Interfacing ARIA with fluorescence microscopy to automate multiplexed tissue imaging

Understanding cell-cell interactions and spatial relationships in healthy or diseased organs represents a technical challenge. Single-cell genomics is powerful but lacks spatial context, however, traditional immunofluorescence enables one to capture only two to six molecular features. Imaging technologies have been developed to address these issues, but each possesses limitations that constrain widespread use.


Radtke AJ.*, Colin CJ*. et al, have established a high throughput immunolabelling protocol recently published in Nature Protocols based on the automation power of our Fluigent ARIA instrument coupled to an image alignment open-source software package. This powerful approach relies on iterative staining and bleaching method to perform high-resolution imaging of more than 65 parameters. This Iterative Bleaching Extends Multiplexing protocol (IBEX) represents a robust and reproducible method to perform deep phenotyping and spatial analysis of cells in complex tissues (healthy organ, infected organs, tumor microenvironment).

Experimental procedure

a) The automated IBEX protocol uses our compact microfluidics system ARIA to deliver multiple solutions to an imaging chamber placed on an inverted microscope stage. Fluids are moved through the system with compressed air and delivered to the imaging chamber based on protocols defined by the user. The inverted microscope and ARIA device communicate via TTL signals using precise timing established by the user with the Aria control software.

b) Samples are sectioned onto coated coverslips and assembled into a closed bath imaging chamber. Following assembly, the imaging chamber is secured to a magnetic platform and mounted onto the microscope stage.

c) Automated IBEX consists of: (1) nuclear labeling with Hoechst, (2) antibody labeling for 1 h at 37 °C using a heated microscope stage, (3) imaging ROIs, (4) bleaching with LiBH4, and (5) repeating Steps 2–4 until the desired number of parameters is achieved, typically 12 h for a six-cycle, 25-plex experiment.

Example of Images obtained using automated IBEX method in human tissues

a) Images from human jejunum (six cycles, 16 of 24 parameters shown). Scale bars: 200 µm (left), 50 µm (cyan box), 25 µm (red box).

b) Images from human skin (five cycles, 15 of 19 parameters shown). Scale bars: 200 µm (left), 25 µm (insets). Keratin 10 (K10), Keratin 14 (K14).


In this paper, the authors have demonstrated the automation power of our sequential perfusion system ARIA together with its ability to be synchronized with a widefield (or other) microscope to perform multiplexed antibody labelling. ARIA can send and receive TTL signals to launch an image acquisition cycle and resume the perfusion protocol once the imaging cycle has been completed. Other applications such as DNA-paint, OligoSTORM, dose/response studies, or dynamic pulse-chase experiments can be automated using ARIA.

To learn more about this ARIA-based high throughput automated immunolabelling protocol, explore the article here:

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