Current Landscape of Advanced Imaging Tools for Pathology Diagnostics

Abstract

Histopathology relies on century-old workflows of formalin fixation, paraffin embedding, sectioning, and staining tissue specimens on glass slides. Despite being robust, this conventional process is slow, labor-intensive, and limited to providing two-dimensional views. Emerging technologies promise to enhance and accelerate histopathology. Slide-free microscopy allows rapid imaging of fresh, unsectioned specimens, overcoming slide preparation delays. Methods such as fluorescence confocal microscopy, multiphoton microscopy, along with more recent innovations including microscopy with UV surface excitation and fluorescence-imitating brightfield imaging can generate images resembling conventional histology directly from the surface of tissue specimens. Slide-free microscopy enable applications such as rapid intraoperative margin assessment and, with appropriate technology, three-dimensional histopathology. Multiomics profiling techniques, including imaging mass spectrometry and Raman spectroscopy, provide highly multiplexed molecular maps of tissues, although clinical translation remains challenging. Artificial intelligence is aiding the adoption of new imaging modalities via virtual staining, which converts methods such as slide-free microscopy into synthetic brightfield-like or even molecularly informed images. Although not yet commonplace, these emerging technologies collectively demonstrate the potential to modernize histopathology. Artificial intelligence-assisted workflows will ease the transition to new imaging modalities. With further validation, these advances may transform the century-old conventional histopathology pipeline to better serve 21st-century medicine. This review provides an overview of these enabling technology platforms and discusses their potential impact.

Keywords: artificial intelligence; histology; microscopy; multiomics; slide-free; stain-free.

Figure 1.

(A) Instrumentation setup for stimulated Raman spectroscopy (SRS) microscopy. Reproduced from Ji et al. (B) SRS imaging, with pseudocoloring, of a resected specimen of a recurrent low-grade oligodendroglioma compared with standard hematoxylin and eosin (H&E). Reproduced from Orringer et al.

Figure 2.

Virtual histology images of several human pathological specimen types, obtained with the Instapath structured illumination microscopy fresh tissue microscopy scanner.

Figure 3.

Top panel. Prostate. Thick formalin-fixed but unsectioned specimen was stained with hematoxylin and eosin and imaged with fluorescence-imitating brightfield imaging. Cycle Generative Adversarial Network mode-converting converting artificial intelligence was used to increase resemblance to conventional hematoxylin and eosin histology. Bottom panel. Fluorescence-imitating brightfield imaging can provides increased insight into three-dimensional surface topography. Left: core-needle biopsy of lung, with alveoli prominently visible. Right: large artery in the stomach, with view of interior wall as the vessel penetrates down from the cut surface.

Figure 4.

Three-dimensional (3D) light-sheet microscopy is used to image prostate specimens stained with an hematoxylin and eosin (H&E) analog (left). The images are converted to synthetic immunohistochemical (IHC) images using a 3D deep learning image-to-image translation approach. A simple threshold-based segmentation is used to volumetrically segment the glands (right). Reproduced from Xie et al.

Figure 5.

Dual-mode emission and transmission images showing novel features revealed in the fluorescence mode (panels C and D) compared with standard brightfield (panels A and B) of the identical field. (A and C) Invasive breast cancer with necrosis and the remains of a blood vessel. Details are hard to discern; however, in panel c, it is easy to distinguish remnants of the elastic lamina (bright) from adjacent necrotic areas. (B and D) An arteriole is clearly shown in the brightfield image, which does not show the details of the internal and external elastic laminae. Interestingly, the internal laminae are multicolored (yellow and blue)—the physiological significance is as yet unknown.