Automated immunolabeling to perform highly multiplexed tissue imaging with ARIA

The Chu Lab is a recently established group at University College Institute of Ophthalmology in partnership with Moorfields Eye Hospital led by Dr. Colin Chu. It is is dedicated to ocular immunology, immune cell imaging, spatial biology and gene therapy.

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. In this study, automated immunolabeling has been applied to obtain a versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues.

“As a clinician and immunologist with an interest in imaging, I was involved in developing a new technique for multiplexed immunohistochemistry. We wanted to automate the process by adding a microfluidic solution, but I had no experience with this technology. The ARIA system fit our needs and was incredibly simple and intuitive to use. The software is well-designed and readily interfaced with our microscope. Fluigent provided support throughout setup and transformed what seemed a large undertaking, into an incredibly simple task.”

Colin Chu, University College London


Dr. Colin Chu has recently established his own lab at University College London (UCL) Institute of Ophthalmology in partnership with Moorfields Eye Hospital. As an ophthalmologist specialized in ocular immunology, his research is focused on using in vivo and ex vivo imaging to better understand the causes of blinding diseases.

While he was a visiting fellow in the lab of Dr Ron Germain at the National Institute of Health (NIH) in Maryland, USA, Dr Chu helped establish an automated highly multiplexed immunohistochemistry approach with his colleague Dr Andrea Radtke. This work was published in Nature Protocols in February 2022.

It uses an automated immunolabeling technique called IBEX, to achieve over 30 distinct antibody markers on the same section of tissue. Previously this had only been performed manually, limiting the throughput, speed and accessibility of the technique. Working together with Fluigent to connect an ARIA microfluidics system to a Leica THUNDER microscope, the two systems were able to interface to deliver automated staining and image acquisition.

Dr. Chu is now setting up the system at UCL to apply the automated immunolabeling technique, IBEX, to the retina in mice, zebrafish and human post-mortem tissue. In addition to multiplexed tissue imaging, his research topics include ocular gene therapy and in vivo immune cell imaging using adaptive optics, optical coherence tomography (OCT) and fluorescence microscopy to better understand ocular inflammation.

Fluigent instrument ARIA coupled to a widefield microscope to perform automated multiplexed tissue imaging

High content imaging of tissue samples was performed using an automated immunolabeling method, consisting of iterative cycles of antibody labelling, imaging, and fluorophore bleaching using lithium borohydride (LiBH4). Image processing and registration were done using the SimpleITK open-source software.

To perform an automated IBEX study, the complete setup comprised several pieces of equipment:

– a pressure source (here our FLPG)

–  a single output sequential perfusion system ARIA

–  a closed bath imaging chamber containing tissue sections and stage heater from Warner Instruments

– a THUNDER microscope from Leica

Using ARIA software, a method containing all the fluid injection and image acquisition steps was created. Once loaded, the sequence can be run automatically. The automated immunolabeling method relies on TTL signals sent and received by ARIA, enabling a cycle of image acquisition to be launched at the end of an injection step. The software  interfaced with the Leica THUNDER microscope.

Once an  image  was acquired, the next fluid injection step started, to bleach the previous round, commence antibody staining with the next panel, then trigger microscope acquisition again. Ultimately, the full sequence of steps gave rise to high-quality, multiplexed protein level data of over 30 markers.

Fixed human tissues including mesenteric lymph node, small intestine and skin were profiled (Figure 1, 2). These tissues are usually very challenging to image due to their delicate structure and high background autofluorescence.

As stated by the authors, the original IBEX method was limited by the requirement of manual execution (manual pipetting for antibody staining and fluorophore inactivation). Here, ARIA provides an affordable automated solution to access high-content imaging as part of the spatial biology revolution.

IBEX automated immunolabeling method: 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.

Fig. 1: Schematic overview of automated IBEX protocol.

Partial Results

Radtke AJ. *, Colin CJ*. et al, have established a high throughput automated immunolabeling 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).

Examples of Images obtained using automated IBEX method in human tissues

automated immunolabeling ibex aria human jejunum
Figure 2. Images from human jejunum (six cycles, 16 of 24 parameters shown). Scale bars: 200 µm (left), 50 µm (cyan box), 25 µm (red box).
automated immunolabeling ibex aria skin
Figure 3. 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 automated and 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.

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