Spectral Imaging and Pathology: Seeing More

Anatomic pathology is a visualdiscipline. Microscopic scenes ofhematoxylin and eosin-stained tissuesyield both low-power spatial clues aswell as insights on the structural andfunctional state of individual cells,thousands of which may be visible at atime. Add to this the fact that assayssuch as immunohistochemistry and insitu hybridizations can provide molecu-lar information within the architecturalcontexts, and it is surprising that digitaland quantitative imaging continues toplay no more than a small role in clini-cal practice. This is still true despiteremarkable improvements in the capa-bilities, cost, and availability of cam-eras (sensors) and computers. Anumber of factors are responsible forthis. The human eye-brain combinationis skilled at perceiving and interpretingvisual scenes and can rapidly evaluatea sample at low magnification and,with facility, switch to high power forvalidation or for further detail. To dothe same electronically may require thecollection and analysis of literally thou-sands of high-resolution digital imagesfrom an entire sample. Secondly,pathology is difficult, and each image-based interpretative system must spe-cialize in the nuances of a particularorgan and disease. Thirdly, current im-aging hardware-softwareconfigurations often require trainedoperator assistance, although graduallythe demands on the user are lessening.Finally, while subjective (“1+, 2+, 3+”)grading or scoring schemes are widelyused, there is little discernible enthusi-asm among practicing pathologists forobjective, quantitative image-based de-scriptors, at least for metrics such asnuclear grade or dysplasia. Reasons forthis include the fact that the case formany quantitative schemes have, untilrecently, not been compelling; thatmulti-institutional image analysis pro-tocols are difficult to implement; andthat recognized standards for imageanalysis have not been established.On the other hand, molecularlybased techniques are being adoptedinto pathology at an ever-increasingrate. Many of these rely on suspensionsor extracts of cells or tissues, but oth-ers, such as immunohistochemistry andfluorescence in situ hybridization, areperformed on tissue sections or isolatedcells and the results evaluated througha microscope. In contrast to conven-tional histopathology, the simplifiedreadout (absence or presence of a cer-tain color) and the (semi-) quantitativenature of the techniques (percent ofcells positive, degree of positivity,number of signals per cell) are morereadily approached through digitalimage analysis.Spectral imaging is a relativelynew technique that acquires an imagealong with an optical spectrumcaptured at every pixel. Its performanceimprovement over visual examinationand conventional grayscale or 3-colorimaging has led to the development ofnovel, qualitative, as well as quantita-tive tools for interpreting convention-ally stained pathology samples,overcoming some, although not all, ofthe obstacles outlined above. In addi-tion, spectral imaging facilitates the useand extension of molecular pathologyapproaches; in particular, permittingthe detection and analysis of multipleprobes simultaneously. Spectral imag-ing can be applied to fluorescence-based assays, but it offers particularpromise for increasing the utility ofstandard brightfield microscopy famil-iar to all pathologists.Multiplexing in BiomedicineThe genomics revolution and thearrival of truly molecular medicinehave led to demands on pathologists toprovide more information on individualpatients’ tumors. An accurate diagno-sis, perhaps accompanied by broad sta-tistical predictions of how the patientmight fare must now be supplementedwith predictive and prescriptive infor-mation specific to the individual pa-tient. While a great deal of effort isbeing applied to DNA-chip-based RNAexpression arrays for molecular profil-ing of tumors,