Frequently Asked Questions

Below you will find answers to the most commonly asked questions regarding LifeCanvas’ products and technology. Don’t see your query? Contact us at info@lifecanvastech.com and we will happily share our expertise.

What does the acronym stand for?

Stabilization to Harsh conditions via Intramolecular Epoxide Linkages to prevent Degradation.

What does SHIELD do?

SHIELD protects tissue samples and biomolecules within the sample from environmental stressors (heat and chemicals) without increasing inherent autofluorescence. SHIELD’s polyepoxy molecules crosslink surface-exposed protein residues with multiple flexible intramolecular bonds to provide structural reinforcement and enhance the stability of proteins.

Why should I use SHIELD over other tissue preservation methods?

SHIELD greatly enhances protein preservation, especially the native fluorescence of endogenous fluorescent proteins. It also allows destaining, restaining, and multiplexed imaging, and does not quench endogenous fluorescent reporters. Additionally, SHIELD results in far less autofluorescence, and preserves mRNAs for detection using fluorescence in situ hybridization (FISH).

What tissues can I preserve?

Any tissues that can be preserved with CLARITY can also be preserved with SHIELD. This includes brain, liver, lung, heart, kidney, pancreas, skin, and other tissues.

With which species is SHIELD compatible?

SHIELD has been most extensively tested on mouse and rat tissue. However, it is also compatible with non-mammalian and other mammalian (e.g. marmoset and human) samples as well as tissue cultures (e.g. organoids).

How long does SHIELD preservation take?

It takes 4-5 days to preserve mouse organs, and is worth the wait! SHIELD samples produce more meaningful data through better preservation of antigenicity and endogenous fluorescence, along with allowing multiplexing of labeling.

What is Clear+ Tissue Clearing?

Clear+ is a next generation clearing technique using newly developed reagents. Clear+ makes no compromises in sample transparency, processing time, and preserving sample architecture.

Why should I use Clear+ over other detergent-based or organic clearing methods?

Clear+ is a simple clearing method that employs aqueous reagents. It has the following advantages:

  • There is no change in tissue size. All other techniques result in either tissue expansion or shrinking.
  • It is very fast. With our patented stochastic electrotransport technology featured in the SmartBatch+, delipidation takes only 24 hours. It can also be done passively in only 7 days.
  • Endogenous fluorescence is preserved. This means there is no need for anti-FP immunostaining.
  • It is very simple. Including SHIELD and EasyIndex, it takes only 5 solution changes to go from a PFA fixed brain to a fully cleared brain.

 

How should samples be prepared?

To ensure that samples maintain their molecular and mechanical integrity we recommend that samples first be preserved with SHIELD. They can otherwise be damaged by detergents and the electrophoresis.

What tissue types can be cleared?

Many of our users clear mouse brains. However, users have also cleared spinal cord, liver, tumor, lung, heart, kidney, and bladder tissues, as well as brains from other species including rat, bird (zebra finch), marmoset and human samples. We can also help  troubleshoot and optimize protocols for specific tissue samples, and have tissue holders available for samples of variable size. Visit the EasyIndex page to see examples of cleared and index-matched tissue types.

How large of a tissue sample can be cleared?

Theoretically, if the sample can fit inside the clearing chamber, it can be cleared. However, larger tissue samples will take more time to clear. Rat brains can take around 3-4 days to clear in SmartBatch+ while larger samples may take much longer. Therefore, if possible, blocking tissue into sections is recommended. Also, imaging quality can be impacted if samples become too thick in either the XY (lateral) or Z/axial dimension, so this should be a consideration when designing experiments and blocking tissue into sections.

Is the SmartBatch+ compatible with iDISCO?

No, the SmartBatch+ is for aqueous clearing methods only. It is compatible with tissues preserved with SHIELD, CLARITY, or SWITCH. Samples that have only been perfused and post-fixed with PFA, and which have not been through one of the preservation protocols mentioned here, are not suitable for clearing. Without SHIELD treatment, PFA-fixed samples lose their fluorescence and often undergo severe structural deformation and loss of antigenicity upon delipidation.

What are the main advantages of the SmartBatch+ labeling function?

  • It is an order of magnitude faster than passive labeling (2 days vs. weeks to months).
  • Its turnkey design allows you to simply load your samples & probes.
  • Our unique sample cup with patent-pending nanoporous membrane eliminates tissue contamination and damage to probes.
  • Unparalleled uniformity of labeling results in consistent signal intensities from sample surface to core by combining SWITCH with stochastic electrotransport.
  • It typically uses 5-10 times less antibody for abundant targets; 2-3 times less for less abundant (e.g. c-FOS).

 

How does the SmartBatch+ labeling work?

The SmartBatch+ is an active labeling device that combines stochastic electrotransport & eFLASH. Samples are loaded into a cylindrical sample holder along with antibodies and labeling buffers. At the beginning of the experiment, high pH prevents the binding of antibodies to their targets. Stochastic electrotransport drives antibodies into the tissue until they equilibrate throughout the sample. Over the course of the labeling reaction, the pH gradually drops inside the sample cup which increases antibody binding affinity and allows antibodies to bind to their targets.

How many samples can I label at once in the SmartBatch+?

Up to 12 whole mouse brains or 4 whole rat brains.

What kinds of antibodies and dyes are compatible with the SmartBatch+?

The SmartBatch+ is compatible with key neuroscience targets including: activity markers such as cFos, neuronal markers like NeuN, GABAergic interneuron subtype markers like parvalbumin (PV), calbindin (CB), and calretinin (CR), and glial cell markers like the astrocyte marker GFAP and microglial marker IBA-1. Please contact us for our full list of validated antibodies and dyes.

Can SmartLabel be used only for labeling the brain or can it be used for labeling other organs as well?

Although the majority of our work is performed with mouse and rat brains, organs that clear well (e.g. liver, kidney, intestine, and pancreas) can still be effectively labeled

What is a rolling shutter?

A rolling shutter on a sCMOS camera is one of the important components in axial sweeping light sheet microscopy. On an sCMOS camera there is an area of active pixels that can register light. The active pixels of the camera sensor can be selectively restricted, behaving like a virtual slit. In the context of an axially sweeping light sheet, the pixels on the sCMOS can be tailored to register specific parts of the light sheet, or more importantly, the waist of the light sheet where one obtains the best quality images. The synchronization of the beam waist sweeping with the column of active pixels effectively forms a shutter that is moving, or “rolling”, by omitting components of the light sheet outside of the beam waist. 

Why is there dual-sided illumination on the SmartSPIM?  

Dual-sided illumination can reduce striping artifacts and allow for homogeneous illumination for large samples (e.g. whole mouse brains).  

How thick is the light sheet illumination in SmartSPIM?

The z-resolution of a light sheet microscope is not only affected by the light sheet thickness but also other factors, including point-spread-function (PSF) of the detection objective and illumination side lobes. Taking these factors into consideration, the system PSF is most essential for determining image quality. For our SmartSPIM system, the PSF in z is ~1.8 μm with our 15X, NA 0.4 objective, and ~4.2 μm with our 3.6X, NA 0.2 objective.

Is SmartSPIM compatible with both aqueous and organic imaging solutions?

SmartSPIM is compatible with all types of aqueous and organic imaging immersion solutions including EasyIndex, ethyl cinnamate, and dibenzyl ether. The imaging chamber can be easily switched out to accommodate tissues cleared via different methodologies.

How important is sample preparation for SPIM?

Sample preparation is absolutely critical for achieving publication-quality images and data. A sample that is poorly cleared will result in low resolution images, even with the best microscope. A tissue that is poorly labeled will result in compromised signal-to-noise ratios and will be difficult to analyze. Here at LifeCanvas Technologies, we are experts in tissue clearing and immunolabeling. We can offer tips for best sample preparation practices as well as gauge the quality of the sample. We also have a complete pipeline for tissue clearing, staining, imaging, and analyses.

What are ideal samples to image with SmartSPIM?

SmartSPIM is ideal for rapid imaging of optically cleared immobilized tissue, whole organs, or tissue slabs.  SmartSPIM is not ideal for imaging adherent cell monolayers (because the lightsheet is thicker than the monolayer) and live specimens (because of movement).  

What is the importance of refractive index matching?

Refractive index matching is achieved by making the refractive index of your sample equivalent to that of the immersion media. Matching the refractive index results in less refraction of light as it passes through interfaces, allowing for better resolution when imaging deep within the sample. Our EasyIndex solution is used for refractive index matching of tissues, allowing light to pass unhindered through the sample during imaging.

How does one account for the heterogeneity of tissue in imaging?

As many tissue samples consist of materials with different absorbing and light scattering properties, what one perceives as “in-focus” for one z-position of the sample will be different for another z-position. To account for these discrepancies, we have a “Focus Compensation” feature, which allows the user to set the detection objective height throughout the z-position of the sample to ensure the FOV is always in focus.

How large is the data after acquisition?

The datasets will range from gigabytes to terabytes. This depends on imaging volume, number of channels acquired, and step sizes of acquisition (where a smaller step size results in more data being acquired). As an example, using the 3.6X objective in our SmartSPIM light sheet microscope, images acquired from a whole mouse brain in three channels with 1.8 μm x1.8 μm x 4 μm (xyz) voxel size generates ~ 1TB of data.

What is your typical turnaround time?

We can typically complete projects with pre-validated targets for processing and imaging within 8-12 weeks with an additional 1-2 weeks for analyses depending on the complexity of the project and the number of samples. Please see our CRO Terms & Conditions for more information.

How are you able to preserve and image endogenous fluorescence?

Our SHIELD tissue transformation method provides exceptional preservation of endogenous fluorescence reporters (e.g. GFPs and RFPs) from transgenic mouse lines or from labeling through viral transfection.

How should I prepare my samples for LifeCanvas 3D histology?

All you need to do is provide PFA-fixed samples. We will send you the sample preparation protocol and shipping instructions and take care of the rest.

What are your deliverables?

We will provide a summary of representative images, stacks of all raw image files, and if requested, analysis outputs (including cell count/densities per brain region, heatmaps, and coronal section images with atlas overlays). We can also offer higher magnification (15X) imaging from our SmartSPIM light sheet microscope for selected ROIs.

What targets have been validated using your technologies?

Upon request, we can provide a list of validated antibodies, dyes, and stains that work effectively in our active labeling devices (SmartBatch+ and SmartLabel). This list covers numerous targets in the fields of neuroscience, development, and disease, and is continually expanded through our ongoing R&D.

How many targets can be achieved for one sample?

Samples can be labelled and imaged for up to 4 channels (at 488, 561, 642 & 785 nm).