New Publication: Multimode Fibre Based Imaging for Optically Cleared Samples

Tissue clearing combined with the use of fluorescent labels has enabled the use of confocal and light sheet microscopy for 3D tissue imaging. However, one of the main issues with many tissue clearing techniques is that the expensive objectives required for deep tissue imaging are not compatible with solvent-based tissue clearing techniques like BABB, 3DISCO, iDISCO, uDISCO and Visikol HISTO which are highly effective. In this publication the researchers show how the use of a   corrected multimode fibre (MMF) can achieve deep tissue imaging while preventing damage to the objective. It was shown that the MMF was not constrained by the refractive index of the immersion medium and was able to provide variable working distances. This MMF was used for the fluorescence imaging of beads and stained neuroblastoma cells through optically cleared mouse brain tissue, as well as imaging in an extreme oxidative environment. Additionally,  Raman imaging of polystyrene beads was performed in clearing media to demonstrate that this approach may be used for vibrational spectroscopy of optically cleared samples.

For the full article click below: 

Multimode fibre based imaging for optically cleared samples

Biomedical Optics Express

Limitations of 3D Cell Culture Imaging

Over the last five years 3D cell cultures have begun to be readily adopted into the drug discovery pipeline due to their improved in vivo relevancy compared to traditional 2D monolayer in vitro models. However, though these models are becoming more commonly used in drug discovery studies, there is currently a major limitation in how they are analyzed as researchers are currently using methods originally developed for 2D analysis. The current paradigm for analyzing these models consists of one of the following techniques which have their own individual limitations:

  • Traditional Pathology: Cell culture models can be analyzed using traditional pathology where they are removed from well plates and processed. Though this method is capable of acquiring pathological information from tissues, the size of these models makes this process super low-throughput, tedious and expensive.
  • Dissolution Based Assays: Many researchers are currently using dissolution based assays where cell culture models are homogenized and analyzed using a fluorescence probe such as Promega's CellTiter-Glo Luminescent kit. While these assays are high-throughput, they do not capture the 3D data held within these models which makes them intrinsically more valuable than traditional 2D cell culture models.
  • Wide Wield Imaging: Well plates can be characterized using wide field imaging where fluorescent markers such as GFP, immunofluorescence and chemical dyes are imaged. Though these assays are high-throughput, they suffer from a  fundamental limitation which is that light attenuation results in an imaging depth of only a few cell layers. Therefore, only data from the periphery of the model is depicted and there is a significant bias to only characterize the periphery of these models which differs substantially from the interior.  
  • Confocal Imaging: Confocal imaging of 3D cell culture models can be high-throughput through use of a high content system such as the Thermo Fisher CX7 LZR, Molecular Devices ImageXpress, Yokogawa CV7000, Perkin Elmer Opera Phenix/CLS Operata or General Electic IN CELL 6000/6500. While these devices allow for a reduction in out of plane light, improved image quality and Z-stacks, they are still limited by light attenuation and thus only the periphery of these models is characterized.

Therefore, the major limitation with the current 3D cell culture imaging paradigm is that none of the current techniques are capable of characterizing these models in their entirety and acquring truly 3D data. To address this problem, we have developed our Visikol HISTO-M reagent which rapidly renders tissues transparent. We have shown that through the application of Visikol HISTO-M reagent to 3D cell culture models that we can dramatically improve both wide field imaging and confocal microscopy. 


Figure 1. In this figure we show NCI-H2170 spheroids of 200 um diameter that have been cleared with Visikol HISTO-M (bottom 2 rows) and not cleared (top 2 rows). It can be seen in the optical Z projections in white boxes that because many researchers portray their data as Z projections that the current lack of complete characterization can be easily missed. 


Figure 2. Visikol HISTO-M also has a substantial benefit for wide-field high content microscopy as tissue clearing allows for a 3-fold increase in cells characertized. An NCI-H2170 spheroid was labeled with SYTOX green and imaged (A) and then cleared with Visikol HISTO-M (B) and imaged. In the uncleared spheroid we were able to count 57 cells whereas we were able to count 151 cells in the cleared spheroid. 

Confocal Microscopy

Spheroid Images.png

Wide Field Microscopy

Visikol HISTO-M Cleared Spheroid

Case Study - NCI-H2170 Spheroids and Cisplatin

SPheroid Graph 2.png

Figure 3. The application of tissue clearing was demonstrated to improve assay sensitivity as all of the cells in 3D models can be characertized instead of just the cells on the periphery. 


Figure 4. Visikol HISTO-M also allows for spatial dose response curves where compound and antibody penetration can be quantatively studied. 



We have demonstrated that conventional assays using confocal microscopy and wide field microscopy have a tendency to bias results by only characterizing the outside of these models and that this effect is substantial. This effect can dramatically alter results for compounds or biologics that have a tendency to only target the periphery of these models as current assays could signficiantly overstate the efficacy of a compound as only the periphery is being characterized. 

Spheroid Graph.png

How can you improve your 3D cell culture assays

We currently sell our Visikol HISTO-M reagent through our online store along with the buffers and reagents required to immunolabel tissues uniformly. However, if you do not have access to a high content confocal imager or have the bandwidth to work on implementation, we run a full service lab where we can run 3D cell culture assays for clients. Check out our services page for more information. 

Come see Visikol at Society for Neuroscience - Booth 1027

From November 11 to 15th Visikol will be attending the Society for Neuroscience conference in Washington, DC. Come and learn about how Visikol HISTO can be used to add a new dimension to your neuroscience research. Visikol will be displaying its Visikol HISTO tissue clearing products that allow for the 3D microscopic visualization of whole mount tissues like entire rodent brains. Visikol is focused on making 3D microscopy easy and is excited to discuss with you how leveraging 3D information can improve your research. At our booth we will describe the following applications of our technologies: 

- Owl brain connectomics

- Mapping vasculature in rodent brains

- Microscopic virtual reality to visualize your data in 3D

- 3D immunolabeling

- Whole brain light sheet microscopy

Visikol Presents at the World Youth Forum in Sharma el Sheikh, Egypt

Dr. Michael Johnson was invited as a key note speaker to speak at the World Youth Forum in Sharma el Sheikh, Egypt. The conference was focused on giving voice to the youth of Egypt and highlighted inspiring experiences from youth around the world. Dr. Johnson was invited to discuss Visikol's journey from an idea to a company that is helping accelerate drug discovery and get therapeutics to patients more quickly, 

Fore more information on the conference: World Youth Forum 

Dr. Michael Johnson Visikol
Michael Johnson World Youth Forum

Visikol Featured in Rutgers Magazine

Visikol was featured this week in the Rutgers Magazine on page 40. The company can trace its roots back to Rutgers where it originally developed its Visikol HISTO technology. The company is now focused on leveraging its proprietary tissue imaging technologies through its 3Screen service to help accelerate drug discovery. Visikol has shown that its 3Screen services dramatically improve the the amount and completeness of data that can be acquired from in vitro cell culture models as well as whole mount tissues. 

Michael Johnson Visikol Rutgers

Tom Villani Featured in Biocompare Article on 3D Cell Cultures

Check out this recent Biocompare article by Lauren Tanabe, Ph.D. where Visikol Inc's CSO Tom Villani discusses 3D cell culture models and their use in the drug discovery process. 3D cell culture models are being adopted in the drug discovery space at a rapid rate due to their in vivo relevancy. Visikol is leveraging 3D cell culture models and their patented Visikol HISTO technology to offer best-in-class 3D drug discovery assays: 

  • Antibody penetration
  • Apoptosis
  • Angiogenesis
  • Cell-cell junctions and cadherins
  • Cell proliferation
  • Cell migration
  • Cell viability
  • Cholestasis
  • Drug induced liver injury

For more information go to: Services

Visikol HISTO Microtissue
  • Fibrosis
  • Immune cell invasion
  • Inflammatory Signaling
  • Mitochondrial Health + Toxicity
  • NASH
  • Neuronal Differentiation
  • Neuronal Function
  • ROS
  • Steatosis



Visikol Presents at Allergan Innovation Forum on 3D Drug Discovery Assays

Recently, Visikol began offering drug discovery assay development services to clients that are focused on 3D in vitro cell culture screening and whole mount tissue analysis. In early September, Tom Villani and Michael Johnson were invited to give a seminar at an Allergan Innovation Forum in Irvine, California about these services and how they fit into the drug discovery process. 

For more information on our services check out Visikol Services or email us at 

Visikol Gets Second Place at Boston Biotech Week Pitch Competition

This past week Visikol participated in Boston Biotech Week and Dr. Michael Johnson pitched during the biotech startup competition and came in second place overall out of twenty companies.


The competition brought out companies from around-the-world and was judged by Investors and Business Development Directors from Abbvie, Atlas Venture, Agent Capital, SR One, MRL Venture Fund and Lightstone Ventures.

Pitch Competition.png
Visikol Pitch Competition Boston

Visikol HISTO Assay Services

Our long-term goals as a company are to improve the drug discovery process and the diagnosis of disease. This quarter, we are excited to launch our offering of assay development services for microtissues in drug discovery by leveraging our Visikol HISTO technology. Currently, high content imaging of microtissues introduces an unavoidable bias, since microtissues are too thick and opaque, and thus only the outermost cells can be detected. The outermost cells are more exposed to nutrients, oxygen, drug compounds, etc. with respect to the innermost cells, and thus characterization of microtissues has been fundamentally limited. Using Visikol HISTO, microtissues are rapidly rendered transparent allowing for the characterization of effects of drug candidates on the complete population of cells contained within microtissues. With Visikol HISTO, you can break down the effects of a drug compound according to populations of cells segregated by cell-type, location in the microtissue, etc.

For more information on our drug discovery services and to see if your research question can be answered with our platform, email us at or check out Visikol Services




Visikol HISTO Tutorial Videos

We keep in contact with our sample testers and customers to understand how we can improve our user experience so that every customer can incorporate the Visikol HISTO technology into their workflow without friction. Through this process, we identified that many of our customers wanted more detail and background on how exactly our products work and a step-by-step overview of how to use them. To address this need, we have launched a suite of tutorial videos to help our customers better use our products.

Visikol HISTO - Getting Started Tutorial

Confocal Microscopy Tutorial

Light Sheet Microscopy Tutorial


Visikol and InSphero App Note

Check out Visikol and InSphero's recent application note that features the use of Visikol HISTO-M with InSphero's microtissue models. It was shown that combining Visikol HISTO-M with confocal microscopy allows for a 3-fold increase in the number of cells detected in microtissues.

Check out the full application note here

Visikol and InSphero Application Note

Teaming Up With Young Talent to Disrupt Bio-imaging

Medical imaging has made remarkable leaps and bounds. In today's world, doctors and scientists can generate rich images from a multitude of sources, such as confocal microscopes and MRI machines. With popular programs such as the open-source, powerful ImageJ editor, these images can be further enhanced and even be used to construct accurate three-dimensional models of the original sample. However, despite breakthroughs in virtual reality technology, these 3-D models and image stacks are still viewed in a two-dimensional context: through a monitor. The low usage of inexpensive VR solutions such as Google Cardboard can be attributed to the time and expertise normally required to create a VR compatible model or video clip. To allow for simple integration into VR technology, Visikol teamed up with Somerville High School student Joe Napoli to write a Stereoscopic 3D Projection plugin for ImageJ.

Visikol HISTO Vasculature Viewer

The Stereoscopic 3D Projection plugin provides a straightforward way to create anaglyph or Google Cardboard-compatible VR projections. Written in Java 8, this plugin takes in an image stack as input. After reading in values such as slice spacing from the image stack's metadata, the user can dictate how many degrees the projection will rotate before generating a new frame, how many degrees the final projection will rotate, the start and stop rotation of the projection, and whether the projection is done in anaglyph or for Googe Cardboard. Here, the user can also adjust the slice spacing, if necessary. To generate these projections, the plugin utilizes ImageJ's powerful innate 3D projection function to create projections for the left and right eyes. The right eye projection is rotated an additional five degrees, to account for the spacing between one's eyes. If the projection is in anaglyph format, the left and right eye projections are colored red and blue, respectively, and then merged into one model. For the Google Cardboard format, however, the image stack retains its original color and the left and right eye projections are placed side by side instead of being merged. Regardless of whether the product is formatted for anaglyph or Google Cardboard, the plugin's output can be saved as a video or directly viewed.

Visikol ImageJ Plugin User Interface


The Stereoscopic 3D Projection plugin's source, along with screenshots of the plugin in action, can be found on GitHub. The source is also attached to the JAR, located on the plugin's official ImageJ site. The Stereoscopic 3D Projection plugin can be installed through Fiji's updater after adding to the list of update sites, or by downloading the JAR file and placing it into ImageJ's plugins folder.

Visikol HISTO Stereoscopic

Governor Chris Christie stops by Visikol headquarters

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. 

CEO and Co-Founder Michael Johnson with Governor Chris Christie 

CEO and Co-Founder Michael Johnson with Governor Chris Christie 

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 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, substitute ethanol wherever methanol appears in the protocol, and perform all steps at 4°C.
  • 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