Tissue clearing may alter emission and absorption properties of common fluorophores

Abstract

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Whole-body and whole-organ 3D imaging of hypoxia using activatable covalent fluorescent probe compatible with tissue clearing

“…Xanthene dyes and cyanine dyes, especially the Alexa Fluor series, have been frequently used in most tissue clearing methods. Recently, however, several fluorescent dyes including Alexa Fluor dyes have been reported to change their emission wavelengths and brightness in RIMSs 25 . In this research, we focused on fluorescein isothiocyanate (FITC), tetramethylrhodamine (TAMRA), and Si-Rhodamine as representative xanthene-type dyes, and cyanine 3 and 5 (Cy3 and Cy5) as representative cyanine dyes to be investigated (Figure 2a, upper line).…”

“…Firstly, there are incompatibilities between regular fluorescent dyes and refractive index matching solutions (RIMSs) in which fluorescently labeled tissue is immersed during observation. The brightness of several fluorescent dyes was reported to be decreased in certain RIMSs [25][26][27] . Secondly, unfixable fluorescent small molecules will be washed out in the process of tissue clearing because the integrity of cell membranes is compromised by detergents and organic solvents for rendering tissue transparent 28,29 .…”

Optical Characterization of Biological Tissues Based on Fluorescence, Absorption, and Scattering Properties

“…For example, as shown in Figure 4 , changing tissue conditions by drying (in the skin) and boiling (in adipose fat) caused an increase in the intensity of the obtained fluorescence emissions. Such an increase may be due to varying the water content in the sample after the applied modification, which may alter the amplitude of the emission spectra and affect the absorption characteristics as well [ 50 ]. Additionally, the photostability of the fluorescent molecules, which induces irreversible degradation or “photobleaching,” is greatly affected by the fluorophore environment and the utilized excitation wavelength [ 51 ].…”

Visualizing the Nucleome Using the CRISPR–Cas9 System: From in vitro to in vivo

“…Hence, tissue clearing can significantly change the properties of fluorophores. For example, optical clearing with a mixture of benzyl alcohol and benzyl benzoate not only changed the fluorescence intensity of DAPI and Alexa Fluor dyes, but also shifted the positions of the maxima in their absorption and emission spectra up to several tens of nm [ 193 ]. This effect should be taken into account when selecting the optical filters for working with biological samples to avoid signal mixing.…”

“…This effect should be taken into account when selecting the optical filters for working with biological samples to avoid signal mixing. The same mixture of benzyl alcohol and benzyl benzoate caused the quenching of eGFP fluorescence to the background level [ 193 ]. In contrast, optical clearing with gadobutrol increased the fluorescence intensity of TagRFP 1.5-fold [ 194 ].…”