Contract Research Services

Delipidating tissue to enable intact-sample labeling and imaging involves exposing samples to harsh treatments like high temperatures and pH changes. If left unchecked, this can cause damage to proteins and overall tissue structure, leading to incomplete or inaccurate image datasets. Therefore, to preserve the sample’s endogenous fluorescence and antigenicity along with tissue architecture, we use a novel tissue preservation technique called SHIELD that forms intramolecular bonds using polyfunctional, flexible epoxides to stabilize tissue architecture.

After SHIELD preservation, the tissue can be delipidated and either refractive index (RI)-matched for imaging or subjected to active immunohistochemical labeling. Notably, SHIELD-preserved tissues are well-suited for antibody multiplexing, i.e., iterative staining and de-staining, without loss of tissue antigenicity, to build up a rich picture of protein expression over repeated rounds of imaging.

Eliminating membrane lipids is crucial to enabling better light penetration for imaging and increasing tissue permeability for active transport of molecular probes deep into intact tissue. Our active clearing device, SmartClear II Pro, employs a patent-pending stochastic electrotransport mechanism to foster rapid delivery of exogenous molecules such as the detergent SDS into tissues, facilitating uniform removal of light-scattering membrane lipids. Thanks to the application of a rotational electric field which minimizes the displacement of structural biomolecules, the tissue is cleared without any damage or deformation.

Attempting to passively immunolabel samples that are a few millimeters to one centimeter thick usually takes many weeks and very high concentrations of expensive antibodies, without any guarantee that staining will extend to the sample’s innermost structures.

Our SmartLabel device combines two technologies – stochastic electrotransport and SWITCH – and allows us to achieve whole-organ antibody staining that is uniform from surface to core. While stochastic electrotransport provides for efficient distribution of antibodies into the organ, SWITCH controls reaction kinetics to ensure that antibody binding isn’t activated until reagent concentration has been homogenized throughout the sample. The result is strikingly uniform labeling that enables you to visualize proteins deep within the organ and study the fine-scale topography of the cells they identify.

Acquiring high resolution, three-dimensional volumetric image data of whole organs using confocal or two-photon microscopy is a time-consuming and expensive process, as these slow line-scanning techniques are best suited to small, localized regions of interest. Light-sheet microscopy overcomes this speed limitation by selectively illuminating distinct focal planes sequentially from the sides of the tissue sample to achieve optical sectioning. LifeCanvas’s own light-sheet microscope, SmartSPIM, offers superior imaging speed and uniform axial resolution across the entire sample, generating datasets with pixels sized 1.8 µm/px in XY and with 4 µm Z-steps (3.6x, 0.2 NA objective; ~4.5 µm tall PSF). With rapid 4-color acquisition (488, 561, 642, & 785 laser lines) of your embedded samples, we can precisely overlay multiplexed immunofluorescence signals and fluorescent protein expression patterns alike.

Ultimately, some level of quantitative data analysis may be necessary to obtain true biological insights. Analysis of fully intact, whole organ datasets requires advanced algorithms to align three-dimensional image volumes to standardized organ atlases (such as the Allen Brain Atlas), followed by object quantification (such as fluorescence intensity quantification or cell counts) within aligned and segmented regions. LifeCanvas Technologies is pioneering these and other quantitative data analysis measures for 3D volumetric datasets.