Point-of-care pathology with miniature optical-sectioning microscopes. Microscopic observation of tissue specimens—prepared according to established protocols in histology for fixation, embedding, sectioning and staining—is regarded by the scientific and medical community as a highly reliable method for the diagnosis and study of disease. The science and art of histopathology is based upon the observation of deviations from normal cellular and nuclear morphology, as well as high-resolution localization of protein or nucleic acid markers of disease with immunohistochemistry and in situ hybridization, respectively.
Despite its acceptance as a “gold standard” for diagnosis, there are limitations to conventional histopathologic diagnosis. First is the need to perform an invasive biopsy or resection procedure. Second is its dependence on the ability of the human observer (typically, the pathologist) to make reliable judgments, both as a single observer over time (“intra-observer variation”) and between different observers (“inter-observer variation”). A third issue relates to possible artifacts introduced during processing that can impair morphological and/or molecular interpretation. These artifacts include sample desiccation, shrinkage caused by fixation and embedding in non-aqueous materials, and differential loss or alteration of molecular constituents. In short, devitalized tissue sections mounted on glass slides provide pathologists with a limited two-dimensional cross-section of what was once living three-dimensional tissue.
In contrast, “point-of-care pathology (POCP)” consists of real-time morphologic examination, at the cellular level, of living tissues in their native context. POCP has the potential to provide immediate assessment of disease status, improve diagnostic accuracy, guide tissue biopsies, and accelerate the diagnostic and therapeutic processes. In conjunction with advanced biomarker-targeted contrast agents, POCP has the potential to provide real-time decision support for many clinical scenarios such as early disease detection and surgical guidance.
A press release on our work to develop a handheld microscope for early cancer detection and surgical guidance:
The photograph below shows a miniature microscope being held above a large tabletop prototype of the same "line-scanned dual-axis confocal" microscope, as described in a recent publication and press release.
A miniature MEMS-scanned optical-sectioning device for clinical use. The dual-axis confocal (DAC) microscope architecture is a unique configuration that possesses much-needed advantages over existing imaging strategies for in vivo imaging. The inspiration for the DAC design was provided by a method to achieve improved resolution through off-axis illumination and collection of light with high-NA (high magnification) objectives [1]. Later, simple low-NA optics were proposed as a method to achieve a long working distance and a large field of view [2]. More recently, we have shown that a dual-axis architecture may be fiber coupled and combined with post-objective scanning to provide scalability of the design to millimeter dimensions [3-5], and still provide the cellular-level spatial resolution that is required for pathological diagnosis. Furthermore, we have shown, through diffraction-theory calculations [6,7], as well as Monte-Carlo tissue scattering simulations and experiments [8-10], that the dual-axis configuration provides superior optical sectioning, through efficient rejection of out-of-focus and multiply scattered light, compared to a conventional single-axis confocal architecture. Finally, because it utilizes simple low-NA optics and inexpensive portable diode laser sources, the DAC microscope is ideally suited for clinical translation and commercialization. As part of NIH-funded projects, we have developed endoscopic and handheld DAC microscopes for early disease detection and surgical guidance [3, 11, 12].
Figure 1. (A) The single-axis architecture uses a high-NA objective to excite and collect light from tissue. (B) The dual-axis configuration uses separate low-NA lenses to excite and collect light off-axis.
Figure 2. Handheld surgical line-scanned dual-axis confocal (LS-DAC) microscope development. (a) Photo of the LS-DAC scan head, with MEMS mirror and alignment prisms. (b) Photo of main body and 3x objective for the LS-DAC microscope. (c) images of fluorescently labeled vasculature in a mouse. The right image is color-coded for depth. (d) Axial response of the microscope to a flat mirror, showing an optical sectioning width (FWHM) of 2 microns. (e) Image of a reflective USAF target, showing the ability to resolve features as small as 1 micron. (f) Image of mouse kidney (fresh) stained with methylene blue, which accumulates preferentially in the nuclei. [12]
[1] E. Stelzer, and S. Lindek, Opt Commun 111, 536 (1994).
[2] R. Webb, and F. Rogomentich, Appl Opt (1999).
[3] J. T. C. Liu et al., J Biomed Opt 15, 026029 (2010).
[4] J. T. C. Liu et al., Opt Lett 32, 256 (2007).
[5] H. Ra et al., Opt Express 16, 7224 (2008).
[6] J. T. C. Liu et al., J Biomed Opt 11, 054019 (2006).
[7] Y. Chen et al., J Biomed Opt 18, 66006 (2013).
[8] J. T. C. Liu et al., J Biomed Opt 13, 034020 (2008).
[9] Y. Chen et al., Opt Lett 37, 4495 (2012)
[10] A.K. Glaser et al., Biomed Opt Exp 7, 454 (2016).
[11] W. Piyawattanametha et al., J Biomed Opt 17, 021102 (2012).
[12] C. Yin et al., Biomed Opt Exp 7, 251 (2016).
Selected recent publications.
L. Wei, C. Yin, and J.T.C. Liu, "Dual-axis confocal microscopy for point-of-care pathology," IEEE J. Select. Topics Quantum Elect. 25 (2019) [PDF]
L. Wei, C. Yin, Y. Fujita, N. Sanai, and J.T.C. Liu, "A handheld line-scanned dual-axis confocal microscope with pistoned MEMS actuation for flat-field fluorescence imaging," Opt. Lett. 44, 671 (2019) [PDF]
C. Yin, L. Wei, S. Abeytunge, G. Peterson, M. Rajadhyaksha, and J.T.C. Liu, "Label-free in vivo pathology of human epithelia with a high-speed handheld dual-axis confocal microscope," J. Biomed. Opt. 24, 030501 (2019) [PDF]
C. Yin, L. Wei, K. Kose, A.K. Glaser, G. Peterson, M. Rajadhyaksha, and J.T.C. Liu, “Real-time video mosaicking to guide handheld in vivo microscopy,” J. Biophotonics, 13, e202000048 (2020) [PDF]