A Photonic Chip-based Microscopy Platform For Super-Resolution Histology of FFPE Tissue Sections

Session

Poster Session

Authors

Luis E. Villegas-Hernández (1), Vishesh K. Dubey (1), Balpreet S. Ahluwalia (1, 2)

Affiliations

1. UiT The Arctic University of Norway
2. Karolinska Institute

Keywords

Chip-based microscopy

Super-resolution microscopy

Histology

FFPE

Abstract text

Introduction:

Histology is the branch of life sciences devoted to the study of the structure and function of tissues using microscopes. Within it, the field of histopathology assists with the identification of structural changes associated with the onset and development of diseases. In these two disciplines, the selection of the right microscopy method and sample preparation are equally important to attain detailed visualizations of the tissues. While modern optical microscopes allow for fast imaging of commonly available formalin-fixed paraffin-embedded (FFPE) tissue sections at the subcellular scale, the resolution capabilities of these instruments fall short for the study of many ultrastructural features in tissues (e.g., tight junctions, synapses, foot processes, microvilli brush-border, among others), which are fundamental both for the advancement of basic histological research and for an accurate diagnosis in clinical settings.

Current status on high-resolution histology:

Traditionally, the ultrastructural observation of tissues has required the resolving capabilities of electron microscopy, at the expense of slow and sophisticated sample preparation, low imaging throughput, and high operational costs. Over the last two decades, however, the advent of fluorescence-based optical super-resolution microscopy (SRM) techniques has facilitated nanoscale visualizations of the biological machinery in living organisms, contributing to significant breakthroughs in life sciences [1]. Particularly in histology, the SRM methods have given us a glimpse of their potential benefits for the study of standard FFPE samples, allowing the identification of ultrastructural changes associated with various pathologies [2-4]. Nevertheless, multiple barriers defer the practical adoption of the existing SRM methods in standard histological laboratories. These include complex and expensive equipment, the need for highly specialized operators along with specialized sample preparations, and insufficient data throughput for routine analyses. Hence, a microscopy platform capable of high-throughput and high-resolution imaging while compatible with standard processing workflows would prove advantageous for the adoption of SRM in histology.

Chip-based microscopy for histology:

Recently, photonic chip-based microscopy has been proposed as a tool for bioimaging applications, enabling SRM over large fields of view [5-7] in live [8, 9], and fixed cells [10, 11], as well as in tissue cryosections [12]. In this work, we propose chip-based microscopy as a multimodal imaging platform for histological assessment of FFPE tissue sections. By following standard preparation protocols on diverse paraffin-embedded specimens (Figure 1a), we demonstrate the suitability of this technique for high-resolution and high-contrast microscopy imaging over large fields of view (Figure 1b) across diverse microscopy modalities including waveguide-based total internal reflection fluorescence, intensity fluctuation-based analysis, and correlative light-electron microscopy [13].

 

Figure 1. Photonic chip-based microscopy for histological investigations of FFPE tissue sections. a)  View of a photonic chip containing an FFPE tissue section. The photonic chip is compatible with standard FFPE preparation steps including paraffin melting (shown here), and subsequent solvent incubations for deparaffinization and rehydration. b) Schematic representation of a photonic chip-based microscope setup. An excitation laser source is coupled onto an optical waveguide for fluorescence excitation of the sample via total internal reflection. The fluorescent signal is collected with arbitrary objective lenses of different magnifications, allowing for high-contrast microscope images over large fields of view.

References

1.    Prakash, K., et al., Super-resolution microscopy: a brief history and new avenues. Philosophical Transactions of the Royal Society A, 2022. 380(2220): p. 20210110.

2.    Artelt, N., et al., Comparative analysis of podocyte foot process morphology in three species by 3D super-resolution microscopy. Frontiers in Medicine, 2018. 5: p. 292.

3.    Xu, J., et al., Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis. Nature Communications, 2020. 11(1): p. 1899.

4.    Ilgen, P., et al., STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue. PloS one, 2014. 9(7): p. e101563.

5.    Diekmann, R., et al., Chip-based wide field-of-view nanoscopy. Nature Photonics, 2017. 11(5): p. 322.

6.    Helle, Ø.I., et al., Structured illumination microscopy using a photonic chip. Nature Photonics, 2020: p. 1-8.

7.    Helle, Ø.I., et al., Nanoscopy on-a-chip: super-resolution imaging on the millimeter scale. Optics Express, 2019. 27(5): p. 6700-6710.

8.    Tinguely, J.-C., Ø.I. Helle, and B.S. Ahluwalia, Silicon nitride waveguide platform for fluorescence microscopy of living cells. Optics Express, 2017. 25(22): p. 27678-27690.

9.    Opstad, I.S., et al., A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view. Journal of Biophotonics, 2020. 13(6): p. e201960222.

10.  Coucheron, D.A., et al., High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip. JoVE (Journal of Visualized Experiments), 2019(153): p. e60378.

11.  Archetti, A., et al., Waveguide-PAINT offers an open platform for large field-of-view super-resolution imaging. Nature Communications, 2019. 10(1): p. 1-9.

12.  Villegas-Hernández, L.E., et al., Chip-based multimodal super-resolution microscopy for histological investigations of cryopreserved tissue sections. Light: Science & Applications, 2022. 11(1): p. 1-17.

13.  Villegas-Hernández, L.E., et al., Super-resolution histology of paraffin-embedded samples via photonic chip-based microscopy. bioRxiv, 2023: p. 2023.06. 14.544765.