Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

Abstract

Millions of archived formalin-fixed, paraffin-embedded (FFPE) specimens contain valuable molecular insight into healthy and diseased states persevered in their native ultrastructure. To diagnose and treat diseases in tissue on the nanoscopic scale, pathology traditionally employs electron microscopy (EM), but this platform has significant limitations including cost and painstaking sample preparation. The invention of single molecule localization microscopy (SMLM) optically overcame the diffraction limit of light to resolve fluorescently labeled molecules on the nanoscale, leading to many exciting biological discoveries. However, applications of SMLM in preserved tissues has been limited. Through adaptation of the immunofluorescence workflow on FFPE sections milled at histological thickness, cellular architecture can now be visualized on the nanoscale using SMLM including individual mitochondria, undulations in the nuclear lamina, and the HER2 receptor on membrane protrusions in human breast cancer specimens. Using astigmatism imaging, these structures can also be resolved in three dimensions to a depth of ~800 nm. These results demonstrate the utility of SMLM in efficiently uncovering ultrastructural information of archived clinical samples, which may offer molecular insights into the physiopathology of tissues to assist in disease diagnosis and treatment using conventional sample preparation methods.

Single molecule localization microscopy coupled with touch preparation for the quantification of trastuzumab-bound HER2

“…Fluorescent microscopy methods can now achieve high resolution and have been extensively used to investigate growth factor receptors 44 . Super-resolution microscopy methods have been used to examine HER2 in tissues qualitatively 29 , 45 . In particular, formalin fixed paraffin-embedded tissue slices were visualized using anti-HER2 antibodies (detecting the intracellular HER2 epitope) and fluorescently labeled secondary antibodies.…”

“…STED images showed HER2 is largely found on the membrane, and that HER2 clusters may be present 45 . Additionally, 2D and 3D SMLM was used to detect HER2 in breast cancer tissues 29 . Fine patterns of HER2 on the membrane were visualized.…”

“…These techniques are also compatible with tissue specimen imaging. SMLM was used to detect HER2 in paraffin embedded tissue sections 29 and to determine how G-protein coupled receptors organize in fresh-frozen human tumor samples 30 .…”

Single-molecule fluorescence microscopy review: shedding new light on old problems

“…For instance, single-molecule localization microscopy in combination with astigmatism imaging applied to formalin-fixed, paraffin-embedded (FFPE) breast cancer samples after immunostaining, has enabled the visualization of proteins in mitochondrial and nuclear membranes as well as a cellular membrane protein (oestrogen receptor Her2) which is overexpressed in one-fourth of the cells [177]. RNA polymerase movements along DNA during the transcription cycle were tracked using 3D super-resolution imaging [178].…”

Quantitative sub-resolution blood velocity estimation using ultrasound localization microscopy ex-vivo and in-vivo

“…Super-resolved images can be generated by accumulating the positions of the source centers in hundreds or thousands of images. In optics, this technique has been used to image intracellular proteins (Betzig et al 2006) and the adhesion of live cells (Shroff et al 2008) at nanometric scales, providing new understanding of cellular architecture (Creech et al 2017). A similar approach can be used in ultrasound, where gas-filled encapsulated microbubble (MB) contrast agents with a number weighted mean diameter on the order of 1-5 µm act as blinking sources.…”