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All-Digital Histopathology by Infrared-Optical Hybrid Microscopy

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All-Digital Histopathology by Infrared-Optical Hybrid Microscopy All-digital histopathology by infrared-optical hybrid microscopy Martin Schnella, Shachi Mittala,b, Kianoush Falahkheirkhaha,c, Anirudh Mittala,b, Kevin Yeha,b, Seth Kenkela,d, Andre Kajdacsy-Ballae, P. Scott Carneyf, and Rohit Bhargavaa,b,c,d,g,h,i,1 aBeckman Institute for Advanced Science and Technology, University of Illinois at Urbana–Champaign, Urbana, IL 61801; bDepartment of Bioengineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; cDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbana– Champaign, Urbana, IL 61801; dDepartment of Mechanical Science and Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; eDepartment of Pathology, University of Illinois at Chicago, Chicago, IL 60612; fThe Institute of Optics, University of Rochester, Rochester, NY 14620; gCancer Center at Illinois, University of Illinois at Urbana–Champaign, Urbana, IL 61801; hDepartment of Electrical and Computer Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and iDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Christian Huck, University of Innsbruck, Innsbruck, Austria, and accepted by Editorial Board Member John A. Rogers December 20, 2019 (received for review July 19, 2019) Optical microscopy for biomedical samples requires expertise in fidelity imaging in both point-scanning (15) and wide-field (16–19) staining to visualize structure and composition. Midinfrared (mid-IR) modalities, IR pixel sizes are ∼100-fold larger than those easily spectroscopic imaging offers label-free molecular recording and achieved in visible microscopy, and data acquisition speed is still virtual staining by probing fundamental vibrational modes of three to four orders of magnitude slower than in visible imaging molecular components. This quantitative signal can be combined (20). The abundance of applications, ease of use, and ubiquity of with machine learning to enable microscopy in diverse fields visible microscopy points to the significant potential of an inte- from cancer diagnoses to forensics. However, absorption of IR grated approach with IR spectroscopy. light by common optical imaging components makes mid-IR light Several transformative applications of a hybrid of visible mi- incompatible with modern optical microscopy and almost all croscopy and IR spectroscopy are apparent. For example, the biomedical research and clinical workflows. Here we conceptualize microscopic examination of stained tissue using visible micros- an IR-optical hybrid (IR-OH) approach that sensitively measures copy has been the standard method for detecting and grading molecular composition based on an optical microscope with wide- most forms of human cancer in research and clinical care (21, 22). CHEMISTRY field interferometric detection of absorption-induced sample expan- Combinations of staining and microscopy with advanced artificial sion. We demonstrate that IR-OH exceeds state-of-the-art IR micros- copy in coverage (10-fold), spatial resolution (fourfold), and spectral intelligence (23, 24) now provide new capabilities, but this avenue consistency (by mitigating the effects of scattering). The combined is ultimately limited by available exogenous labels. Complemen- impact of these advances allows full slide infrared absorption im- tarily, IR imaging (25) offers detailed molecular contrast without ages of unstained breast tissue sections on a visible microscope the need to stain tissue, and its combination with machine learning – platform. We further show that automated histopathologic segmen- is being used to augment and automate histopathology (26 28), tation and generation of computationally stained (stainless) images discover cell physiology (29), inform therapeutic decisions (30), and MEDICAL SCIENCES is possible, resolving morphological features in both color and spa- aid discovery of new biomarkers (31). Making visible microscopy tial detail comparable to current pathology protocols but without stains or human interpretation. IR-OH is compatible with clinical and Significance research pathology practice and could make for a cost-effective alternative to conventional stain-based protocols for stainless, This study reports the ability to provide label-free molecular all-digital pathology. information from infrared (IR) spectroscopy via the ubiquitous optical microscope. Modeling the thermal-mechanical coupling infrared spectroscopy | imaging | quantum cascade laser | pathology | of samples, we design, build, and validate an IR-optical hybrid breast cancer (IR-OH) microscope that uses optical interferometry to measure the dimensional change in materials arising from spectral ab- ptical microscopy is ubiquitous in biomedical research for sorption. We show that the seamless compatibility of IR-OH with Othe examination of microscopic structure of tissues and forms routine optical microscopy and with emerging computational a cornerstone of all development and disease studies and much ubiquity enables all-digital pathology with applications across medical decision-making (1). Visualizing structure and chemical the spectrum of biomedical science. IR-OH microscopy provides a composition in biomedical samples, however, requires the use of means to retain the ease of use and universal availability of stains or labels. Label-free optical techniques have been proposed optical microscopy, add a wide palette of IR molecular contrast, to visualize molecular content without dyes or labels to monitor and utilize emerging computational capabilities to change how processes unperturbed, to observe composition that is not easily we routinely handle, image, and understand microscopic tissue amenable to staining (2). Molecular spectroscopy through more structure. than 125 y of progress (3) enables the absorption of midinfrared (mid-IR) light to be used as an identifying molecular signature (4). Author contributions: M.S., P.S.C., and R.B. designed research; M.S., S.M., K.F., A.M., K.Y., S.K., A.K.-B., and R.B. performed research; M.S., S.M., K.F., K.Y., and A.K.-B. contributed Extant applications of mid-IR microscopy span biomedical tissue new reagents/analytic tools; M.S., S.M., K.F., A.M., S.K., P.S.C., and R.B. analyzed data; and diagnostics (5) and cell biology (6) to polymeric (7), plant (8), and M.S., S.M., P.S.C., and R.B. wrote the paper. forensic samples (9) and interstellar analyses (10) over nearly 70 y The authors declare no competing interest. (11). IR imaging, however, is incompatible with visible microscopy. This article is a PNAS Direct Submission. C.H. is a guest editor invited by the Detectors used in visible and near-IR imaging are nonresponsive, Editorial Board. and most glasses are highly absorbing over the IR spectral band Published under the PNAS license. (2 to 12 μm), precluding the use of commonly available imaging 1To whom correspondence may be addressed. Email: [email protected]. components that have advanced other spectroscopic approaches This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (12). Despite exciting recent advances in quantum cascade lasers doi:10.1073/pnas.1912400117/-/DCSupplemental. (QCL) (13, 14) providing a powerful impetus for rapid, high- www.pnas.org/cgi/doi/10.1073/pnas.1912400117 PNAS Latest Articles | 1of9 Downloaded by guest on September 25, 2021 and IR imaging compatible could thus yield tremendous benefits. IR-OH microscope as shown schematically in Fig. 1C.Detailedin Using visible imaging components for recording IR absorption has Materials and Methods, our instrument consists of a pulsed mid-IR recently been the focus of several approaches using up-conversion QCL that is used to illuminate a large area on the sample at the of light or secondary sample effects. Strong absorption causes repetition rate, Ω. We probe the induced sample deformations secondaryeffectssuchasrefractiveindexchangesforultra- by illuminating with visible light from a narrowband LED sensitive measurements (32) or local photothermal expansion ðλ0 = 660 nmÞ and collecting backscattered light from the sample for photoacoustic imaging (33), which provide a means to over- with a Mirau interference objective. While the sample is vertically come the extant limitations of IR imaging. The concept of dynamic (z) translated, we capture interferograms, IxyðzÞ,atahighcamera photothermal changes in morphology (34, 35), force (36, 37), or frame rate of F = 500 Hz (Fig. 1 D and E). Demodulation at near-field coupling (38–40), using an atomic force microscope frequency Ω − S of the interferograms, Ixy, yields a signal pro- cantilever as local probe, has been reported for point-by-point IR portional to the first-order harmonic of the surface deformation, measurements. Noncontact optical photothermal microscopy is eðΩÞ,whereS is the fringe frequency as determined by the rate at more recent (41–50) and typically utilizes a local IR illumination which interference fringes pass by as a function of the vertical coincident with a highly focused visible probe beam to measure sample position z (61). Subsequent normalization of the demodu- local refractive index change by beam scattered out of the angular lated signal to the incident IR intensity, IIR, constructs the IR acceptance of the objective lens. This method has been applied to sðΩÞ chemical imaging of tissue and live cells (44, 46, 47), bacteria (48), photothermal image, p . High SNR is provided by a high-intensity and pharmaceutical tablets (49). These
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