2.1.

Sample Handling

All experiments were conducted under the protocols approved by the institutional review boards of the Massachusetts General Hospital (MGH) and the University of Massachusetts at Lowell (UML). Normal and neoplastic brain specimens were gathered from resection surgeries performed at the Department of Neurosurgery at MGH, Boston, and sent to UML Advanced Biophotonics Laboratory for imaging.

Fresh samples were delivered to UML on ice within 1 to 12 hours of their acquisition from the operating room. The sizes of the specimens were between $2\mathrm{mm}\times 2\mathrm{mm}\times 2\mathrm{mm}$ and $40\mathrm{mm}\times 40\mathrm{mm}\times 40\mathrm{mm}$. Upon arrival to UML, the specimens were stained for 2 to 5 min, depending on the size of the specimen, in $0.05\mathrm{mg}/\mathrm{ml}$ Phosphate Buffered Saline (Mediatech Inc. Manassas, VA) solution of MB (Methylene Blue Injection, 1%, American Regent Inc., Shirley, NY). The concentration of $0.05\mathrm{mg}/\mathrm{ml}$ MB was empirically selected to provide optimal contrast of the confocal images. After staining, excessive dye was quickly rinsed off the samples. The samples were then moistened with saline solution (pH 7.4), covered with a microscopic cover slip, mounted in the sample holder [Fig. 1(a)], and imaged.

Fig. 1

(a) Image of brain tissue mounted in the sample holder. (b) Schematic diagram of the multimodal confocal system.

After imaging, the brain tissues were fixed in 10% formalin (Ricca Chemical Company, Arlington, TX) and processed for paraffin-embedded H&E histopathology.

2.2.

Confocal Imaging

The schematic of the multimodal confocal system is presented in Fig. 1(b).^14,15A 642 nm diode laser (Micro Laser Systems, Garden Grove, CA) was used as the light source. The total power on the sample did not exceed 2.5 mW. Two photomultiplier tubes (PMT R9110 Hamamatsu, Bridgewater NJ) were employed for detection of reflectance and fluorescence signals. A polygon mirror (Lincoln Laser, Phoenix AZ) and galvanometric mirror (General Scanning Inc., Billerica MA) scanned the imaged point in the $x$ and $y$ directions, respectively. The scanning rate was set to 9 frames per second. The fluorescence signal was deflected by the 12-deg dichroic mirror (Iridian Spectral Technologies, Ottawa, Ontario) with a reflection band between 670 and 740 nm, and focused by a lens onto a 200-μm pinhole of the fluorescence PMT. To reject excitation light, a 690-nm bandpass filter with full width at half maximum of 40 nm (Chroma Technology Corp., Bellows Falls, VT) was installed in the fluorescence channel. A nonpolarizing beam splitter (CVI MellesGriot, Albuquerque, NM) and a lens were used to send elastically scattered signal into the 200-μm pinhole of the reflectance PMT. We employed two water immersion objective lenses, including $40\times /\mathrm{NA}1.15$ and $60\times /\mathrm{NA}1.2$ (Olympus, Melville, NJ), which provided a field of view of 370 and 250 μm, respectively. The system yielded lateral resolution better than 0.9 μm and axial resolution of 3 μm.

2.3.

Histopathology

After imaging, tissue was formalin fixed and paraffin embedded in a plane corresponding to the plane of imaging to enable side-by-side correlation of optical images and histopathology. Sections 5 μm thick sections were cut and stained by standard H&E method.¹⁶ H&E sections were digitized using a Zeiss Axioskop microscope (Zeiss, Stuttgart, Germany) equipped with a $40\times$ objective lens, NA 1.0 (Zeiss) with the resolution of 0.36 μm. The resulting optical images were correlated with histopathology obtained from the same depth within the tissue.

3.1.

Normal Brain

Example images of white and gray brain matter are shown in Fig. 2. These images were taken from two different areas of a cross-section of the right temporal lobe. Comparison of white and gray matter images demonstrates clear differences in appearance, both in reflectance [Figs. 2(a) and 2(c)] and in fluorescence [Figs. 2(b) and 2(d)]. Reflectance images of both white and gray matter demonstrate lattice-like architecture of normal brain, with evenly spaced and well-organized processes. Myelin, a lipid with a high index of refraction that surrounds the axons in white matter, produces this dense, white, mesh-like appearance. Due to the presence of the myelinated axons, the reflectance images of white matter [Fig. 2(a)] are brighter, as compared with gray matter [Fig. 2(c)]. The fluorescence image of normal gray matter [Fig. 2(d)] presents neuronal cell bodies (dashed arrow), which in our images were approximately 27 μm in size with exiting nonmyelinated axons (solid arrow) that stretch about 52 μm. Glial cells, shown with dotted arrow, are also present and are approximately 9 μm in size. Normal white matter tissue is made up of glial cells and myelinated axons.¹⁷ Glial cells can be detected by the fluorescence imaging [Fig. 2(b), dotted arrow], but the dielectric properties of myelin surrounding the axons in white matter prevent uptake of MB, and as a result axons are not apparent in the fluorescence image of white matter. These results are in good correlation with previously reported observations.¹⁷

3.2.

Meningiomas

Meningiomas arise from the arachnoid layer of the meninges, the membranes that surround the brain and the spinal cord. They most commonly present with seizures; focal neurological deficits, if the tumor is near regions of the brain such as the motor or language cortex; or signs and symptoms of elevated intracranial pressure.^18,19 These tumors take the shape of the area they occupy, forming circles around one another.¹⁹ Figure 3 shows typical meningothelial whorls that can be seen in both reflectance [Fig. 3(a)] and fluorescence [Fig. 3(b)] confocal images. In the reflectance image, the fibrous outer layer of the meningeal whorls (dashed arrows) can be clearly distinguished. Interestingly, meningiomas do not exhibit the dense lattice like structure of the normal brain tissue [Figs. 2(a) and 2(c)]. In contrast to reflectance, the fluorescence image [Fig. 3(b)] demonstrates cellular inner parts of the whorls (solid arrows). It should also be noted that the combination of reflectance and fluorescence images provides complementary information about the structure of meningiomas. Comparison with respective H&E histopathology demonstrates good correlation with both reflectance and fluorescence images.

3.3.

Glial Neoplasms

Gliomas are the most common and deadly type of intrinsic brain tumors.²⁰ They originate from the macroglial cells. The glial cells provide nutrients and oxygen to the neurons, electrochemically insulate them from one another, destroy pathogens, and remove debris. Three types of glial cells, including astrocytes, oligodendrocytes, and ependymal cells, can give rise to glioma. Astrocytes serve to regulate blood flow and chemical balance of neurons in the brain and are the most abundant of the macroglial cells. Oligodendrocytes form the myelin sheath that insulates the central nervous system axons. Ependymal cells create and regulate the flow of cerebrospinal fluid.²¹ According to their biological behavior, gliomas are graded in four categories by the WHO. Grade I tumors are typically well encapsulated and may be cured with a complete resection. Grades II through IV are typically diffuse and infiltrative, and recur as a rule. Associated survival times shorten with increasing grade. Grade II tumors are also known as low-grade gliomas (LGG). Grade III (anaplastic) and IV (glioblastoma) are high-grade tumors.²² Optical images were registered for all of the above-mentioned glioma grades.

3.4.

Metastases

Metastatic brain tumors account for about one-fourth of all cancers that metastasize. They are much more common than primary brain malignancies and occur in approximately 10% to 30% of adult cancers. These tumors often retain morphological features of their primary cancer, and their appearance is remarkably different from that of normal brain, meningioma, or any of the glial neoplasms. Out of cancers that metastasize to the brain, the most common are lung, breast, renal carcinomas, and melanomas.²⁴ Example reflectance, fluorescence, and histological images of a metastatic lung adenocarcinoma are presented in Figs. 8(a), 8(b), and 8(c), respectively. As expected, clear differences can be revealed between the metastatic tumor and all the primary brain neoplasms imaged. In all the images, carcinoma nodules are shown with solid arrows. They are surrounded by dense bands of fibrous tissue shown with dotted arrow, whereas dashed arrows point to necrotic tissue. Dash and dotted arrows point toward the macrophages that can be seen in fluorescence images and in corresponding histopathology. Similarly to the other tumors, reflectance [Fig. 8(a)] and fluorescence [Fig. 8(b)] combine to provide essential information on the morphology and architecture of the specimen. Both optical images correlate well with respective H&E histopathology and enable clear differentiation among viable carcinoma, necrosis, and mesenchymal tissue.