How Does CLARITY Work?
CLARITY tissue processing involves the incubation of a tissue sample with a cocktail that may include acrylamide, bisacrylamide, formaldehyde and thermal initiator. Formaldehyde mediates cross-linking of biomolecules to acrylamide monomers via amine groups (see figure below). Hydrogel polymerization is initiated by incubating the perfused tissue at 37 °C, resulting in a meshwork tissue-gel hybrid that preserves biomolecules and structural integrity of tissue at the ultrastructural level. Lipid membranes are removed by passive thermal, or active electrophoretic, clearing. The resulting intact tissue-hydrogel hybrid can undergo multiple rounds of molecular and structural interrogation using immunohistochemistry for proteins, and in situ hybridization for nucleic acids, using a variety of light microscopy methods.
Obtaining high-resolution information from solid tumors, while maintaining the global perspective needed to understand the complex tumor microenvironment, represents a key challenge for preclinical and clinical cancer applications. Technologies currently utilized for preclinical as well as diagnostic, prognostic, and predictive clinical cancer research and standard of care practice are dependent on 2-dimensional analysis of thin tissue sections (5-10 micron) in the format of formalin fixed paraffin embedded tissue (FFPE). This is primarily due to the limitations of visualizing thick tissues with conventional light microscopy techniques. Other technologies utilizing molecular technologies have advanced the preclinical and clinical cancer field; however these suffer from the inability to link molecular information to tumor and microenvironment architecture and morphology. Preserving the spatial information at the same time as capturing high resolution quantitative information, could lead to a better understanding of the role and types of cells within the solid tumor microenvironment. A current revolution in disease tissue processing and imaging addresses this challenge with a suite of new optical methods based on forming and imaging transparent tissue-gel composites.
The Clarity Chemistry Process
What is COLM?
COLM (CLARITY Optimized Light Sheet Microscopy) is based on light sheet microscopy, a fluorescence microscopy technique where the sample is illuminated perpendicularly to the direction of observation by a laser light-sheet, i.e. a laser beam which is focused only in one direction. As only the observed section is illuminated, this method reduces the photodamage and stress induced on a living sample. Also the optical sectioning capability reduces the background signal and thus creates images with high contrast. Because light sheet microscopy scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1000 times faster than those offered by point-scanning methods. Historically this method has been limited in resolution to cellular scales; however COLM (specifically developed for CLARITY samples) has succeeded in enabling subcellular resolution with light sheet even deep within intact tissue.
The COLM Optical Layout
Additional Resources (PubMed)
Multiplexed intact-tissue transcriptional analysis at cellular resolution.
Sylwestrak E, Rajasethupathy P, Wright MA, Jaffe A & Deisseroth K. Cell 2016 64:792-804.
Optimization of CLARITY for Clearing Whole-Brain and Other Intact Organs.
Epp JR, Niibori Y, Hsiang HL, Mercaldo V, Deisseroth K, Josselyn SA & Frankland PW eNeuro 2015 May 25; 2(3).
Functional Targeted Brain Endoskeletonization.
Deisseroth, K. and Gradinaru, V. 2012 USPTO Patent App. 13/980,842.
Basomedial amygdala: mediates top-down control of anxiety and fear.
Adhikari A, Lerner T, Finkelstein J, Pak S, Jennings JH, Davidson TJ, Ferenczi E, Gunaydin LA, Mirzabekov JM, Ye L, Kim SY, Lei A & Deisseroth K. Nature 2015 527:179-85.
Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits.
Lerner TN, Shilyansky C, Davidson TJ, Evans KE, Beier KT, Zalocusky KA, Crow AK, Malenka RC, Luo L, Tomer R, Deisseroth K. et al. Cell. 2015 Jul 30;162(3):635-47.
Advanced CLARITY for rapid and high-resolution imaging of intact tissues.
Tomer R, Ye L, Hsueh B, Deisseroth K. et al. Nat Protoc. 2014 Jul;9(7):1682-97.
CLARITY for mapping the nervous system.
Chung K, Deisseroth K. et al. Nat Methods. 2013 Jun;10(6):508-13.
Structural and molecular interrogation of intact biological systems.
Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K. et al. Nature. 2013 May 16;497(7449):332-7.