High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

Summary

Spectral Iterative Bleaching Extends Multiplexity (IBEX) builds upon the base IBEX technique by adding heparin blocking to minimize nonspecific binding and leveraging spectral detection with computational unmixing to suppress autofluorescence. This approach accelerates image acquisition while reducing sources of background, enabling robust multi-round, high-parameter spatial proteomic analyses.

Abstract

Highly multiplexed imaging enables the study of functionally diverse cells and their niches within their native tissue environments. Iterative Bleaching Extends Multiplexity (IBEX) is a cyclic immunolabeling and fluorophore inactivation technique that allows for multiple markers to be visualized on the same tissue section. Captured images can be subsequently analyzed to acquire single-cell data to define cell clusters, their localizations, and neighboring cell types. Interpreting these data relies on the ability to distinguish true marker expressions from sources of background inherent to fluorescence microscopy. Spectral IBEX, an adaptation of the IBEX protocol, integrates spectral confocal detection with computational unmixing and incorporates heparin blocking to reduce charge-based off-target binding. This combination improves the signal-to-background ratio, suppresses tissue autofluorescence, and minimizes bleed-through while also reducing acquisition time compared to conventional multi-track confocal imaging. Application to human nasal polyp tissue, a model characterized by high eosinophil content and strong autofluorescence, demonstrated reliable imaging of 26 markers across structural, immune, and cell state compartments over six imaging rounds. The resulting workflow generates high-dimensional, spatially resolved proteomic information that captures complex tissue architecture and cellular niches. Together, this optimized approach provides a robust and broadly applicable strategy for multiplexed imaging, particularly suited to tissues where autofluorescence and non-specific staining limit conventional approaches.

Introduction

Tissues are comprised of a diverse set of cells with specialized functions that organize into three-dimensional structures and, through interaction, coordinate the biological processes that are necessary for life. Pathology arises when disruptions to the cellular composition or tissue architecture interfere with these interactions and compromise tissue homeostasis¹. Knowledge of how the numerous specialized cell types localize and interact within our tissues is required in order to understand the biology that underlies human health and disease.

Advances in single-cell profiling technologies at the transcriptomic, epi....

Protocol

Nasal polyp tissues used in this study were collected during sinus surgery at Hamilton Health Sciences with ethical approval from the Hamilton Integrated Research Ethics Board. Informed consent was obtained from all participants.

1. Sample collection

1. Obtain samples during routine polypectomy procedures performed on patients with chronic rhinosinusitis. Upon surgical removal, place the samples in a vial containing medical saline on ice for transport and immediately process them for imaging to preserve tissue integrity.

2. Reagent preparation

1. Prepare fixation and permeabilization solu....

Results

Two fundamental challenges faced by immunofluorescence imaging techniques are the non-specific binding of fluorophore-conjugated antibody probes and tissue autofluorescence. This protocol is designed to help distinguish a true signal from background to optimize multiplexed immunofluorescence techniques.

Heparin blocking reduces non-specific fluorophore binding while preserving signal

Discussion

Spectral IBEX, an adaptation of the IBEX protocol, successfully addresses two key issues in immunofluorescence imaging: non-specific binding and autofluorescence. Treating highly eosinophilic tissues with heparin and using spectral unmixing to eliminate autofluorescence and spectral overlap between channels significantly increased the detection of true signal above background. Capturing all markers in a single pass reduces acquisition time, which may increase the rate of IBEX cycling. While conventional confocal acquisit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2N sulfuric acid (H2SO4) solution	N/A	N/A	Whatever is available/used in the lab
500 mL beaker	N/A	N/A	Whatever is available/used in the lab
6-well plate	N/A	N/A	Whatever is available/used in the lab
Bcl-6 - Alexa Fluor 647	BD Biosciences	561525	Dilution factor 1:100
BD Cytofix/Cytoperm Fixation/Permeabilization Kit	BD Biosciences	554722
Benchtop dissection microscope	N/A	N/A	Whatever is available/used in the lab
Bovine Serum Albumin (BSA)	SIGMA-ALDRICH	A4503
CD11c - Alexa Fluor 700	BD Biosciences	561352	Dilution factor 1:100
CD138/Syndecan-1 - PE	BioLegend	356503	Dilution factor 1:200
CD20 - Alexa Fluor 488	Invitrogen	53020282	Dilution factor 1:100
CD21 - Alexa Fluor 532	NovusBio	NBP2-60733AF532	Dilution factor 1:100
CD23 - PE	BioLegend	338507	Dilution factor 1:200
CD3 - iFluor 594	AAT Bioquest	100320C0	Dilution factor 1:100
CD31 - Alexa Fluor 700	BioLegend	303134	Dilution factor 1:100
CD34 - iFluor 594	AAT Bioquest	103400C0	Dilution factor 1:100
CD38 - Alexa Fluor 700	BioLegend	303523	Dilution factor 1:100
CD4 - Alexa Fluor 647	BioLegend	300520	Dilution factor 1:100
CD45 - Alexa Fluor 532	ThermoFisher Scientific	58045942	Dilution factor 1:100
CD54/ICAM-1 - Alexa Fluor 647	BioLegend	353113	Dilution factor 1:100
Chrome Alum-Gelatin Adhesive	Newcomer Supply	1033A
Cryomold	Fisher Scientific	NC9511236	Also, Catalog No.50-465-347 for bigger samples
DAPI calibration slide	Agar Scientific	AG2273
DAPI solution, 1 mg/mL	ThermoFisher Scientific	62248	Dilution factor 1:3000
E-cadherin/CD324 - Alexa Fluor 647	BioLegend	147307	Dilution factor 1:100
EpCAM/CD326 - Alexa Fluor 594	BioLegend	324228	Dilution factor 1:100
Eppendorf/falcon tubes	N/A	N/A	Whatever is available/used in the lab
Fluoromount-G	SouthernBiotech	0100-01
Forceps	N/A	N/A	Whatever is available/used in the lab
FoxP3 - eFluor 660	ThermoFisher Scientific	50577382	Dilution factor 1:100
Frosted end microscope slides	ChemScience	GEW45-2575-W
GL7 - Alexa Fluor 488	ThermoFisher Scientific	53590282	Dilution factor 1:100
Heparin sodium salt	SIGMA-ALDRICH	H3393	10KU
HLA-DR - PE	NovusBio	NB100-77855PE	Dilution factor 1:200
Human TruStain FcX (Fc Receptor Blocking Solution)	BioLegend	422302	Dilution factor 1:100
Humidity chamber	N/A	N/A	Whatever is available/used in the lab
Ice bucket with lid	N/A	N/A	Whatever is available/used in the lab
IgD - Alexa Fluor 488	BioLegend	348215	Dilution factor 1:100
IL4Ra/CD124 - PE	BioLegend	144803	Dilution factor 1:200
Imaris File Converter	OXFORD instruments	N/A	Coverting any image file format to .ims format (https://imaris.oxinst.com/microscopy-imaging-software-free-trial#file-converter)
Isopentane (2-Methylbutane)	N/A	N/A	Whatever is available/used in the lab
Ki-67 - Alexa Fluor 488	BD Biosciences	558616	Dilution factor 1:100
Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-Ply	Kimtech Science	06-666
Large (i.e., 25 cm long) forceps	N/A	N/A	Whatever is available/used in the lab
Liquid Nitrogen	N/A	N/A	Whatever is available/used in the lab
Lithium borohydride (LiBH4)	SIGMA-ALDRICH	222356
MECA-79 - Alexa Fluor 488	Invitrogen	53603680	Dilution factor 1:100
Microscope Slide Cover Glass No.1 22x50 mm	Fisher Scientific	12-542A
Milli-Q water	N/A	N/A	Whatever is available/used in the lab
MUC2 - Alexa Fluor 488	NovusBio	NB120-11197AF488	Dilution factor 1:100
Nunc Lab-Tek Chambered Coverglass	ThermoFisher Scientific	155380
PAP pen	abcam	AB2601
Phosphate buffered saline (PBS) solution	N/A	N/A	Whatever is available/used in the lab
Scalpel	N/A	N/A	Whatever is available/used in the lab
SimpleITK Imaris Extensions	N/A	N/A	Software used to align images from different rounds (https://github.com/niaid/imaris_extensions)
Sucrose	SIGMA-ALDRICH	S0389
Tissue-Tek O.C.T. Compound	Sakura Finetek USA	4583
Triton X-100	SIGMA-ALDRICH	X100
Zeiss LSM 980	ZEISS Microscopy	N/A	The imaging system used in this study
Zen Blue	ZEISS Microscopy	N/A	The software used for post acquisition image processing