Single point vs. mapping approach for spectral cytopathology (SCP)

Abstract

In this paper we describe the advantages of collecting infrared microspectral data in imaging mode opposed to point mode. Imaging data are processed using the PapMap algorithm, which co-adds pixel spectra that have been scrutinized for R-Mie scattering effects as well as other constraints. The signal-to-noise quality of PapMap spectra will be compared to point spectra for oral mucosa cells deposited onto low-e slides. Also the effects of software atmospheric correction will be discussed. Combined with the PapMap algorithm, data collection in imaging mode proves to be a superior method for spectral cytopathology.

Figure 1

(A) Visual image of a 2 mm × 2 mm area on a sample spot collected on the PE Spotlight microscope showing desirable cell density, and (B) its corresponding binary mask constructed using the PapMap algorithm.

Figure 2

Enlarged region of a small area (approximately 170 µm × 130 µm), displaying the (A) visual image collected on the PE Spotlight microscope, (B) its corresponding binary mask constructed using the PapMap algorithm, and (C) the amide I intensity in each of the contiguous “raw cell” regions where the bright yellow hues exhibit pixels with the highest amide I intensity values.

Figure 3

(A) Distortion of the spectral band shapes and positions in the fingerprint region (1800–900 cm−1), as the result of R-Mie scattering effects. The most significant changes are in the intensity ratio of the amide I to amide II bands, but there are distortive changes throughout the spectral region. (B) R-Mie effects are minimized in imaging mode using PapMap since the algorithm scrutinizes which pixels are co-added by subjecting them to various constraints. The spectrum of the same cell collected in imaging mode and processed using PapMap is almost identical to the spectrum collected in single point mode.

Figure 4

PCA scores plot of oral mucosa cells collected via imaging mode with atmospheric water correction on (red) and off (blue) where each symbol represents the spectrum of an individual cell.

Figure 5

Second derivative vector normalized spectra for the spectral range 1800–900 cm−1 where (A) represents a small cell ca. 20 pixels and (B) represents a large cell ca. 80 pixels. The blue trace is the spectrum calculated using the PapMap algorithm. The green and red traces are of spectra collected in point mode using two aperture sizes: 25 µm and an adjusted aperture to encompass the inside of the cell, respectively. High resolution images of a representative small cell and a large cell are depicted with each respective second derivative trace.

Figure 6

(A) PCA scores plot of 12 oral mucosa cells from one volunteer collected via imaging mode (red) and point mode using an adjusted aperture (blue). (B) Integrated loading vector 1 (black trace) for PC1 of the scores plot shown in A. This spectrum is severely affected by R-Mie scattering: the amide I and amide II bands are located at 1642 and 1532 cm−1, respectively. The red and blue traces are from individual cells on either side of PC1 as indicated in A.