We always love heading out of the lab to chat with researchers in person to learn about the work that they are doing and how we can potentially help. Today, Michael presented about Visikol at New Jersey Medical School to a group of PhD students and Post Docs.
We are excited to announce that our publication on pairing Visikol TOX with Optical CT imaging was just published in Birth Defects! Thanks to Dr. Mark Pierce from Rutgers BME, Alex Magsam, and John Miller from Modus QA!
Chris Christie came by the Visikol lab today to chat about innovation and biotech in NJ. Michael got the chance to introduce the Visikol technology and talk about how we are working to improve bio-imaging.
What a great three months for Visikol! I am incredibly proud of the progress that we have made in reaching our milestones and shifting the paradigm of histology from 2D to 3D. Over the last few months we have focused entirely upon improving our Visikol HISTO technology and are looking forward to the second half of the year with many exciting announcements to come. Below you can read about what we have been focusing on for the last three months.
Improved User Experience:
In launching any new product whether it is Airbnb or Visikol HISTO, the user experience is critical as building a product that works in your hands is not enough – The product needs to perform flawlessly in the hands of ALL your users. With Visikol HISTO, we have focused a lot of our time on improving the customer experience so that any researcher regardless of background or skill set can successfully drop the technology into their existing bio-imaging workflow. We have done this through an extensive beta test and partnering with researchers in diverse fields. Through this process, we gained an intimate understanding of how our customers use the Visikol HISTO technology and in Q2 launched the Visikol HISTO protocol builder to help improve the overall customer experience. This web app was built by Nick Crider and addressed our biggest shortcoming with Visikol HISTO which was that customers wanted customized protocols for THEIR specific applications. Moving forward, our focus will be on continually improving the user experience so that customers can more quickly and successfully drop the Visikol HISTO technology into their workflow. In Q3, we will begin launching a suite of tutorial videos on 3D bio-imaging as well as application specific Visikol HISTO kits, image processing plugins and a webinar series.
Build out of Visikol HISTO bio-imaging platform as a service (i.e. HISTOPlex):
One of the long-term goals for Visikol is to develop and launch a 3D bio-imaging service business that can assist pharmaceutical companies with the drug discovery process. Specifically, we plan to develop several bio-imaging assays that will allow pharma companies to extract actionable 3D insights from tissues. We are tentatively calling this platform HISTOPlex and it will be a high throughput system that leverages the Visikol HISTO technique. We began developing this platform in Q1 of 2017 and plan to begin offering it in late 2018 to customers. We have shown that for tissues like spheroids commonly used in drug discovery that we can acquire more accurate and complete information than is acquired today.
Big Data Analysis:
While we will continue to build on the Visikol HISTO technology and improve the user experience, it is essentially complete. This is an exciting time as we are now able to transition Visikol from focusing on building tools for acquiring data to focusing on HISTOPlex to analyze the data. This is a major shift as most publications and work in the tissue clearing space have focused solely on data acquisition from tissues. With HISTOPlex, we are able to generate hundreds of gigabytes of data from tissues and are beginning to develop a platform for storing this data and extracting features of interest. We see HISTOPlex shifting the paradigm of how tissues are characertized and look forward to accelerating the drug discovery process.
I look forward to updating you on our progress in Q3 - stay tuned.
We have introduced a trouble shooting guide below that address common problems and feedback that we have received from beta testers and customers. The trouble shooting guide describes the sources of each problem and potential solutions.
I can’t image past 500-800 µm. Labeling appears uneven, and drops off significantly at this depth.
- Antibody concentration too high: ring of intense staining near surface, drops off significantly after that.
- Solution: Reduce antibody concentration, if signal is too weak, incubate in lower concentration for half of time, and then re-incubate in higher concentration.
- Antibody concentration too low: signal drops off into middle of tissue.
- Solution: Increase antibody concentration.
- Optical attenuation due to absorption of photons by upper layers of tissue causes “shadow” to tissues below, even with perfect staining
- Solution: Increase laser power and gain as depth increases. Caution: increased laser power increases rate of photobleaching, be sure samples contain no air bubbles. Leica SP5 and SP8 can automate laser power and gain corrections. Compare intensity loss to nuclear stain intensity, since nuclear stain diffuses very fast into tissue. Can use this signal to correct for signal loss in image processing.
Intense band of labeled tissue at surface, then significant drop-off afterwards.
- Antibody concentration too high.
- Solution: reduce concentration by increasing dilution factor. See our antibody optimization protocol.
My tissue didn’t clear!
- Plastic Incompatibility
- Visikol HISTO-2 will degrade polystyrene. For processing tissues with the Visikol HISTO approach we suggest moving away from polystyrene and towards polypropylene and glass where possible. Plastic leaching into your sample may affect the clearing ability of Visikol HISTO.
- Incomplete Dehydration/Clearing
- Most of the time a lack of tissue transparency is simply due to not completely dehydrating a sample. If you use methanol or ethanol for dehydration that has water in it and is not pure, you will not remove all the water from your tissue, resulting in tissue cloudiness. This can also be caused by not sealing the vessels containing your sample when clearing, as Visikol HISTO-2 is hygroscopic. Additionally, not using enough volume of Visikol HISTO-1 and Visikol HISTO-2 for your tissue size can cause inadequate clearing.
For a mouse brain that is not completely clear, we suggest placing the brain back into 7-10 mL of Visikol HISTO-1 for 24 hours, followed by transfer to 25 mL methanol for 2 hours. Then transfer back to 7-10 mL Visikol HISTO-1 for 24 hours, followed by 7-10 mL of fresh Visikol HISTO-2 for 24 hours.
Fluorescent protein quenched
- To visualize fluorescent protein, samples must be dehydrated using t-Butanol instead of methanol.
- Keep cleared samples in the dark, and protect your specimens with aluminum foil as fluorescent proteins photobleach rapidly when exposed to ambient light.
- Do not treat fluorescent protein labeled samples with H2O2 bleaching step; this step will oxidize fluorescent protein and signal will be lost.
My antibody didn’t label the tissue
- Some antibodies are not compatible with 3D immunolabeling. Validate the specificity of your antibody on small tissue sections before proceeding to larger tissues. Contact us if you have any questions about your specific antibody.
- Only purchase antibodies that have been validated for use in IHC.
- Please follow our antibody optimization protocol.
Center of tissue appears dark
- Insufficient antibody concentration
- Solution: Increase antibody concentration. A range of concentrations should be explored on a small section of tissue prior to scaling to large tissues.
- Optical attenuation leads to diminished signal at increasing depths depending on several factors, such as concentration of label bound in upper layers of tissue, level of autofluorescence, type of objective, and laser power.
Tissue has become yellow
Visikol HISTO will cause tissues to become slightly yellow during the clearing process. To reduce this effect, conduct all tissue processing steps at room temperature. This tissue yellowing will effect the orange color channel (TRITC) the most.
For the full Visikol HISTO Guidebook click here.
Visikol was featured this week at the NSF/NIH Innovation Zone at the BIO conference in San Diego. The Innovation Zone features biotech startup companies that have received SBIR grant funding.
Not all antibodies are made equal and not all will work for 3D tissue imaging with Visikol HISTO. Recently we have compiled a list of the best antibodies that our team and our collaborators have used so far with Visikol HISTO. Check it out here.
We have assembled all of our knowledge about using Visikol HISTO with various tissues into an easy-to-use tool that will generate a step-by-step protocol specifically for your tissue, label, and microscope. The Protocol Builder web app allows you to select parameters specific to your goals and delivers a printable and shareable protocol so you will be sure to get the best results from Visikol HISTO. Thanks so much to our beta testers for all the feedback we have incorporated into this tool.
To try out the protocol builder click below. Let us know what you think and if you have any feedback or questions.
Over the past six months, we have been compiling all the feedback, considerations, and advice about using Visikol HISTO into our comprehensive Visikol HISTO Guidebook. Our new and improved guidebook has detailed descriptions of every consideration that is important to labeling and tissue clearing. To read through our updated guidebook click below.
Seeing Deeper into 3D Tissue Cultures
Microtissues and organoids are too thick to be fully imaged using confocal microscopy. In PBS, only the outer layers of cells may be imaged. After treatment with Visikol HISTO-M, cells can be imaged on every plane through the center, allowing you to extract more information from your tissue models.
3D Tissue Culture in PBS
3D Tissue Culture with Visikol HISTO-M
Visikol was selected to exhibit at the NSF/NIH Innovation Zone at this years BIO2017 International Conference in San Diego. The company was awarded an NSF Phase I SBIR in 2016 and has submitted several other SBIR grants in the last year that are being considered for funding.
Visikol HISTO can be applied to a wide-range of applications from mapping the neurons within a mouse brain to characterizing a breast biopsy in its entirety. Here we show Visikol HISTO being used to visualize Ki67 expression within a 960 um thick piece of mouse mammary tumor tissue.
Over the last year we have developed the Visikol HISTO tool which can acquire huge quantities of 3D data from tissue - Michael chatted last week on a panel at the NJ Tech Council HealthTech Conference about the platform and big data in the healthcare space. We are working hard to turn our data into actionable insights for researchers and pharmaceutical companies.
As researchers optimize the Visikol HISTO process for their immunolabels of interest, it is important to use smaller tissues before proceeding to whole organs like the brain. This saves the researcher a lot of time and money through the optimization process and ensures that they achieve the best results possible. To help researchers with cutting their tissues into smaller pieces, we have added coronal and sagittal 2mm brain slicers to the Visikol store.
When we first launched Visikol HISTO in late 2016 we did not have a reliable protocol for use with fluorescent proteins and the technique would result in FP quenching. To address this problem we have developed two new protocols that allow for Visikol HISTO to be used with FP and immunolabeling at the same time.
See Visikol HISTO in action with a tdTomato mouse brain below that was imaged with a LaVision BioTec Ultramicroscope II
After launching Visikol HISTO we got a lot of questions from researchers on how best to image their tissues and to mount them. To address this problem we have introduced the ClearWell™ which is a silicon well that sticks to glass microscope slides and allows for Visikol HISTO to be used with a wide array of tissue thicknesses and objectives. We currently have three different ClearWell sizes to allow for imaging of all tissue thicknesses.
In a recent publication from Kyung Hee Univeristy in Korea, researchers used Visikol for Plant Biology to study seed shattering using GUS staining with tissue clearing.
Xena Flowers and Dr. Jeffrey Goodman from the Department of Developmental Neurobiology, NYS Institute for Basic Research and the College of Staten Island have been using Visikol HISTO to evaluate traumatic brain injury (TBI) in rat brains in 3D. Specifically, they have been characterizing the impact of TBI on the blood brain barrier. Stay tuned for a publication describing their work!
Visikol was recently selected by the National Council of Entrepreneurial Tech Transfer as one of the best Univeristy Startups in the country. The University Startups Conference and Demo Day brings together a diverse audience of university Startup Officers, university startups, entrepreneurs, Fortune 1000/Global 1000 corporations, VCs, angel investors, policy leaders, and federal government agencies. The event provides a venue to form and maintain robust transaction networks among the participants with the aim to align university IP and startups with investor and corporate needs.