Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

Summary

Multiphoton microscopy of whole mouse organs is possible by optically clearing the organ before imaging, but not all protocols preserve the fluorescent signal of fluorescent proteins. Using an optical clearing method with ethanol-based dehydration and benzyl alcohol:benzyl benzoate clearing, we show high-resolution multiphoton images of whole mouse brain expressing YFP.

Abstract

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the organ before imaging.^1,2 However, for organs that contain fluorescent proteins such as GFP and YFP, optical clearing protocols that use methanol dehydration and clear using benzyl alcohol:benzyl benzoate (BABB) while unprotected from light³ do not preserve the fluorescent signal. The protocol presented here is a novel way in which to perform whole organ optical clearing on mouse brain while preserving the fluorescence signal of YFP expressed in neurons. Altering the optical clearing protocol such that the organ is dehydrated using an ethanol graded series has been found to reduce the damage to the fluorescent proteins and preserve their fluorescent signal for multiphoton imaging.⁴ Using an optimized method of optical clearing with ethanol-based dehydration and clearing by BABB while shielded from light, we show high-resolution multiphoton images of yellow fluorescent protein (YFP) expression in the neurons of a mouse brain more than 2 mm beneath the tissue surface.

Protocol

1. Animal Perfusion5 and Whole Mouse Brain Clearing The entire length of the procedure can vary depending on the length of time used per dehydration step, but in total the whole process can be conducted in two days. Weigh YFP mice and then deeply anesthetize with an intraperitoneal injection of ketamine/xylazine (100 mg/kg:10 mg/kg). Confirm a surgical plane of deep anesthesia before proceeding to surgery. Check the animal every 5 min to see if it reacts to a firm toe or tai…

Discussion

While standard organic dyes are compatible with a range of organic solvents, and therefore do not pose a particular challenge for clearing protocols, fluorescent proteins are often less tolerant of changes in solvent.⁴ The goal of the present work was to overcome a serious limitation of previous optical clearing protocols where fluorescence of XFPs was lost or severely degraded. The images that are presented here demonstrate that fluorescence from YFP was preserved. The optical clearing technique described …

Materials

Name of the reagent	Company	Catalog number	Comments
Phosphate Buffered Saline	Sigma-Aldrich, Inc.	D8537	500 ml, pH 7.2
Paraformaldehyde	Electron Microscopy Sciences	15710	10 x 10 ml, 16% paraformaldehyde
Ethyl alcohol	American Bioanalytical	AB00515-00500	500 ml, 200 proof
Ethyl alcohol	Pharmco Products, Inc.	111000190	1 gal., 190 proof
Benzyl alcohol	Sigma-Aldrich, Inc.	402834	500 ml, 99+%
Benzyl benzoate	Sigma-Aldrich, Inc.	B6630-IL	500 ml, ≥ 99%
5X/0.5 NA objective	Nikon	AZ Plan Flour 5X	15 WD