Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

Abstract

Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.

Fig. 1

(a) Schematic of the LS-DAC microscope. A cylindrical lens “C” is inserted in the collimated region of the illumination path to transform a point focus into a line focus. The focal line is scanned by the scan mirror in the $x$ direction to create a 2-D en face image of the sample (in the $x\text{-}y$ plane). The hemispherical SIL acts as a sample holder that is translated along the axial ($z$) direction by a motorized stage (not shown) to enable volumetric imaging. (b) Zoomed-in view of the LS-DAC microscope near the sample.

Fig. 2

Workflow of the study with example images. (a, b) Glioma patients were orally administered 5-ALA prior to PpIX-fluorescence-guided surgery. (c) A brain biopsy (grade-III glioma) obtained during the surgical procedure was then imaged with optical-sectioning fluorescence microscopy to obtain images of PpIX expression with subcellular resolution. (d) Corresponding images from histology slides (both H&E staining and PpIX fluorescence) were obtained to validate the optical-sectioning results. (e, f) Example of wide-field intraoperative images from a high-grade glioma (HGG) and a low-grade glioma (LGG) case, respectively, showing that the tumor resembles the surrounding normal tissues under white light imaging in both cases. (g) Photograph of a biopsy specimen placed on the sample holder of a tabletop LS-DAC microscope. (h) Image of an H&E-stained histology section at $40\times$ as a confirmation of the presence of glioma cells in the biopsy specimen. (i, j) Intraoperative wide-field images of PpIX fluorescence (pink color) from the regions shown in (e) and (f), showing that wide-field surgical fluorescence microscopy was capable of detecting PpIX fluorescence from the HGG but not from the LGG. (k) Optical-sectioning microscopy image of the biopsy, showing subcellular PpIX expression. (l) PpIX fluorescence histology image of the same biopsy imaged in (k), showing a similar pattern of PpIX expression. All scale bars represent $50\mu \mathrm{m}$.

Fig. 3

The same quantification algorithm was used for both LS-DAC images and histology images. (a) Example image of PpIX-expressing glioma tissue obtained with the LS-DAC microscope, the performance of which is standardized with the method detailed in Sec. 2.2. The scale bar represents $50\mu \mathrm{m}$. (b) Histogram of the raw image in which the intensity distribution of the background is approximated as an exponential decay. The dotted line denotes the threshold for segmentation in which pixels with intensities above this threshold are considered “positive” for PpIX expression. (c) A binary image of (a) obtained after segmentation, utilizing a 99.5th percentile threshold to the exponential fit.

Fig. 4

(a) A maximum-intensity depth projection (along the $z$ axis) of a volumetric image of the alignment phantom is shown for a misaligned LS-DAC microscope. (b) A maximum intensity projection of a volumetric image of the phantom (Video 1) is shown from a well-aligned system in which the intensity of the fluorescent beads is uniform across the FOV with $<15\%$ deviation (center to edge) and all beads are well-resolved across the entire FOV of $350\mu \mathrm{m}$ ($x$) by $520\mu \mathrm{m}$ ($y$) by $150\mu \mathrm{m}$ ($z$) (Video 1, MPEG, 2.6 MB [URL: http://dx.doi.org/10.1117/1.JBO.22.4.046005.1]). (c) Example cross-sectional views of one bead. The FWHM dimensions of the microsphere allow for the assessment of the spatial resolution of the system. (d, e) Alpha-blending volume renderings of (a and b), respectively, illustrate the uniformity of the detected fluorescence signal from the beads as well as the uniformity of the spatial resolution across the entire FOV. The scale bar represents $50\mu \mathrm{m}$.

Fig. 5

Quantitative comparison of LS-DAC microscopy images versus corresponding fluorescence histology images in terms of PpIX density. (a) Representative binary images of two of the biopsy samples, one with low PpIX density (blue) and the other with high PpIX density (red). (b) Scatter plot of all 14 glioma specimens obtained over 15 months. Each point on the scatter plot represents the average PpIX density at three randomly chosen locations within each biopsy sample, with the error bars indicating the standard deviation from the measurements. Note that a volumetric dataset (Video 2) was collected at each tissue location, but only a $10\text{-}\mu \mathrm{m}$ optical section at the surface was quantified to simulate a $10\text{-}\mu \mathrm{m}$ slide-mounted histology section. The scale bars represent $50\mu \mathrm{m}$. (Video 2, MPEG, 1.4 MB [URL: http://dx.doi.org/10.1117/1.JBO.22.4.046005.2]).