Brain cells: A 3-D visualization of fluorescently-labeled brain cells within an intact brain tissue.
Brain cells: A 3-D visualization of fluorescently-labeled brain cells within an intact brain tissue.

Deeper Insights in Transparent Animals

Interview with Viviana Gradinaru – co-developer of CLARITY and developer of PARS

Transparent organisms help us to identify spatial arrangements and connections of cells and tissues, especially neuronal circuits can easily be identified and characterized. CLARITY is on everyone’s lips. Viviana Gradinaru, assistant professor at the California Institute of Technology, has further developed the method and recently caused a stir by rendering whole rodent bodies transparent. In this interview she comments on the developments of clearing strategies and their impact on modern science.

Brain cells: A 3-D visualization of fluorescently-labeled brain cells within an intact brain tissue.



You have been involved in the initial CLARITY publication with Karl Deisseroth. What was your role in the development of CLARITY?

I started the project that led to CLARITY early 2010 after conversations with Karl Deisseroth. It was the only non-optogenetic project in the lab and it therefore required a different infrastructure. In parallel to preliminary experiments, I setup a special CLARITY lab in a new building, which is now the home of the CNC program (including biochemistry, surgery, and microscopy capabilities).

In the beginning, only Charu Ramakrishnan, a research assistant and lab manager, and I worked on the project. But it required highly interdisciplinary skills. So Karl looked through his postdoctoral applicants to find someone with chemical engineering expertise (in particular with hydrogels, which we thought could be used to support cleared tissue), and found an applicant to his lab who wanted to work on a stem cell and optogenetics project, but who also had chemical engineering expertise. This was Kwanghun Chung from Georgia Tech, who joined us as a postdoc after about one year into the project. And Logan Grosenick, for example, joined us as specialist for imaging, both hardware and software wise. Kwanghun was then the driving force to develop the first generation of CLARITY [1].

How did the idea of clearing a tissue by introducing a hydrogel come up in the first place?

Initially we wanted to make the neuronal networks durable und remove elements distracting for observation. Therefore we intended to instruct neurons to create an endoskeleton, e.g. by expressing keratins or chitins, that should stabilize the cellular structure after the brain had been removed. A hydrogel was needed for support. Both the idea of hydrogel embedding and removing elements for easier visualization of specific neuronal circuits have been described in the primary patent by Karl Deisseroth and me [2]. At that time we considered organ structure to be digested away from the targeted endoskeleton structures using hypotonic shock, enzyme digestion, heat, and the like. We thought that additional three-dimensional support should be supported by any suitable matrix, e.g. collagen, resins or hydrogels. During our work we noticed, that the hydrogel itself, which ended up being acrylamide, was sufficient to stabilize the tissue structure and better (deeper) visibility could be obtained by removing lipids.

Did you notice the impact CLARITY would have on science at that time?

Yes. We’ve learned a great deal from naturally-transparent organisms before: think about all the research on the nematode worm, or the zebrafish, that allows for easy identification of gene mutations and their consequences on organ development and survival. Some of this work was recognized with a Nobel Prize in 2002. Unfortunately, looking through mammalian tissue is not an equally easy task due to the light-scattering lipids that are present throughout thick tissues, including the cell membrane, making organs opaque.

Can you please describe the advances PACT made to CLARITY? What were the challenges to overcome?

PACT [3] is just Passive CLARITY, with adjustments to make the hydrogel pores larger to speed up passive lipid removal and increase antibody penetration. We were trying to clear the tissue without using an electrical field, which the original method [1, 4] used, but was challenging to implement and could damage tissue.

How did you develop RIMS?

Our initial paper used FocusClear. However, many labs trying to implement the method were worried about lack of availability and cost. We developed RIMS (refractive index matching solution) to make our and others work easier and affordable [3]. RIMS is an affordable alternative to FocusClear that researchers can make in the lab and can also be used to store samples over months without damaging fluorescence. This being said, our samples also gave good results when imaged in a simple sorbitol solution (we call it sRIMS and we give the recipe in the paper as well).

How does whole body clearing by PARS work?

The current report in Cell builds on the CLARITY method to achieve whole adult organism clearing and staining. This was possible due to the realization that the intrinsic circulatory systems (such as the vasculature) could be used to achieve clearing rather than passive diffusion of detergents and labels, which would be prohibitively slow for large organs or whole organisms. This is the PARS method, short for Perfusion-assisted Agent Release in Situ.

The vascular routes of the rodents are used to introduce and distribute the necessary solutions: fixing agents, washing buffers, hydrogel solutions and SDS detergent and buffers. Even antibody staining is achieved by cardiac perfusion. For clearing of the brain and the spinal chord, we recommend intracranial perfusion into the cerebrospinal fluid routes (PARS-CSF). The whole procedure can be achieved in 2 weeks time maximum. Most organs clear within days. And this method could also be applied to other organisms – for example human corpses – although imaging and data analysis can become a bottleneck with increasing volumes for investigation.

What were the major challenges to overcome?

There were three main issues we had to take care of: Preserving tissue integrity, which includes preventing organ deformation and fine axons or epitope damage, achieving a minimum protein and nucleic acid loss and retain, when applicable, endogenous fluorescence – all these while still clearing the tissue faster than what passive clearing would allow. Although transparency to the eye is a rough indicator of clearing – complete transparent tissue was not the main goal, as overclearing can also happen at the cost of tissue integrity. We cleared our samples sufficiently to detect molecular markers, including RNA transcripts at single molecule resolution.

What are the possibilities these novel optical clearing methods give to science? Which new areas of application do you see in the future?

A transparent whole-organism is particularly useful to map the distribution and molecular identity of peripheral nerves at their target organs throughout the body; such nerves could then be modulated with optogenetics in animal models of disease to understand what needs tuning to improve symptoms and the resulting knowledge could facilitate better therapies that rely on, for example, electrically stimulating nerves for better organ function or for decreasing chronic pain. While it is difficult to predict how quickly research will progress to reach the stage of clinical trials, there is lots of momentum and enthusiasm. Pharmaceutical companies are becoming interested in creating optogenetics units and we also had interest in PARS clearing, which can even be used in human tissue. We have collaborators who are using a passive form of CLARITY (PACT) on human brain tissue from patients who died of Alzheimer’s disease or dementia. And we have also shown that it works to detect cancer cells in a biopsy from a human skin-cancer patient. Looking through tissues with cell and protein resolution can teach us a great deal about pathology progression – which is the first step towards better cures.

Your main research focus is on optogenetics. How do optical clearing and optogenetics combine?

PARS complements optogenetics, in that it can reveal circuit-wide effects of optogenetic manipulations and also aid in mapping novel circuits that need tuning in disease.

In optogenetics we modify neurons in the rodent brain so that they respond to light and induce a certain behavior in the model organism. When you shine a light, the neurons fire. When you turn it off, the neurons stop firing. We hope to learn from optogenetics more about how the brain functions. Studying brain functioning in animal models, such as rodents, increases our understanding of how human brains work – and, with it, our ability to design better treatments for brain-related conditions.

A major challenge is to identify the circuits which show a therapeutic effect after treatment. We do not have detailed maps of connectivity across the brain. This can be a problem, as our prior optogenetic study on deep brain stimulation showed [5]. That electrical deep brain stimulation might act fundamentally through white matter away from the electrode site highlights the need for better brain maps. It is however difficult to create such maps for fine axons that run in bundles throughout the brain when the common method to do this is sectioning the tissue in paper-thin slices, then imaging each one, and putting it all back together with imaging software: it is slow, tedious, costly, and error prone. Clearing the tissue allows us to visualize and identify cellular components and their molecular identity without slicing.

This video shows step-wise cross-sectional images at different depths throughout fluorescently labeled intact intestine tissue. In these images, blood vessels are labeled green, cells are labeled red, and cell nuclei are labeled blue. Images were taken using the PARS technique (Credit: Bin Yang and Viviana Gradinaru).

You have been selected as one of the "40 under 40" from Cell. Congratulations! What does motivate and inspire you?

Learning something new about the biological system both motivates and inspires me. And I see technology development as a facilitator to get that new understanding.

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