bottom row of Fig. 4, better illustrate the intensity variation across the pouch. Also, as discussed here, the negative of the intensity distribution can be used as a grayscale filter that can be used to provide a more uniform light source.

Fig. 4

(Top) Grayscale photographs of light intensity from the pouch for concentrations of 20% (left), 30% (middle), and 40% (right) silica by weight. (Bottom) Same images shown in inverse to highlight regions of small intensity variation. Inverse images can also be used as filters to make the light distribution more uniform (see text).

Even with the 30% concentration, the region near the end of the delivery fiber exhibits a higher than average brightness. Significant improvements in uniformity can still likely be made, and the detailed analysis and optimization of the device scattering properties are opportunities for future research.

To determine the absolute intensity distribution of the pouch a Tenma 72-6693 light meter was used (Tenma, Centerville, Ohio). The 30% concentration pouch was placed in a dark room, and the light meter was used to record the total brightness, $L_{\mathit{tot}}$ from the pouch (in lux). The pouch was also imaged during this time with a Canon PowerShot SD-600 at an image resolution of 6.0 megapixels. To make the data processing more manageable, the image was down-sampled in Adobe Photoshop to a total of $N=52,275\text{pixels}$ and converted to an $8\text{-}\text{bit}$ grayscale image with each pixel intensity, $p_j$ , ranging from 0 (darkness) to 255 (saturation). The brightness of each pixel, $B_j$ , is then computed from the pixel intensity as

Fig. 5

Absolute intensity distribution in the pouch on a per-pixel basis. (Top) 2-D image. Illuminating fiber enters from bottom. Notic the saturated region near the fiber end. (Bottom) 3-D image of same, showing intensity distribution in the pouch.

4.3.

Temperature Distribution

A key design factor is to ensure that the pouch does not generate appreciable heat when in use. To assess the temperature rise of the device during operation, the system was turned on for $15\min$ . A $0.5\text{-}\mathrm{mm}$ -diam. K-type thermocouple (Omega Engineering, response time $\sim 10\mathrm{ms}$ ) was then placed in contact with different areas of the top of the pouch, and the temperature was recorded once the reading stabilized. Results are shown in Fig. 6 expressed as the measured temperature increase above ambient temperature, which was $27.2°\mathrm{C}$ $(81.1°\mathrm{F})$ measured using the same thermocouple setup. The temperature rise is highest near the fiber end and decreases away from it. In all cases, the temperature rise is less than $2.2°\mathrm{C}$ $(4.0°\mathrm{F})$ , which meets the $5°\mathrm{C}$ design requirement stated earlier. Furthermore, in actual use, the pouch will be surrounded by tissue, which has a significantly greater cooling capability than air, and thus the temperature rise in the pouch will be even less than that measured in ambient air.

Fig. 6

Measured temperature increase above ambient for different regions of the pouch. The thermocouple was placed in contact with the pouch and left in place until the temperature reading stabilized.

4.4.

In Vivo Optical Microscopy

Visual comparisons of the same microvascular location (Fig. 7 ) provide evidence that the transmitted light is collimated with our standard bright-field light source (as expected) and diffuse with the fabricated pouch device (also as expected). Fine detail of the tissue structure is better seen with the collimated light; thus, the pouch light system is not likely to replace standard microscopy for histological work. Unlike histological microscopy, however, we sought a means to evaluate physiological function in a noninvasive manner in developing the light source. The main criterion for a suitable light source to determine function in intravital microscopy is the ability to measure distances accurately. To this end, we compared the measured blood vessel diameter using both standard bright-field microscopy and the fabricated pouch device. Two blinded observers measured $N=32$ vessel diameters at the same vascular locations using both bright-field and pouch-transmitted light sources. The average diameter of arterioles and venules from all measurements is shown in Fig. 8a for each light source. The difference in diameter as measured with the two light sources for each measurement location is shown in Figure 8b. As can be seen, measured values are all within $\pm 2\mu \mathrm{m}$ (about 10%), with over half $(18∕32)$ within $\pm 0.5\mu \mathrm{m}$ $(\sim 2\%)$ . Thus, despite the fact that qualitative images can appear considerably different (Fig. 7), measured functional parameters such as diameter are in very good agreement compared to traditional bright-field microscopy.

Fig. 7

Video images of the same vascular location in the hamster cheek pouch taken with (a) the pouch device transmitted light and (b) bright-field transmitted light. Arrows denote the walls of the in-focus arteriole. Scale bars are 20 microns.

Fig. 8

(a) The $\text{mean}\pm \mathrm{stddev}$ of the $n=16$ imaged arteriole and venule diameters using bright-field or the pouch device transmitted light. Four vessels of each type were observed in each of four hamsters. (b) A histogram of the difference between the pouch and bright-field diameter measurements (pouch–bright-field) for all vessels $(n=32)$ . The inset shows the $\text{mean}\pm \mathrm{stddev}$ of the diameter difference ( $p=0.90$ ).