AOTF Spectral Imaging for Cancer Detection

Alex Fong, VP, Life Sciences

High throughput screening of multiply stained clinical pathology samples is currently a difficult and laborious task requiring expert review. In examining a patient slide, pathologists rely primarily on examination of cellular morphology and tissue architecture. While this approach has been the backbone of pathology for many decades, the discovery of numerous molecular biomarkers of disease has lent increasing importance to the role of biomarkers in detecting abnormalities and determining proper treatment. However, individual biomarkers generally do not have sufficient sensitivity and specificity to identify disease and there has been growing recognition that the use of multiple biomarkers is highly desirable. Optimally the pathologist would like to see the multiple biomarkers of interest and the morphology of interest simultaneously throughout the slide. But multiply stained slides can be very difficult to interpret by eye. We have therefore applied our Acousto-Optic tunable filter (AOTF) based HSi-440C Hyper-Spectral Imaging system to imaging traditionally stained clinical pathology samples to which additional transmission stains labeling multiple specific biomarkers have been added.

For spectrally imaging a multiply stained sample the number of different wavelengths required is in principal the same as the number of independent stains, and therefore spectra, in the sample. Our experience has been, however that that many more wavelengths than this are often needed for highest quality results. The actual number required depends on the number of stains used, the degree to which they spectrally overlap, and the complexity of the sample being imaged.

The figure below illustrates this important point. On the far left is a color image of Pap-stained cervical cells co-stained for the biomarker p16 with Deep Space Black. The Pap stain itself is actually a combination of four separate stains (Hematoxylin, Eosin, Fast Green, and OG6), each of which stains different parts of different types of cells, for a total of five stains in this sample. Hematoxylin is the main nuclear stain of primary importance for morphologic evaluation. The best possible unmixed Hematoxylin image for various number of unmixing wavelengths is shown in the four images to the right. It can be seen that analysis results using 6 wavelengths is far from adequate, and approximately 30 wavelengths are required for best results

Pap-stained cervical cells co-stained for the biomarker p16 with Deep Space Black
Pap-stained cervical cells co-stained for the biomarker p16 with Deep Space Black

Spectral imaging of Hematoxylin in a traditional Pap slide requires nearly 30 wavelengths to be accurate. The high speed and flexibility of the AOTF make this practical.

A clinical sample often requires many hundreds of fields of view in order to cover an entire slide under a microscope, even at low magnification. A multi-stained sample therefore requires dozens of wavelengths and thousands of wavelength changes per slide. In high-throughput clinical applications, it is essential that the slide scan time be reduced to a minimum. For multispectral high-throughput scanning, it is therefore necessary that the wavelength switching be accomplished fast enough so as not to slow down the native frame rate of the camera. The AOTF’s ~100 microsecond switching time and spectral flexibility together with their superior imaging quality make them an ideal choice for this application.

The AOTF based HSi-440C has now been applied to cervical cancer detection and shown to provide the pathologist with significant additional information to aid in more accurate interpretation. More details of these results will be in the next newsletter. For more information please contact