Method enables protein labeling of tens of millions of densely packed cells in organ-scale tissues

In this figure extracted from the research study, staining of proteins highlights various cell type markers: neurons overall (cyan), and cells specifically involved with neurotransmitters dopamine (yellow) and acetylcholine (magenta). Credit: Chung Lab/MIT Picower Institute

A new technology developed at MIT enables scientists to label proteins across millions of individual cells in fully intact 3D tissues with unprecedented speed, uniformity, and versatility. Using the technology, the team was able to richly label whole rodent brains and other large tissue samples in a single day.

In their new study in Nature Biotechnology, they also demonstrate that the ability to label proteins with antibodies at the single-cell level across whole brains can reveal insights left hidden by other widely used labeling methods.

Profiling the proteins that cells are making is a staple of studies in biology, neuroscience and related fields because the proteins a cell is expressing at a given moment can reflect the functions the cell is trying to perform or its response to its circumstances, such as disease or treatment.

As much as microscopy and labeling technologies have advanced, enabling innumerable discoveries, scientists have still lacked a reliable and practical way of tracking protein expression at the level of millions of densely packed individual cells in whole, 3D intact tissues such as an entire mouse brain or a full region of a human brain.

Often confined to thin tissue sections under slides, scientists therefore haven't had tools to thoroughly appreciate cellular protein expression in the whole, connected systems in which it occurs.

"Conventionally, investigating the molecules within cells requires dissociating tissue into single cells or slicing it into thin sections, as light and chemicals required for analysis cannot penetrate deep into tissues," said study senior author Kwanghun Chung, associate professor in The Picower Institute for Learning and Memory, the Departments of Chemical Engineering and Brain and Cognitive Sciences, and the Institute for Medical Engineering and Science at MIT.

"Our lab developed technologies such as CLARITY and SHIELD, which enable investigation of whole organs by rendering them transparent, but we now needed a way to chemically label whole organs to gain useful scientific insights."

"Imagine marinating a thick steak by simply dipping it in sauce. The outer layers absorb the marinade quickly and intensely, while the inner layers remain largely untouched unless the meat is soaked for an extended period. The same principle applies to chemical processing of tissues: if cells within a tissue are not uniformly processed, they cannot be quantitatively compared.

"The challenge is even greater for protein labeling, as the chemicals we use for labeling are hundreds of times larger than those in marinades. As a result, it can take weeks for these molecules to diffuse into intact organs, making uniform chemical processing of organ-scale tissues virtually impossible and extremely slow." 

A mouse brain hemisphere stained with various cell type markers: neurons overall (cyan), and cells specifically involved with neurotransmitters dopamine (yellow) and acetylcholine (magenta). Credit: Chung Lab/MIT Picower Institute

The new approach, called "CuRVE," represents a major advance—years in the making—toward that goal by demonstrating a fundamentally new approach to uniformly processing large and dense tissues whole.

In the study, the researchers explain how they overcame the technical barriers via an implementation of CuRVE called "eFLASH," and provide copious vivid demonstrations of the technology, including how it yielded new neuroscience insights.

"This is a significant leap, especially in terms of the actual performance of the technology," said co-lead author Dae Hee Yun, a former MIT graduate student and now a senior application engineer at LifeCanvas Technologies, a startup company Chung founded to disseminate the tools his lab invents.

The paper's other lead author is Young-Gyun Park, a former MIT postdoctoral researcher now an assistant professor at KAIST in South Korea.  

by Massachusetts Institute of Technology

Source: New method enables protein labeling of tens of millions of densely packed cells in organ-scale tissues