Robot-Guided Atomic Force Microscopy for Mechano-Visual Phenotyping of Cancer Specimens

Abstract

Atomic force microscopy (AFM) and other forms of scanning probe microscopy have been successfully used to assess biomechanical and bioelectrical characteristics of individual cells. When extending such approaches to heterogeneous tissue, there exists the added challenge of traversing the tissue while directing the probe to the exact location of the targeted biological components under study. Such maneuvers are extremely challenging owing to the relatively small field of view, limited availability of reliable visual cues, and lack of context. In this study we designed a system that leverages the visual topology of the serial tissue sections of interest to help guide robotic control of the AFM stage to provide the requisite navigational support. The process begins by mapping the whole-slide image of a stained specimen with a well-matched, consecutive section of unstained section of tissue in a piecewise fashion. The morphological characteristics and localization of any biomarkers in the stained section can be used to position the AFM probe in the unstained tissue at regions of interest where the AFM measurements are acquired. This general approach can be utilized in various forms of microscopy for navigation assistance in tissue specimens.

Keywords: atomic force microscopy; robotic microscopy; serial sections registration; virtual microscopy; whole-slide imaging.

Figure 1

Unstained tissue imaged with atomic force microscope (AFM). a: 4 × view (without probe) of a tissue section, showing the limited field of view presented by hardware configuration of the AFM. b: 10 × view of a 0.6 mm tissue microarray disc with AFM probe.

Figure 2

Aligning multiple whole-slide images. Above: a conceptual diagram showing that the registration, i.e., generation and tracking of the global transformation, between two large whole-slide images enables synchronized visualization of the specimens at any chosen scale, rotation, and translation. Arrows show transformations being tracked by software. Below: a clinically relevant example of aligning two immunostained serial sections of lung fine needle aspirate specimens showing differential diagnosis of a prostate adenocarcinoma metastasized to lung. Arrow heads indicate where nuclear positivity of thyroid transcription factor 1 (TTF1) characterized lung epithelial cells. Arrows indicate that cytoplasmic staining of prostate-specific membrane antigen (PSMA) as well as the absence of TTF1 staining revealed prostate originality of cancer cells.

Figure 3

Software system architecture illustrating hierarchy of hardware and software components of the system and the relevant communications in between. AFM, atomic force microscopy; WSI, whole-slide imaging.

Figure 4

Frame stitch of specimen creating more visible space for registration. Left: reconstruction/stitching of scanned frame images. Right: software eliminates the artifacts created from the atomic force microscopic probe and creates a more useful display of the stitched frames.

Figure 5

Extending the scheme shown in Figure 2, the system maintains transformations among coordinate systems across various mechanical and optical components at software level in order to register the atomic force microscopic specimen with related whole-slide images.

Figure 6

Atomic force microscopic (AFM) experimental setup. a, the glass slide with TMA placed on the XY-stage; b, the AFM head attached with AFM probe placed over the glass slide for tissue indentation; c, the magnified view of AFM probe aligned at the start point of the ROI in the tissue. Illustrated by circled numbers: (1) AFM charge-coupled device camera, (2) AFM inverted microscope, (3) vibration isolation table, (4) MP-285 micromanipulator, (5) tissue slide, (6) slide holder, (7) AFM head, (8) AFM probe, (9) tissue, (10) probing region of interest (ROI), and (11) tracking ROI.

Figure 7

Schematic of atomic force microscopic probing on tissue.

Figure 8

Registration of the whole-slide images of hematoxylin and eosin (H&E) (left) and P63/smooth muscle actin (SMA) (right) by overlaying threshold outlines from the H&E specimen to the P63/SMA specimen.

Figure 9

An example procedure of registering panels of images based on known landmarks of the specimen. Left: simulated specimen on atomic force microscope. Middle: hematoxylin and eosin image. Right: P63/smooth muscle actin double stain.

Figure 10

Example procedure of sampling a morphologically interesting feature. a: Overlaying a hand-drawn necrotic region to simulated atomic force microscopic (AFM) specimen to guide navigation of the AFM microscope to general location of the interested feature. b: Smooth muscle actin signal on the right panel guided the selection of the region-of-interest, which was illustrated in black on all panels. c: Two-dimensional elastic modulus map of the scan region showing the distinction of elastic features between soft necrotic tissue and rigid desmoplastic region, although the epithelial region in between is not as prominent. The color at each sampling point indicates the elastic modulus computed from the AFM curve at the location.

Figure 11

Example of region of interests (ROIs) examined. ROI1: tumor region that displayed a mixture of elastic signals. ROI2: (also shown in Fig. 10), component intersection between necrosis, epithelium, and desmoplastic stroma. ROI3: breast tissue with normal glandular structure located at tumor vicinity. The pairs of panels shown on the left are atomic force microscopic (AFM) image with registered hematoxylin and eosin-stained serial section. The pairs on the right show enlarged ROI with the AFM elastic maps.