Label-free Super-resolution Imaging Enabled by Vibrational Imaging of Swelled Tissue and Analysis

Summary

By combining sample-expansion hydrogel chemistry with label-free chemical-specific stimulated Raman scattering microscopy, the protocol describes how to achieve label-free super-resolution volumetric imaging in biological samples. With an additional machine learning image segmentation algorithm, protein-specific multi-component images in tissues without antibody labeling were obtained.

Abstract

The universal utilization of fluorescence microscopy, especially super-resolution microscopy, has greatly advanced knowledge about modern biology. Conversely, the requirement of fluorophore labeling in fluorescent techniques poses significant challenges, such as photobleaching and non-uniform labeling of fluorescent probes and prolonged sample processing. In this protocol, the detailed working procedures of vibrational imaging of swelled tissue and analysis (VISTA) are presented. VISTA circumvents obstacles associated with fluorophores and achieves label-free super-resolution volumetric imaging in biological samples with spatial resolution down to 78 nm. The procedure is established by embedding cells and tissues in hydrogel, isotropically expanding the hydrogel sample hybrid, and visualizing endogenous protein distributions by vibrational imaging with stimulated Raman scattering microscopy. The method is demonstrated on both cells and mouse brain tissues. Highly correlative VISTA and immunofluorescence images were observed, validating the protein origin of imaging specificities. Exploiting such correlation, a machine learning-based image-segmentation algorithm was trained to achieve multi-component prediction of nuclei, blood vessels, neuronal cells, and dendrites from label-free mouse brain images. The procedure was further adapted to investigate pathological poly-glutamine (polyQ) aggregates in cells and amyloid-beta (Aβ) plaques in brain tissues with high throughput, justifying its potential for large-scale clinical samples.

Introduction

The development of optical imaging methods has revolutionized the understanding of modern biology because they provide unprecedented spatial and temporal information of targets across different scales, from subcellular proteins to whole organs¹. Among them, fluorescence microscopy is the most well-established, with a large palette of organic dyes with high extinction coefficients and quantum yields², easy-to-use genetic-encoded fluorescent proteins³, and super-resolution methods such as STED, PALM, and STORM for imaging nanometer-scale structures^4,5. In addition, recent advancements in sample engineering and preservation chemistry, which expand specimens embedded in swellable polymer hydrogels^6,7,8, enable sub-diffraction limited resolution on conventional fluorescence microscopes. For instance, typical expansion microscopy (ExM) effectively enhances the image resolution by four times with fourfold isotropic sample expansion⁷.

Despite its advantages, super-resolution fluorescence microscopy shares limitations that originate from fluorophore labeling. First, photobleaching and inactivation of fluorophores compromise the capacity for repetitive and quantitative fluorescence evaluations. Photobleaching is an inevitable event when light keeps pumping electrons into electronically excited states⁹. Second, labeling the fluorophores to the desired targets is not always a straightforward task. For instance, immunostaining demands a long and laborious sample preparation process and hinders imaging throughput¹⁰. It could also introduce artifacts due to inhomogeneous antibody-labeling, especially deep inside tissues¹¹. Moreover, proper labeling strategies that target fluorophores for the desired proteins might be underdeveloped. For example, extensive screenings were required to find effective antibodies for Aβ plaques¹². Smaller organic dyes, such as Congo red, often have limited specificity, only staining the core of the Aβ plaque. It is, therefore, highly desirable to develop a label-free super-resolution modality that circumvents the drawbacks of fluorophore-labeling and provides complementary high-resolution imaging from cells to tissues, and even to large-scale human samples.

Raman microscopy provides label-free contrast for chemical-specific structures and maps out the distribution of otherwise invisible chemical bonds by looking at the excited vibrational transitions¹³. In particular, stimulated Raman scattering (SRS) imaging on label-free or tiny-labeled samples has been demonstrated to have similar speed and resolution to fluorescence microscopy^14,15. For example, healthy brain region has been readily differentiated from tumor-infiltrated region in human and mouse tissues^16,17. Aβ plaques were also clearly imaged by targeting protein CH₃ vibration (2940 cm⁻¹) and amide I (1660 cm⁻¹) on a fresh-frozen brain slice without any labeling¹⁸. Raman scattering, therefore, offers robust label-free contrast that overcomes the limitations of fluorophores. The question then became how one can accomplish super-resolution capacity using Raman scattering, which could reveal nanoscale structural details and functional implications in biological samples.

Although extensive efforts have been made to achieve super-resolution for Raman microscopy with elegant optic instrumentations, the resolution enhancement on biological samples has been rather limited^19,20,21. Here, based on the recent works^22,23, we present a protocol that combines a sample-expansion strategy with stimulated Raman scattering for super-resolution label-free vibrational imaging, named Vibrational Imaging of Swelled Tissues and Analysis (VISTA). First, cells and tissues were embedded in hydrogel matrixes through an optimized protein-hydrogel hybridization protocol. The hydrogel tissue hybrids were then incubated in detergent-rich solutions for delipidation, followed by expansion in water. The expanded samples were then imaged by a regular SRS microscope by targeting CH₃ vibrations from retained endogenous proteins. VISTA, owing to its label-free imaging feature, bypasses photobleaching and inhomogeneous labeling arising from fluorophore labeling, with much higher sample processing throughput. This is also the first sub-100 nm (down to 78 nm) label-free imaging reported. No additional optical instrumentation besides typical SRS setup^22,24 is required, making it readily applicable. With correlative VISTA and immunofluorescence images, an established machine-learning image-segmentation algorithm was trained^25,26 to generate protein-specific multiplex images from single-channel images. The method was further applied to investigate Aβ plaques in mouse brain tissues, providing a holistic image suited for sub-phenotyping based on the fine views of the plaque core and peripheral filaments surrounded by cell nuclei and blood vessels.

Protocol

All animal procedures performed in this study were approved by the California Institute of Technology Institutional Animal Care and Use Committee (IACUC), and the protocol procedures complied with all relevant ethical regulations. 1. Preparation of stock solutions for fixation and sample expansion Prepare 40 mL of fixation solution by first dissolving 12 g of acrylamide (30% w/v) solid in 26 mL of nuclease-free water. Then, add 10 mL of 16% PFA stock solution to th…

Representative Results

After establishing the working principle of the imaging and analysis method, image registration was done to evaluate the expansion ratio and to ensure isotropic expansion during sample processing (Figure 1A,B). Both untreated and VISTA samples were imaged while targeting the bond vibration at 2940 cm−1, which originates from CH3 of endogenous proteins. In untreated samples, the protein-rich structures like nuclei were dark due to the overwhelming …

Discussion

In summary, we present the protocol for VISTA, which is a label-free modality to image protein-rich cellular and subcellular structures of cells and tissues. By targeting endogenous CH₃ from proteins in hydrogel-embedded cell and tissues, the method achieves an effective imaging resolution down to 78 nm in biological samples and resolves minor extrusion in Huntingtin aggregates and fibrils in Aβ plaques. This technique is the first instance to report sub-100 nm resolution for label-free imaging modalities…

Materials

1.0 M Tris pH 8	Sigma-Aldrich	648314
16% Paraformaldehyde	Electron microscopy science	15710	diluted to 4% in PBS
25x water immersion objective	Olympus	XLPLN25XWMP2	NA 1.05
5XFAD Mice	Mutant Mouse Resource and Research Centers and the Jackson Laboratory	B6SJL-Tg (APPSwFlLon, PSEN1*M146L*L286 V) 6799Vas/Mmjax	Alzheimer brain
60x water immersion objective	Olympus	UPLSAPO60XWIR	NA 1.2
Acrylamide	Sigma-Aldrich	A9099
ammonium persulfate	Sigma-Aldrich	A3678
anti-MAP2	Cell Signaling Technology	8707
anti-NeuN	Cell Signaling Technology	24307
borosilicate coverslip #1.5	Fisher Scientific	1254581
C57BL/6J Mice	Jackson Laboratory (JAX)	664	Normal mice
D2O	Sigma-Aldrich	151882	for SRS calibration
DAPI	Thermo Fisher	D1306
DMEM	GIBCO	10566-016
FBS	GIBCO	A4766
glass slide 3" x 1" x 1 mm	VWR	16004-430
goat anti-chicken IgY, Alexa Fluor 647	Invitrogen	A-21449
goat anti-mouse IgG, Alexa Fluor 647	Invitrogen	A-21236
goat anti-rabbit IgG, Alexa Fluor 488	Invitrogen	A-11034
goat anti-rat IgG, Alexa Fluor 568	Invitrogen	A-11077
Grace Bio-Labs Press-To-Seal silicone isolators	Sigma-Aldrich	GBL664108	microscope spacer
Htt-97Q-GFP Plasmid	Gift from Prof. R. Kopito and Prof. F.-U.Hartl.
Laser scanning microscope	Olympus	FV3000	laser scanning confocal microscope
lipofectamine 3000	Thermo Fisher	L3000001	transfection agent
Lycopersicon Esculentum Lectin DyLight®594 (lectin)	Vector Laboratories	DL-1177-1
Microscope spacer	Grace Bio-Labs	621502
N,N′-methylenebisacrylamide (BIS)	Sigma-Aldrich	M1533	bought as 2% solution in water
Nuclease free water	Thermo Fisher	10977-015
Penicillin-Streptomycin	GIBCO	15140-122
poly-strene beads	Sigma-Aldrich	43302	for resolution characterization
Sodium Acrylate	Sigma-Aldrich	408220
sodium dodecyl sulfate	Sigma-Aldrich	71725
soft-wool paint brush #3	TANIS	000333
SRS Laser	A.P.E	picoEmerald	2ps pulse width
tetramethylethylenediamine	Sigma-Aldrich	T9281
Tissue culture flask 25 cm2	Corning	430639
Triton X-100	Sigma-Aldrich	T8787
Tween-20	Sigma-Aldrich	P9416
tweezer	Fine Science Tool	11295-51
Vibrotome	Leica	VT1200S	the vibratome