Q2 Quarterly Review

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. 

Michael 

Michael speaking at Rutgers Univeristy about Visikol. 

Michael speaking at Rutgers Univeristy about Visikol. 

Visikol HISTO Trouble Shooting Guide

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.

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 HISTO Guidebook

 

 

 

Launching the Visikol HISTO Protocol Builder

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. 

Visikol HISTO Protocol Builder

Updated Visikol HISTO Guidebook

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. 

Visikol HISTO Guidebook

Introducing Visikol HISTO-M

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

Figure 1. Confocal z-stack montage of NCI-2170 Organoids stained with SYTOX green, in PBS. Notice that only outer layer of cells is visible to imaging. 20 um z-step.

Figure 1. Confocal z-stack montage of NCI-2170 Organoids stained with SYTOX green, in PBS. Notice that only outer layer of cells is visible to imaging. 20 um z-step.

3D Tissue Culture with Visikol HISTO-M

Figure 2. Confocal z-stack montage of Visikol MICRO-treated NCI-2170 organoids stained with SYTOX green. The full plane of cells is visible in each image. 20 um z-step.

Figure 2. Confocal z-stack montage of Visikol MICRO-treated NCI-2170 organoids stained with SYTOX green. The full plane of cells is visible in each image. 20 um z-step.

Visikol HISTO for Breast Cancer Visualization

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.

Mouse brain slicers now available from the Visikol store!

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. 

Visikol HISTO now compatible with fluorescent protein!

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. 

Fluorescent Protein Only Protocol

FP and Immunolabeling Protocol

See Visikol HISTO in action with a tdTomato mouse brain below that was imaged with a LaVision BioTec Ultramicroscope II

ClearWells now available from the Visikol store for deep tissue imaging

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.

ClearWell.png

Xena Flowers and Dr. Jeffrey Goodman use Visikol HISTO to evaluate traumatic brain injury in rat models

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!

Xena Flowers.png

Visikol ranked one of the top university startups in the country!

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.

Visikol NCET2

Visikol wins "Best in Class" at NJEN Posters, Pitches and Prize event at Princeton Univeristy

Once a year, the New Jersey Entrepreneurial Network provides the entrepreneurial community with an afternoon of posters and networking for startups in New Jersey. Poster presenters introduce their businesses to important contacts among New Jersey’s research universities, small and large business, potential funding sources and professional service providers.

The event was held at Princeton Univeristy and this year’s keynote speaker at the event was Jesse Treu, PhD of Domain Associates

Michael Johnson represented Visikol at the event and was awarded “Best in Class” amongst many other highly successful startups from New Jersey. 

Michael Johnson - Visikol - NJEN Poster

Visikol at Society of Toxicology

Each year, Visikol attends the Society of Toxicology (SOT) Annual Meeting with experts in toxicology from around the world. At SOT this year, the Visikol team discussed their Visikol TOX platform for DART studies as well as their Visikol HISTO platform for the 3D visualization of spheroids. 

This year Michael Johnson presented a poster on the Visikol TOX process during the conference, comparing Visikol TOX skeletal processing for DART studies to traditional KOH-based processing. The Visikol TOX technology platform has the potential to accelerate DART studies by up to 30%, allowing contract research organizations to get studies back to pharmaceutical companies more quickly.  For more, check out the poster here.

Over the last few months Visikol has launched the Visikol HISTO platform for the 3D histological visualization whole tissues. The team has applied this technology in the toxicology and drug development space to specifically improve the characterization of spheroids. Spheroids are 3D cell culture models that are starting to be used frequently for the drug development process as they more accurately mimic in vivo tissues than traditional 2D in vitro models. However, until Visikol HISTO, the characterization of spheroids was limited to 2D and 1D assays that limit the utility of spheroids and the information that can be obtained.

With Visikol HISTO, we are starting to move beyond traditional single end-point spheroid assays and towards whole spheroid characterization. Instead of just receiving a single data-point from a spheroid, Visikol HISTO allows researchers to better understand how drugs are interacting in 3D with spheroid models. We started offering Visikol HISTO kits and reagents in the fall of 2016 and are currently expanding our capacity for spheroid-specific characterization assays. 

Visikol TOX for DART Studies

When we first devised the Visikol TOX platform in 2014, it was not entirely clear to us the value that it would have in the toxicology field. However, through the last three years of development we have transformed this technology into a robust skeletal visualization platform for developmental and reproductive toxicology studies that increases throughput by up to 30%, reducing costs and allowing therapeutics to get to market more quickly. We recently completed the development of this platform and are now offering it for use in developmental and reproductive toxicology (DART) studies, at a cost of $650 per study.

DART studies are one of the most expensive and time-consuming components of the regulatory data required to bring a new chemical entity into the marketplace. In these studies, pregnant animals (rats, rabbits, mice) are dosed with chemicals of interest and the impact of these chemicals on the development of fetuses is investigated. While these studies entail several concurrent processes, the biggest bottleneck in this process is the gross visualization of fetuses for skeletal abnormalities.

For over 90 years, scientists have visualized the gross skeletal morphology of animals using a process called diaphonization, where bones are stained with a red dye called Alizarin (sometimes with blue Alcian cartilage staining) and soft tissues are digested away with a strong base (potassium hydroxide) to reveal the stained bones. While this process is the gold standard for skeletal visualization in all DART studies, it can take up to two weeks for fetal rabbits, can destroy tissues, and requires consistent oversight.

With Visikol TOX we have replaced this destructive and slow process with a rapid and non-destructive process that leverages the Visikol tissue clearing technology. Instead of digesting the soft tissues to visualize bone, the tissue is rendered transparent, resulting in the same endpoint in 2-4 days instead of the traditional 7 to 14 days. We recently demonstrated in a 3rd party GLP equivalency study with Product Safety Labs (Dayton, NJ) that the Visikol TOX approach was equivalent in end-point to the traditional approach while drastically reducing tissue processing time.

We have designed Visikol TOX to be a drop in replacement to traditional KOH-based skeletal processing and are currently working with several CRO’s to integrate it into their DART workflow. All of the Visikol TOX reagents to conduct a 480 fetal rat study cost $650 and the technique can accelerate overall DART study time by up to 30%. 

Visikol TOX - At SOT

Q1 Quarterly Review

Building Visikol continues to be an amazingly fun, challenging, and rewarding endeavor. Every morning, I find myself checking my email right away to see what feedback we have gotten from our customers, and what complex research problems researchers are using our products to investigate. This feedback continually reminds me of why we launched Visikol in the first place – to accelerate innovation and to allow researchers to ask and answer new questions.

Over the last few months we have celebrated many firsts – On February 24th we celebrated our first anniversary as Visikol, Inc. and in January we brought on our first new team member: Dr. Graeme Gardner from Rutgers University. We are thrilled by the progress that we have made so far and are excited to continue to grow the company:

Visikol HISTO Progress:

To date, 300 research labs in 30 countries have used Visikol HISTO, and the feedback that we have received so far has been just fantastic. Bringing an enabling technology into the marketplace is a challenging process fraught with many roadblocks. Through beta-testing Visikol HISTO with researchers in new applications and soliciting feedback, we have been able to uncover these roadblocks and find strategies to mitigate them. To help our users get the most out of Visikol, we continually update and improve our product guidance with all the feedback received from researchers. If you are interested in learning more, please see our updated Visikol HISTO Guidebook.

Visikol TOX Progress:

Of all of the things that we have completed thus far, I am most proud of the Visikol TOX product for developmental and reproductive toxicology studies. Since inventing Visikol TOX in 2014, we have turned it into a valuable bio-imaging platform that not only accelerates the drug discovery process, but also reduces the number of animals required in toxicology studies. Visikol TOX represents to me the great synergies of our team as Tom, Nick, and I all worked on different components that came together to create a valuable and highly impactful platform.

Grant Progress:

Our team has absolutely no shortage of great ideas and we find ourselves continually submitting grants for new applications of the Visikol HISTO technology as well as other novel bio-imaging ideas. We are very fortunate that there are programs like the NIH and NSF small business innovation research grants (SBIR) that fund innovative and cutting edge technology development. In December and January we submitted for six Phase I SBIR grants with collaborators and expect to hear back on most of these grants in the near future. We are optimistic about these projects as we have teamed up with some great collaborators! We also submitted our Phase II NSF SBIR after the successful completion of our NSF Phase I project, and are very excited at the prospect of continuing our NSF funded research. Our goal for 2017 is to further expand our R&D efforts through the use of SBIR grants, and we have placed significant time and effort on these. Since launching Visikol we have submitted thirteen, been rejected from five, been awarded one, and are waiting to hear back on seven.

Things to come:

In the next few months you will see significant developments from our team around Visikol HISTO assays for spheroid visualization. Drug discovery is undergoing a paradigm shift where in-vitro work is moving from 2-D cell based assays to 3D cell based assays as they more accurately depict in-vivo tissues. However, researchers are still using 2D (histology) and 1D assays (total ATP) to evaluate these 3D spheroids, ignoring the spatial features that make them intrinsically more valuable than 2D cell cultures. We are developing 3D assays for spheroids that will allow researchers to move beyond traditional endpoints (IC50) and towards evaluating the spatial heterogeneity of spheroids. For example, we will enable companies to understand where their antibodies are targeting within tumors (necrotic core, periphery) and the type of cell these drugs target.

As we are now gaining traction in the marketplace with Visikol HISTO and Visikol TOX, our focus is shifting from pure research and development, to commercialization and the application of the Visikol HISTO platform to several areas including spheroids. We are excited to apply the Visikol HISTO technology to new problems and will be shifting to spending considerable resources on commercialization in the second half of the year.

I look forward to sharing more great news with you in Q2! Thanks for following our progress.

-Michael