Confocal Microscopy for the Three-Dimensional Imaging of Cleared Tissues

Since the introduction of the microscope, tissues have been characterized according to the traditional histological paradigm that relies upon sectioning whole tissues into ultra-thin 2D slices. These slices are then placed on glass microscope slides, labeled and qualitatively interpreted by a researcher or pathologist. While this approach is foundational to all life science fields, it is limited in its ability to characterize complex and heterogeneous tissues. Therefore, since the advent of traditional 2D microscopy, researchers have worked to develop 3D microscopy techniques that characterize tissues in their entirety.

Tissue Clearing

One of the major challenges with the 3D microscopic imaging of tissues is that due to their opacity, optical imaging is limited to a depth of a few cell layers. Therefore, researchers have focused on developing techniques for increasing imaging depth that reduce opacity and these techniques are referred to as tissue clearing techniques. There are currently over a dozen tissue clearing techniques including CLARITY, CUBIC, Scale, SeeDB, Visikol® HISTO™, FocusClear™, i/3/uDISCO and BABB. Each one of these tissue clearing techniques has its own specific advantages and disadvantages and none are suited for all applications.

Fluorescent Labeling

3D microscopy depends equally on tissue transparency as well as fluorescent labeling. Fluorescent proteins (e.g. GFP, RFP, YFP, tdtomato), immunolabels, chemical dyes and various other techniques can be paired with tissue clearing to allow for protein specific imaging. However, a researcher needs to thoroughly understand the compatibility of their tissue clearing technique with labeling approaches as each technique will have different protocol considerations for tissue types and labels. The most common problem that researchers have in imaging tissues in 3D is achieving uniform labeling and it is highly suggested that a researcher starts small and optimizes labeling as they work their way up in tissue thickness.


Once a tissue has been cleared and labeled, the last step before data processing is to image the tissue using either light sheet, 2-photon or confocal microscopy. Confocal microscopy is the most ubiquitous type of 3D imaging technique and is ideal for high resolution/small volume imaging. Larger volumes such as whole rodent brains can be imaged using confocal microscopy, but it will take considerably longer than light sheet microscopy. Prior to imaging, there are a couple of important features of your confocal microscope to take note of. The first of which is whether the confocal microscope is inverted or upright. If the confocal is inverted, then the maximum imaging depth of the system will be limited to approx. 2 mm due to mismatches in refractive index.

If the system is upright, then it is possible the system can be used to conduct whole rodent brain imaging. However, whole brain imaging requires the use of dipping objectives which may or may not be compatible with your tissue clearing technique as many will be destroyed by solvent based techniques (e.g. i/3/uDISCO, Visikol HISTO, BABB). If instead of using dipping objectives you choose to use common air objectives, then your imaging depth will be limited to approx. 2 mm. It is important to note that the working distance of the objective is also very important as oil dipping objectives will be highly limited in imaging depth due not to light attenuation but their working distance.  

Principles of Microscopy - Visikol

Confocal Microscopy Overview

The purpose of confocal microscopy and other 3D microscopy technique are to only capture data from a single z-plane in a tissue at a time. The way confocal microscopes achieve this is through the use of a pinhole mechanism where photons of light outside of the desired z-plane are rejected by a narrow pinhole of approx. 30-70 um. Confocal microscopes illuminate a cleared tissue sample with either a laser or LED at a specific wavelength where the light source passes through all z-planes in the tissue. The resulting fluorescence which is a different wavelength than the excitation wavelength is then passed through the pinhole mechanism by use of a dichroic mirror. By moving the tissue through the focal plane of the objective, numerous optical z-planes can be acquired from a cleared tissue. These z-planes can then be stitched together with other z-stacks to create 3D histological data sets for multiple markers from whole tissues.

Confocal microscope - pinhole - Visikol
Michael Johnson