2.1.

Instrumentation

Figure 1 shows an optical schematic of the MDM. The ${\mathrm{OPMI}}^{\circledR }$ picodental microscope (Carl Zeiss, Jena, Germany) is used as the core optical component of the MDM. The magnification setting is adjusted by turning a lens turret to one of five positions. Light is routed from the source to the microscope head by a fiber optic light guide. The working distance of the microscope is $25\mathrm{cm}$ . The FOV of the device is dependent on the choice of magnification setting and ranges between 1 and $4\mathrm{cm}$ in diameter. With a monitor size of $17\mathrm{in.}$ , the system magnification ranges from 8.5 times to 34.7 times.

Fig. 1

(a) MDM in the Head and Neck Clinic at the MD Anderson Cancer Center and (b) system optical schematic.

The original light source was replaced with an air-cooled 100-W mercury arc lamp (Photon Technology International, Birmingham, New Jersey), housed in a custom light box. The lamp and ellipsoidal reflector produce a converging beam which is collimated using a 25-mm planoconvex lens (Lambda Optics, Costa Mesa, California). To protect both the patient and optical components from excessive heating, a cold mirror (Barr Associates, Westford, Massachusetts) is used to eliminate unwanted IR radiation, and an absorbing glass filter removes UV light at wavelengths below $350\mathrm{nm}$ . The beam then passes through a 10-position excitation filter wheel (Sutter Instruments, Novato, California) and through a second planoconvex lens, which focuses the beam onto the proximal face of a 5-mm-diam fused quartz light guide (Fiberoptic Systems, Simi Valley, California), which routes the light to the microscope head. Since the microscope was not originally designed to support near-UV excitation, a custom-machined adaptor was implemented to bypass the original illumination optics and to hold the distal end of the light guide and a fused quartz focusing lens (Newport Optics, Stratford, Connecticut). This adaptor provides a significant increase in near-UV excitation power and subsequently reduces exposure times required for fluorescence imaging. The adaptor also holds a linear polarizer (Chroma Technologies, Rockingham, Vermont), which is servo controlled and engaged by the LabVIEW (Austin, Texas) interface.

The illumination filter wheel contains narrow-band illumination filters at 420, 430, 530, and $600\mathrm{nm}$ for NB imaging. These wavelengths were selected to match hemoglobin absorption features, and also to provide multiple wavelengths throughout the visible spectrum to study the effects of wavelength on penetration depth. The illumination filter wheel also contains illumination filters centered at 365, 380, 405, and $450\mathrm{nm}$ for autofluorescence excitation. These excitation wavelengths were chosen based on previous work by Heinzelman ¹³ and Utzinger ²² The full width at half maximum (FWHM) transmission bandwidth for the excitation filters ranges from $20\mathrm{nm}$ for NB imaging to $50\mathrm{nm}$ for UV illumination (Chroma Technologies, Omega Optics). A 1.0 neutral density filter is used to attenuate the white light illumination power. A light-blocking disk occupies one position in the excitation filter wheel and is used for measurement of the background light level.

The emission arm of the MDM consists of a Zeiss objective lens and 50/25/25 beamsplitter, which passes 50% of the emission light to the microscope eyepieces and 25% each to two CCD detectors. An emission filter wheel (Oriel Optics, Stratford, Connecticut) is located in one detection arm and contains long-pass filters and a linear polarizer. A long-pass filter with a cut-on at $410\mathrm{nm}$ is used with the 365- and 380-nm excitation, one with a cut-on at $430\mathrm{nm}$ is used with 405-nm excitation wavelength, and one with a cut-on at $475\mathrm{nm}$ is used with 450-nm excitation. An additional linear polarizer is placed in the opposing detector arm, as shown in Fig. 1. In one arm, the polarizer is oriented parallel to the excitation polarizer and in the other, the polarizer is oriented perpendicular to the excitation polarizer. The opposing polarizers enable simultaneous OPR and parallel polarization imaging; this feature is useful for reducing changes in FOV that can occur due to patient movement.

Two Retiga Exi (QImaging, Burnaby, BC, Canada) 12-bit color-cooled cameras are used to collect image data. This camera model was chosen because of its low-light sensitivity and compatibility with the LabVIEW programming environment. The sensor is a Sony ExHAD ICX285 progressive-scan high-sensitivity CCD chip, providing adequate spectral sensitivity at chosen wavelengths of interest, from 400 to $650\mathrm{nm}$ . The chip is covered by a Bayer Mask mosaic for color imaging. Image data are saved in raw Bayer format and color interpolation is performed in postprocessing based on color-balancing standards.

Control of the MDM system is accomplished by a custom LabVIEW graphical interface. The interface provides the operator with the ability to image with one excitation/emission wavelength pair at a time, or to collect a rapid sequence of image data over the range of the MDM’s capabilities. The interface also enables the user to store information such as study number and patient data, which are linked and saved with the optical data. Imaging parameters are recorded for all data collected, and background measurements are taken with each sequence.

The MDM takes about $1\min$ to collect a complete image sequence. The sequence is composed of white light reflectance, fluorescence, NB, OPR, parallel polarization reflectance, and background data sets. A predefined set of exposure and gain settings was established for each image in the sequence based on measurements of normal volunteers. To account for interpatient variability, measurement site variability, and changes due to disease, the predefined set of exposure times could be manually scaled up or down in 20% intervals during imaging. Additionally, four duplicate images were taken for each illumination/emission setting at lower and higher exposure times relative to the initial choice. Table 1 shows typical exposure times based on the most commonly used exposure settings used in the pilot clinical trial.

Table 1

Irradiance and typical exposure times for each illumination condition.

A set of standards was chosen to quantify the performance of the MDM as well as to track any changes occurring in the device over time. Positive reflectance standards include 99, 75, and 50% reflectance spectralon disks (Labsphere, North Sutton, New Hampshire), and red, green, and blue portions of a Macbeth Color Chart (GretaMacbeth LLC, New Windsor, New York). A 2% spectralon disk serves as a negative reflectance standard. Positive fluorescence standards include a set of four fluorescent slides (Microscopy/Microscopy Education, microscopyeducation.com) and the negative fluorescence standard is a frosted quartz disk (Mark Optics, Santa Ana, California).

For white light illumination, color balance was achieved by imaging a white balance sheet and adjusting the RGB ratio in software so that equal pixel intensity values were obtained in the red, green, and blue channels. For fluorescence imaging, a constant color balance was used throughout the study to maximize qualitative diagnostic ability and to reflect the natural blue-green tissue fluorescence color. The fluorescence color slides were used as a standard to track any changes in color response of the camera over time in fluorescence mode.

2.2.

Pilot Clinical Study

Several sites within the oral cavity of normal subjects were imaged using the MDM as part of an Institutional Review Board (IRB)-approved protocol at Rice University. In a separate, ongoing pilot clinical study conducted at the University of Texas M. D. Anderson Cancer Center (MDACC), the MDM was used to image clinically abnormal and normal oral mucosal sites in patients. This protocol was approved by the IRBs at both Rice University and MDACC.

The measurements were performed either in the outpatient clinic or in the operating room on patients under general anesthesia prior to surgery. The areas to be imaged were chosen by the clinician and included areas that had previously been identified as abnormal by clinical examination and/or biopsy. The physician first evaluated these targeted areas using the MDM under white light illumination, adjusting the focus and FOV. Several measurement sequences were then taken from each subject including abnormal sites, and when possible, a corresponding contralateral normal site. Excess saliva was suctioned prior to imaging. Because teeth exhibit particularly strong autofluorescence, attempts were made to cover any teeth in the FOV with low-fluorescence gloves or a mouth guard. The head of the patient was positioned and held by the physician during imaging to reduce motion artifacts. In patients seen in the clinic, biopsies were obtained from clinically abnormal sites and a contralateral normal site. In patients who underwent surgical excision, the resected specimens were histologically examined. A clinical diagnosis for normal and abnormal-appearing areas (graded as normal, abnormal but not suspicious for neoplasia, suspicious for neoplasia, or cancer) was rendered by the clinician. The histopathology diagnosis of biopsy sites and resected tissue from imaged areas were considered the gold standard for diagnosis.

Four observers (DR, CK, AG, RRK) examined the images to identify features that differed in abnormal sites compared to clinically normal areas, and which might be further explored for diagnostic relevance. Qualitative observations of image data were correlated with clinical observations regarding peripheral extent of abnormal changes and clinical diagnostic category. Pathology results from resected tissue and biopsies enabled comparison between MDM image data and histopathologic diagnosis of lesions.

3.1.

Instrument Performance

The resolution of the MDM was determined by imaging a U.S. Air Force (USAF) spatial resolution target, as shown in Fig. 2 . A line spacing of $15.6\mathrm{\mu }\mathrm{m}$ (group 5, element 1) can clearly be discriminated at the highest magnification. The appearance of shadowing behind each element is due to the finite thickness of the glass substrate, and is not intrinsic to the MDM system. Measurements of white light illumination on a 99% reflectance spectralon standard (Labsphere, North Sutton, New Hampshire) show a 4-cm-diam illumination spot size. This is adequate to illuminate all but the edges of the FOV at the most commonly used magnification. The illumination pattern is Gaussian due to light guide coupling.

Fig. 2

USAF resolution target imaged at high magnification. Element 5 group line spacings of $15.6\mathrm{\mu }\mathrm{m}$ (shown in the dashed box) are easily discriminated in the zoomed-in image on the right.

To confirm the absence of excitation filter leakage and adequacy of performance of longpass filters, a 2-in.-diam frosted quartz disk was imaged in fluorescence mode. Exposure times and gain settings matching or exceeding those used for normal tissue fluorescence imaging were used at each excitation/emission wavelength pair to confirm the absence of signal from the nonfluorescent frosted quartz disk. The ratio of autofluorescence signal from tissue to frosted quartz was always greater than 10:1.

Intrasequence image registration was affected by patient movement, generally causing some changes in the FOV during the course of the image sequence. Figure 4 (in Sec. 3.3) shows several images taken from a typical sequence in a clinical setting at the most common magnification setting. Using user-defined selection of points common to both images, the linear translation was less than 60 pixels (less than 5% of the FOV) between any two of the images in the sequence in both the $x$ and $y$ directions.

Fig. 4

Images of leukoplakia lesion on right gingiva: (A) white light illumination image, arrows indicate area of leukoplakia; (B) parallel polarization image; (C) OPR image; and (D) 420-nm NB image. Note that the OPR (C) enables visualization of tissue below the thin leukoplakia and the 420-nm OPR image (D) accentuates areas of leukoplakia. Scale bars indicate 0.25 cm.

UV radiation during all human measurements is well below the American Conference of Governmental Industrial Hygienists maximum allowed rating.²³

3.2.

MDM Images of Normal Oral Sites

Sites imaged in the oral cavity from four normal volunteers included tongue, buccal, lip, gingiva, hard palate, soft palate, and floor of mouth. Fine vasculature was clearly identifiable in images acquired from the lip, floor of mouth, hard palate, and soft palate using white light, NB, OPR and fluorescence techniques. These images serve as further examples of the resolution capabilities of the MDM. Figure 3 shows an image of the lower lip of a normal volunteer under several illumination conditions. The white light image [Fig. 3A] shows microvasculature from a variety of depths beneath the epithelial surface. The OPR image illustrates similar vascular patterns; specular reflection is no longer visible in the image, and spatial resolution is somewhat reduced because the OPR technique selectively records photons that have undergone more scattering events in the tissue. The NB image obtained with 420-nm illumination [Fig. 3B] shows only the superficial, fine vasculature due to the reduced penetration of this wavelength; vessel contrast is also increased because this wavelength matches the Soret absorption band of hemoglobin. As the illumination wavelength is increased from green [Fig. 3D] to red [Fig. 3F], vessels deeper within the tissue are visible in the image. The NB image obtained with 600-nm illumination shows a loss of contrast and spatial resolution due to the increased ratio of scattering to absorption and increased mean free path between scattering events at this wavelength. Vasculature was not as apparent in the buccal and tongue.

Fig. 3

Images of normal volunteer inner lip: (A) white light illumination image, (B) 420-nm NB image, (C) OPR image, (D) 530-nm NB image, (E) 365-nm excited fluorescence image, and (F) 600-nm NB image. Note increased contrast of vasculature in (C) compared to white light image. Fine vasculature is visible in (B), whereas deep, larger diameter vasculature is visible in (F). Scale bars indicate $0.3\mathrm{cm}$ .

Tissue autofluorescence is predominantly blue at UV excitation wavelengths [Fig. 3E] and blue-green at the longer $450\mathrm{nm}$ excitation. The hard palate, soft palate, floor of mouth, and buccal mucosa provided a higher fluorescence signal than the tongue, gingiva, and lip. The midline of the hard palate was particularly bright. Teeth were highly fluorescent and tooth fluorescence could be seen through portions of the gingival mucosa. Blood vessels appeared dark under fluorescence mode compared to the surrounding tissue. Red fluorescence occasionally appeared on the dorsal tongue.

3.3.

MDM Images of Oral Dysplasia and Cancer

Images of a premalignant lesion and an invasive cancer are presented as representative examples to demonstrate the capabilities of the MDM system for examination of the oral cavity. Some images were cropped to only show relevant areas.

Figure 4 shows images obtained from a thin leukoplakia lesion (indicated by arrows) on the right mandibular gingiva. The thin leukoplakia in the OPR image [Fig. 4C] is less evident demonstrating the ability of OPR imaging to selectively detect photons that have scattered more deeply into the tissue. The blue NB image [Fig. 4D] shows the leukoplakia as brighter in reference to surrounding tissue compared to the white light image.

Figure 5 shows images acquired from a subtle lesion on the right lateral tongue. The clinical impression of the lesion was leukoplakia, but not overly suspicious for dysplasia or cancer. Following imaging, a $2.4\times 1.0\times 0.4{\mathrm{cm}}^3$ volume was surgically resected and histopathology showed moderate squamous dysplasia [Fig. 5H]. The standard white light image [Fig. 5A] shows some patchy irregularities in the mucosal surface. Using the green NB [Fig. 5E] and the OPR imaging conditions [Fig. 5C], an apparent increase in visual contrast was observed between the lesion and surrounding normal areas (indicated by arrows in each case). A decreased blue/green autofluorescence (DA) was observed in the area of the lesion in fluorescence images at 365-, 380-, and 405-nm excitation [Figs. 5B, 5D, 5F]. An image of a contralateral normal area imaged using white light is shown in Fig. 5G for comparison.

Fig. 5

Images from tongue of patient with leukoplakia, (A) to (F) show a FOV containing areas along the lateral aspect of the tongue, which were resected and found to contain moderate dysplasia: (A) white light illumination image, (B) 356-nm excited fluorescence image, (C) OPR image, (D) 380-nm excited fluorescence image, (E) 530-nm NB image, (F) 405-nm excited fluorescence image, (G) white light illumination image of contralateral normal site, (H) hematoxylin and eosin (H&E)-stained tissue from abnormal area indicating moderate dysplasia. Arrows indicate areas with loss of fluorescence or increased contrast. Scale bars indicate $0.5\mathrm{cm}$ in (A) to (G) and $200\mathrm{\mu }\mathrm{m}$ in (H).

Figure 6 shows images acquired from a premalignant lesion on the left lateral tongue. The clinical impression was erythroplakia; a reddish lesion associated with a high risk of dysplasia or early carcinoma. Histopathology from a biopsy of the lesion indicated severe squamous dysplasia with a focal ulceration and chronic inflammation [Fig. 6H]. Both the OPR image [Fig. 6C] and the green NB image [Fig. 6E] showed an area of abnormality (appearing darker red on OPR and darker on NB images as indicated by arrows), which is more extensive in peripheral extent and has increased contrast as viewed against the surrounding mucosa. In the images obtained using all four fluorescence excitation wavelengths a comparative decrease or loss of blue or green fluorescence was seen in the abnormal site. The total area of DA was larger at 405-nm excitation [Fig. 6F] than at 365-nm [Fig. 6B] and 380-nm excitation [Fig. 6D]. In addition, a striking ring of increased red fluorescence surrounding the lesion was observed in the fluorescence images, and is most apparent at 405-nm excitation. This red fluorescence is consistent with the presence of endogenous porphyrins. A white light image obtained from a contralateral normal area is shown in Fig. 6G for comparison.

Fig. 6

Images from ventral tongue of patient with erythroplakia, (A) to (F) show a FOV of the left lateral tongue containing erythroplakia from clinical appearance and severed dysplasia from histophathology of biopsy site: (A) white light illumination image, (B) 356-nm excited fluorescence image, (C) OPR image, (D) 380-nm excited fluorescence image, (E) 530-nm NB image, (F) 405-nm excited fluorescence image, (G) white light illumination image of contralateral normal site, and (H) H&E-stained tissue from abnormal area indicating severe dysplasia. Arrows indicate areas with loss of fluorescence or increased contrast. Scale bars indicate $0.5\mathrm{cm}$ in (A) to (G) and $200\mathrm{\mu }\mathrm{m}$ in (H).

Figure 7 shows images from the right lateral tongue in a subject with histologically confirmed carcinoma. A previously biopsied and ulcerative lesion is shown in the center of the FOV. In the OPR image [Fig. 7B], the blue NB image [Fig. 7C], and the green NB image [Fig. 7E], increased contrast is noted by arrows in the tissue surrounding the ulcer. The blue NB image [Fig. 7C] shows larger dark areas in the ulcerative lesion and in the bottom right compared to the white light image [Fig. 7A]. The red NB image [Fig. 7G] shows smaller dark areas compared to white light. The fluorescence images show a decrease of blue/green fluorescence surrounding the ulcerative lesion, and this DA is more apparent for 405- and 450-nm excitation [Figs. 7F, 7H] than at 365-nm excitation [Figs. 7D, 7E]. Following imaging, a $3.2\times 2.5\times 1.0{\mathrm{cm}}^3$ portion was resected and was determined by histopathology to contain invasive squamous carcinoma centrally, with dysplasia near the margins of resection.

Fig. 7

Images from cancer on right lateral tongue: (A) white light illumination image, (B) OPR image, (C) 430-nm NB image, (D) 365-nm excited fluorescence image, (E) 530-nm NB image, (F) 405-nm excited fluorescence image, (G) 600-nm NB image, and (H) 450-nm excited fluorescence image. Arrows indicate increased contrast or decreased autofluorescence. Scale bars indicate $0.25\mathrm{cm}$ .

In these representative examples, we observe one or more of the following features in areas histologically determined to be abnormal: a decrease of blue/green fluorescence, increased red fluorescence, and an increase of contrast in highly vascular regions. Blood on the surface of the tissue also appeared dark compared to white light under blue and green NB illumination. Teeth and gloves occasionally interfered with fluorescence imaging of tissue due to their autofluorescence, as can be seen in the lower left corner of the fluorescence frames in Fig. 5.