Spectral Imaging with Scattered Light: From Early Cancer Detection to Cell Biology

Abstract

This article reports the evolution of scanning spectral imaging techniques using scattered light for minimally invasive detection of early cancerous changes in tissue and cell biology applications. Optical spectroscopic techniques have shown promising results in the diagnosis of disease on a cellular scale. They do not require tissue removal, can be performed in vivo, and allow for real time diagnoses. Fluorescence and Raman spectroscopy are most effective in revealing molecular properties of tissue. Light scattering spectroscopy (LSS) relates the spectroscopic properties of light elastically scattered by small particles, such as epithelial cell nuclei and organelles, to their size, shape and refractive index. It is capable of characterizing the structural properties of tissue on cellular and sub-cellular scales. However, in order to be useful in the detection of early cancerous changes which are otherwise not visible to the naked eye, it must rapidly survey a comparatively large area while simultaneously detecting these cellular changes. Both goals are achieved by combining LSS with spatial scanning imaging. Two examples are described in this article. The first reviews a clinical system for screening patients with Barrett's esophagus. The second presents a novel advancement in confocal light absorption and scattering spectroscopic (CLASS) microscopy.

Fig. 1

Clinical EPSS instrument. The EPSS instrument is shown in the endoscopy suite before the clinical procedure, with the scanning probe inserted into the working channel of an endoscope. The insets show details of the scanning probe tip and the control box.

Fig. 2

Bovine lower-portion esophagus epithelium. (a) H&E staining. (b) Comparison of the nuclear size distributions extracted from the EPSS instrument measurements on intact epithelium (solid curve) and histological examination of the corresponding H&E stained sections (dots).

Fig. 3

Nuclear size distributions for one high grade dysplasia site and one non-dysplastic site in BE of one of the patients. Dark (red and pink online) regions of the map indicate areas suspicious for dysplasia based on nuclear size distributions extracted from the backscattering spectra for each individual spatial location. Non-dysplastic BE sites had nuclear size distributions centered about 5–6 μm diameter while sites marked as suspicious for dysplasia have nuclear size distributions with a main peak centered from 9 to 15 μm. The arrows indicate the specific locations on the esophageal surface from which the size distributions extracted from the polarized LSS data.

Fig. 4

Biopsies taken during the initial and follow-up endoscopy procedures for patient A, overlaid on the EPSS map acquired during the initial procedure (left panel). Three follow-up biopsies were guided by the EPSS map and pathology confirmed HGD for each (indicated at 360°). High-resolution endoscopic (HRE) image of a location with invisible HGD (right panel) with narrow band imaging (NBI) enabled. Video capture was acquired in subject A at one of the locations where invisible dysplasia was missed by visual examination by HRE with NBI, but located by EPSS, and later confirmed by pathology. The site is marked by an arrow. Note that the site is visually indistinguishable from the surrounding nondysplastic BE tissue.

Fig. 5

Incoherent (dotted lines, color online) and coherent (solid lines, color online) CLASS spectra versus inverse size parameter 1/x = λ/(πδ) plotted on a log-log scale. Coherent and incoherent CLASS spectra are virtually identical for backscattering geometry or NA = 0 objectives. However, while I-CLASS spectrum for the large angle objective (NA = 0.98) is virtually structureless, C-CLASS spectrum preserves the majority of the structure and thus information.

Fig. 6

Schematic of the coherent CLASS scanning microscope.

Fig. 7

Calculated SNR for different size scatterers.

Fig. 8

Numerical simulation of particle sizing accuracy for I-CLASS and C-CLASS as a function of the measurement SNR. Dotted lines are provided to guide the eye.

Fig. 9

Experimental results. (a) Typical experimental CLASS spectra (dots) and model fits (solid curves) for a polystyrene microsphere in glycerol. Manufacturer’s nominal diameter for microsphere is 1053 nm ± 10 nm. Model fit for I-CLASS data gives 1029 nm diameter; model fit for “C-CLASS Concept” data gives 1052 nm diameter. Refractive indices of microsphere and surrounding medium reconstructed from “C-CLASS Concept” data are 1.59 and 1.44, which agree with the known refractive indices of polystyrene and aqueous glycerol solution. (b) Experimental values of particle sizing accuracy (squares) overlaid on calculated values from Fig. 8.

Fig. 10

Diameters of 16 polystyrene microspheres with manufacturers’ nominal diameter of 1053 nm ± 10 nm, extracted from I-CLASS and C-CLASS measurements. (a) I-CLASS measurements: average size is 1046 nm, standard deviation is 99 nm; (b) C-CLASS measurements: average diameter is 1058 nm, standard deviation is 5 nm. Dashed line is provided to guide the eye and is at the manufacturers’ nominal diameter of 1053 nm.