As medical research becomes increasingly complex, the need for a more comprehensive understanding of the biological processes underlying disease has become more important than ever. This has led to the development of new technologies and techniques that allow for more detailed and accurate analysis of tissue samples. One such technology is multiplex immunohistochemistry, which has revolutionized how researchers analyze tissue samples.

Multiplex Immunohistochemistry

Multiplex immunohistochemistry is a technique that allows researchers to simultaneously visualize and quantify multiple proteins within a single tissue sample. This is achieved by using multiple fluorescently labeled antibodies that bind to specific proteins of interest. Each antibody is labeled with a different fluorescent dye, which allows for the visualization of multiple proteins within the same tissue section.

One of the challenges of multiplex immunohistochemistry is the need to validate each antibody to ensure that it binds specifically to its target protein. This can be a time-consuming and expensive process, but it is critical to ensure the accuracy and reproducibility of the results. To address this challenge, Visikol offers validated antibody panels as part of their highly-multiplexed immunohistochemistry services.

Validated Antibody Panels

Visikol’s validated antibody panels are designed to provide actionable insights in an easy-to-read report, allowing clients to make decisions and push forward their research efforts. Each panel is designed to target a specific set of proteins that are relevant to a particular disease or biological process. By using a validated panel, researchers can be confident that each antibody in the panel has been thoroughly tested and validated, which saves time and resources.

These validated antibody panels are also highly versatile and can be customized to suit the specific research needs of scientists working in pharma or medical research. This flexibility allows researchers to design a panel that is tailored to their specific research question, which can lead to more accurate and relevant results.

Advanced Imaging Services

In addition to the validated antibody panels, Visikol also offers a range of advanced imaging services, including digital slide imaging. Digital slide imaging allows for the high-resolution imaging of tissue samples, which can be analyzed using custom-built image processing algorithms. This allows for the creation of a highly-multiplexed fluorescence image stack, which can be analyzed using quantitative analysis to measure cell populations at unprecedented detail within a single sample.

Multiplex immunohistochemistry is a powerful technique that allows for the simultaneous visualization and quantification of multiple proteins within a single tissue sample. Visikol’s validated antibody panels provide a streamlined and cost-effective way to perform this technique while ensuring the accuracy and reproducibility of the results. The flexibility of the panels also allows for customization to suit the specific research needs of scientists working in pharma or medical research. With Visikol’s highly-multiplexed immunohistochemistry services and advanced imaging capabilities, researchers can gain a more comprehensive understanding of the biological processes underlying disease, which can lead to the development of new and more effective treatments.

To learn more about Visikol’s Highly-Multiplexed Immunohistochemistry Services, please reach out to a member of our team today.

Embark on a unique voyage as we leverage the fundamental steps of Cell Population Analysis and count stars over 7,800 light years away. While Visikol’s expertise primarily resides in the realms of microbiology and drug discovery, our capabilities transcend the boundaries of the microscopic world. In the below blog post, we demonstrate how methodologies developed through our multiplexing workflow seamlessly translate into the investigation of the Godzilla Nebula, captured by NASA’s Spitzer Space Telescope in October of 2021 (Figure 1, left).

Step 1: Primary Object Detection and Segmentation

Similar to any multiplexing project, the image of the Godzilla Nebula consists of several channels, each conveying specific information. This particular image comprises three infrared wavelengths, colorized as blue, red, and green (Figure 1,right). Just as we would use the DAPI or Hoechst channel to identify nuclei, the blue image (λ=3.6µM) can be utilized to identify the primary objects, as stars predominantly emit wavelengths within this range. Achieving accurate object detection and segmentation is crucial, and this can be improved by adjusting parameters of various thresholding techniques. In this case, we employed parallel algorithms where the first creates boundaries for the largest and brightest stars, while the second repeats this step for the remaining objects that can be distinguished above noise (Figure 2).

Step 2: Determining Positivity Per Label

Once the primary objects are segmented, we proceed to determine the positivity for the remaining labels. In this image set, the red channel (λ=24µM) is closely associated with the temperature of space dust and serves as an indicator of the supernova state of stars. By assessing the intensity of each object on this channel, we can determine the channel positivity and identify stars that meet the criteria (Figure 3).This process is analogous to classifying positivity for nuclear labels in microscopy images, such as HIF1A, γH2AX, or pHH3.  For cytoplasmic markers that fluoresce outside of the nuclei, positivity would be determined after establishing the boundary of each cell body.

Step 3: Defining Boundaries of Interest

Lastly, we define the boundaries of interest by applying thresholding to markers that aid in distinguishing region types. In this image set, we employ the green channel to outline the boundary of the nebula, as this particular wavelength (λ=8.0µM) acts as a marker for dust, organic molecules, and hydrocarbons (Figure 4). This approach aligns with the methodology used in microbiology to identify markers for tumor tissues, such as PanCK, facilitating differentiation between healthy and cancerous cells. Once the boundaries of the regions are established, specific endpoints can be reported and normalized according to their respective region types (Table 1).

Table 1: Results of population analysis. The percentage of stars in supernova state are normalized by their star population in each respective region.

By applying tactics derived from extensive research centered around cellular imaging, we have successfully conducted population analyses of the Godzilla Nebula. While cell counts and positivity are the most requested deliverables, our pipelines can be tailored to accommodate endpoints aligned with specific hypotheses. Figure 5 exemplifies this approach by showcasing the colocalization of individual channels of supernova state stars, suggesting its prospective utility in photometric or spectroscopic investigations.

Irrespective of the scale, whether investigating the microscopic domain or venturing into the vastness of space, our unwavering commitment lies in unearthing crucial insights and contributing to groundbreaking scientific discoveries. Please feel free to reach out to our team today to discuss your prospective project.  

For comprehensive details regarding the Godzilla Nebula, please consult the following resource: https://www.spitzer.caltech.edu/image/ssc2021-09a-a-monster-star-forming-region

Co-registering DAPI images is a critical step in the multiplexing workflow, particularly after tissues have undergone stripping and re-labeling. While visual inspection suffices for projects with manageable image sizes and sample numbers, it becomes impractical for quality control (QC) when dealing with a large volume of pixels. Therefore, an automated solution is required. This blog post will explore the procedure developed to automate the QC process for co-registering over 900 DAPI images from TMA cores.

Assessing the Alignment Between Images

To accurately assess the alignment between the fixed and moving images, we conducted calculations of the Similarity Metric and Pearson’s Colocalization Coefficient for all 900 image pairs. These metrics quantify the extent of overlap and colocalization between the two images, providing valuable insights into the quality of the co-registration process. The Similarity Metric, specifically using Mattes Mutual Information, is calculated iteratively during the registration process. As the algorithm approaches a solution, this value progressively decreases until the maximum number of iterations is reached or converges to a nearly constant value. A Similarity Metric less than -1 indicates a good alignment between the images. The Pearson’s Coefficient reflects the pixel overlap between the fixed and moved images, accounting for differences in overall intensity. A Pearson’s value close to 1 signifies a strong colocalization between the images. By plotting these two metrics against each other, we not only established a failure threshold but also gained valuable observations regarding potential imaging irregularities.

Successes and Failures

Let’s first study the most significant success and failure. Figure 1 displays the core that achieved the highest success, with a Similarity Metric of -1.7 and a Pearson’s Coefficient of 0.91. Before applying the transformation, a noticeable offset exists between the fixed (red) and moving (green) images. However, after the moving image is transformed, the composite image exhibits a bright yellow color, indicating successful overlap between the green and red signals. On the other hand, the core that experienced the most significant failure (Figure 3B), returned a Similarity Metric of -0.01 and a Pearson’s Coefficient of -0.04, due to inconsistent region selection during image acquisition..

After plotting the Similarity Metric against the Pearson’s Colocalization Coefficient for all pairs (as shown in Figure 2), a clear trend emerges between the two extremes, identified as core A and core B. Now, let’s shift our attention to core C, which lies between these two extremes. Despite having a satisfactory Similarity Metric close to -1, the Pearson’s value falls below 0.4. Despite this, the images for both panels in this core (Figure 3C) would still be deemed acceptable for image analysis. The decrease in the Pearson’s value can be attributed to missing sections within the core and the presence of an illumination artifact in the top-left region of the fixed image. This evidence suggests that cores positioned to the left of this data point also meet the criteria for acceptability. As we delve further into the investigation of cores positioned to the right of core C, it becomes increasingly evident that a distinct boundary can be drawn between the two clusters. These cores of interest are alphabetically labeled in Figure 2 and are classified as either success (green) or failure (red). Representative images of these cores can be observed in Figure 3.

By quite literally drawing a line between the acceptable and failed cores and extrapolating its function, the quality control (QC) constraints have been effectively established (Figure 4). Overall, only 5.3% of all registrations encountered failure, primarily due to tissue tearing, warping, or sample detection issues during the acquisition process. Importantly, registrations were still considered successful even when small sections of the cores were missing, or illumination artifacts were present. These metrics offer significant potential for further optimization of the co-registration algorithm, enabling expedited quality control procedures.

Visikol is home to a team of creative experts who possess extensive expertise in developing innovative and tailored solutions for large-scale data projects. If you want to discuss your current work and explore collaboration opportunities, we invite you to contact us. Our experts are eager to assist you in achieving your objectives.

Fluorescence in situ hybridization (FISH) is a molecular technique used to detect and localize specific DNA or RNA sequences within cells, tissues, or whole mount organisms. This technique is based on the use of fluorophore-labeled probes that hybridize complementary DNA or RNA target sequences, allowing for the visualization and identification of specific genetic loci or transcripts. FISH has become an essential tool for both research and diagnostic applications, allowing for the identification of genetic aberrations and disease-associated biomarkers in a variety of tissues and cell types.

FISH Tissue Labeling

The process of FISH tissue labeling with a fluorescent probe involves several key steps. The first step is the selection and design of the appropriate probe sequence. The probe is typically a short, single-stranded DNA or RNA molecule that is complementary to the target sequence of interest. For FISH, the probe is labeled with a fluorophore allowing for its detection and visualization under a fluorescence microscope. The selection of the probe sequence is critical, as it determines the specificity and sensitivity of the FISH assay.

Once the probe sequence has been selected, the next step is to prepare the tissue sample for labeling. This involves fixing the tissue in a solution of formaldehyde, paraformaldehyde, or other fixatives, which preserve the tissue morphology and prevent the degradation of the nucleic acids within the sample. The fixed tissue is then permeabilized with proteases to allow the probe to penetrate the cell membrane and hybridize to the target sequence. Protease treatment also increases hybridization efficiency and reduces background fluorescence of the sample. In the case of whole mount FISH, the organism may need to be cleared, or made transparent, prior to fixation by using clearing agents meant for large tissues, such as Visikol® HISTO™-1 or HISTO™-2. In addition to these two reagents, Visikol® HISTO™-M can be utilized for cell culture models and tissues less that 1mm thick.

After the tissue has been fixed and permeabilized, the next step is the hybridization of the fluorophore-labeled probe to the target sequence. The probe hybridizes to the complementary target sequence within the tissue, allowing for its detection and localization under a fluorescence microscope. After hybridization, the tissue sample is washed to remove any unbound or nonspecifically bound probes. This step is critical to minimize background fluorescence and increase the signal-to-noise ratio of the FISH assay. The washed tissue sample is then ready for imaging using a fluorescence microscope.

FISH and Immunofluorescence

Immunofluorescence imaging is a complementary technique that can be used in conjunction with FISH to visualize specific proteins or other cellular structures within the tissue sample. This technique involves the use of antibodies that are labeled with fluorophores, which recognize and bind to specific protein antigens within the tissue. The tissue sample is incubated with the labeled antibody, allowing for the specific detection and localization of the protein of interest. Both techniques can utilize multiple fluorophores to create multiplex labeling panels to visualize multiple target sequences at once. Here at Visikol, we specialize in highly multiplexed immunofluorescence imaging techniques involving 10+ different antibodies, made possible by our proprietary EasyPlex™ antibody stripping reagent and co-registration software.

Fluorescence in situ hybridization (FISH) is a powerful technique for the visualization and identification of specific genetic loci or transcripts within cells or tissues. The process of tissue labeling with a fluorescent probe involves several key steps, including the selection and design of the appropriate probe sequence, tissue fixation and permeabilization, probe hybridization, washing, and imaging. Immunofluorescence imaging can be used in conjunction with FISH to visualize specific proteins or other cellular structures within the tissue. The combination of these techniques has a wide range of applications in both research and clinical settings, allowing for the identification of disease-associated biomarkers and rapid diagnosis and treatment.

For more information on Visikol’s HISTO™ clearing reagents or our multiplex immunohistochemistry services, please reach out to a member of our team.

References

Egger D, Troxler M, Bienz K. 1994. Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization–immunocytochemistry. J Histochem Cytochem 42:815–822.

Jin L, Lloyd RV. 1997. In situ hybridization: methods and applications. J Clin Lab Anal 11:2–9.

Rautenstraub BW, Liehr T. 2002. FISH Technology. Berlin: Springer-Verlag.

Understanding intermolecular reactions and how they affect antibody-antigen interactions is necessary during immune based labeling, such as immunohistochemistry, immunofluorescence and multiplex labeling. The antibody-antigen binding complex is dictated by several different intermolecular reactions and is the key concept behind therapeutic analysis when using this method. These interactions can affect the specificity, strength, and respective quality which is obtained during microscopy. Visualizing and imaging dozens of different proteins and antigens on a single tissue slice utilized during the multiplex labeling platform requires multiple antibody labels and subsequent rounds of stripping. This removal of antibody labels from the specimen requires an understanding of these intermolecular reactions. These reactions include Ionic Bonds, Covalent Bonds, Hydrogen (H-Bonds) and van der Waals interactions. Ionic and Covalent bonds are not themselves intermolecular, but these intramolecular forces must be understood first to characterize intermolecular interactions.

Electron Distribution

Electron distribution is at the heart of any intermolecular reaction and is related to the whole spectrum of these reactions. Uniform distributions are associated with nonpolar molecules, and uneven distributions, associated with polar molecules. These characteristics of polar and nonpolar can be seen as opposite ends of the same spectrum. Ionic bonds are at the extreme end of polar molecules and have defined charges that exhibit the strongest bonds. Molecules with these strong, polar, ionic bonds are usually in salt form and are readily soluble in water. It is rare for antibody-antigen complexes to involve ionic bonds directly. Rather, ionic characteristics define the environment which the antibody-antigen complex finds itself in. Depending on the salt concentration of the environment, partial charges of other biomolecules can be neutralized or weakened.

Covalent Bonds

Covalent bonds can span the whole range of the polarity spectrum. Covalent bonds are strong bonds involving the overlap of electron orbitals between atoms to stabilize their valences. Covalent bonds can be used to construct both polar and nonpolar molecules, which presents a wide variety of possible configurations. The polarity of these bonds is determined by the electronegativity of atoms involved. For instance, a carbon atom bound to another carbon atom contains a nonpolar covalent bond because they have equal electronegativities and therefore an equal distribution of electrons. However, a carbon atom bound to an oxygen atom contains a polar covalent bond because oxygen has a higher electronegativity and therefore has a denser electron distribution around it. Molecules with polar covalent bonds have partially charged regions. These regions will orient themselves in space with other molecules that have similar characteristics according to these partial charges. Antibody-antigen complexes are not covalently bound, but partial charges due to the polar covalent bonds of the antibody and the antigen create this electrostatic interaction.

Hydrogen Bonds

The polarity of some covalent bonds also defines Hydrogen bonds. Hydrogen bonds are commonly described as a hydrogen atom bound to either nitrogen, oxygen, or fluorine. The electronegative differences between hydrogen and these other elements generate a noticeable, and strong, partial charge of the molecules they are a part of. When multiple molecules that contain these covalent bonds come together, the large partial charges snap these molecules together in space electrostatically but not covalently. This is the Hydrogen bond. This is the basis behind capillary action in plants and is the main driver of antibody-antigen complexes and interactions. They are stronger than nonpolar covalent bonds, but not as strong as ionic bonds. They are impermanent and must be considered during immune based labeling.

Van der Waals Interactions

On the weak end of molecular bonds and interactions are van der Waals interactions. These interactions are associated with nonpolar, or weakly polar, covalent bonds. Nonpolar molecules have covalent bonds with relatively even electron distributions. However, sporadic changes in the electron distributions caused by environmental conditions can generate very small partial charges especially in larger hydrocarbons. When long fatty acid molecules come in contact with each other, these sporadic charges cause the molecules to conglomerate in a weak electrostatic manner. These are van der Waals interactions and work closely with Hydrogen Bonds to stabilize the antibody-antigen complex.

If you’re interested in working with Visikol, please reach out to a member of our team.

Here at Visikol, we specialize in a diverse range of scientific applications, though our focus can be broken down into two main areas – advanced cell culture and advanced imaging. We offer a variety of 3D in vitro models – including but not limited to – liver fibrosis, hepatotoxicity, immune cell infiltration, and blood brain barrier assays. On the advanced imaging front, we are consistently working to provide our clients with more actionable insights to their tissues through our multiplex IF and IMC services. As the demand for multiplex services rises, we strive to streamline the quoting process by lowering barriers to obtaining data and optimizing turnaround time to better meet our clients’ timelines. We are accomplishing this through the launch of our multiplex project designer webpage, which allows our clients to avoid the wait time associated with multiple meetings and long email chains. With the help of this new feature, your requests will transform from Statements of Work to active projects in the lab in no time.

1.) Navigate to one of the Multiplex Project Designer locations on our website.

2.) Fill out your basic contact information so that we can reach out when your SOW is complete.

3.) Enter in details about your samples (number, type, fixative, control tissue provided, etc.).

1. 1. If you are unsure about any of these details at the time of submission, we are happy to iron these out in the 15-minute meeting we host upon receipt of your request.
      1. i) As a note, any information not matching the SOW may result in change order and price change.

4.) Many of our clients ask if there can be more than one section per slide – and the answer is yes! We just ask that you indicate how many sections per slide and tissue size (or if it is a TMA) in the ‘Additional Information’ section.

Our standard section thickness is 5μm. If your sections are anything outside of this, please make note of this as well.

5.) As for image analysis, we offer standard cell counts and positivity for markers as a standard service. Our other available analysis services include area quantification, cell population analysis, spatial analysis, morphometric characterization, phenotypic analysis, bioinformatics, traditional machine learning and deep learning, and custom pipeline development.

Once you press “Submit,” our team will be notified of your request and all the details provided above. We will get to work generating your SOW and reach out within 48 hours to set up a 15-minute meeting to review your project design together. Not only does this eliminate the need for an initial introductory meeting, but it also ensures we have all the information required to generate a fully comprehensive SOW for our clients. If you have a project in mind that you would like Visikol’s help with, give our multiplex project designer a try!

SOUTH SAN FRANCISCO, Calif., October 11, 2022 (GLOBE NEWSWIRE) — Standard BioTools Inc. (Nasdaq:LAB), driven by a bold purpose – Unleashing tools to accelerate breakthroughs in human health™ – today announced a collaboration agreement with Visikol® for service offerings using Imaging Mass Cytometry™.

The agreement is an expansion of the relationship established in 2020 under which Visikol will offer IMC services to clients from its site in Hampton, NJ. Visikol, a contract research services company focused on advanced tissue imaging and advanced cell culture assays, services all 20 of the top pharmaceutical companies. Visikol will leverage the Standard BioTools™ Hyperion+™ Imaging System, based on Imaging Mass Cytometry (IMC™), in its multiplex tissue imaging services. This initiative is supported by Visikol’s digital pathology services, which aim to transform large imaging-based datasets into actionable insights.

Recently, the demand for tools to evaluate the complex immune cell landscape has grown dramatically as researchers, biotech companies, and pharmaceutical companies look to develop immuno-oncology therapeutics and better understand the intricate interplay between the immune system and disease.

Multiplex tissue imaging is a key tool that researchers can use to characterize a wide range of immune cell and tissue-specific biomarkers simultaneously. Standard BioTools has developed a version of this technology, IMC that currently allows for 40-plus markers to be detected on a single slide with an unprecedented level of sensitivity, quantification, and absence of autofluorescence. In addition, IMC’s rapid panel design capability is particularly well suited to applications within contract research organizations.

“As a company that is focused on advanced tissue imaging, we are excited to add IMC to our portfolio of tools and to work closely with Standard BioTools, as it will allow us to better meet the needs of our clients,” said Visikol CEO Michael Johnson, PhD. “IMC provides our clients with the unique ability to image many markers simultaneously while providing an unparalleled level of image quality.”

“IMC is one of the essential technologies that Standard BioTools provides,” said Michael Egholm, PhD, President and Chief Executive Officer of Standard BioTools. “The valuable relationship with Visikol broadens the use of IMC for spatial omics studies, offering a vital tool in the acceleration of drug discovery and development in particular when dealing with precious and scarce patient biopsies.  We look forward to working with the Visikol team to help their pharma clients uncover important spatial relationships within the tissue microenvironment.”

Forward-Looking Statements
This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, including, among others, statements regarding the potential benefits of research conducted using Standard BioTools products and technologies and anticipated benefits to Standard BioTools of an expanded collaboration. Forward-looking statements are subject to numerous risks and uncertainties that could cause actual results to differ materially from currently anticipated results, including but not limited to risks relating to interruptions or delays in the supply of components or materials for, or manufacturing of, Standard BioTools products; potential product performance and quality issues; intellectual property risks; competition; uncertainties in contractual relationships; and reductions in research and development spending or changes in budget priorities by customers. Information on these and additional risks and uncertainties and other information affecting Standard BioTools’ business and operating results is contained in its Annual Report on Form 10-K for the year ended December 31, 2021, and in its other filings with the Securities and Exchange Commission. These forward-looking statements speak only as of the date hereof. Standard BioTools disclaims any obligation to update these forward-looking statements except as may be required by law.

About Standard BioTools Inc.
Standard BioTools Inc. (Nasdaq:LAB), previously known as Fluidigm, is driven by a bold purpose –Unleashing tools to accelerate breakthroughs in human health. Standard BioTools has an established portfolio of essential, standardized next-generation technologies that help biomedical researchers develop medicines faster and better. As a leading solutions provider, the company provides reliable and repeatable insights in health and disease using its proprietary mass cytometry and microfluidics technologies, which help transform scientific discoveries into better patient outcomes. Standard BioTools works with leading academic, government, pharmaceutical, biotechnology, plant and animal research, and clinical laboratories worldwide, focusing on the most pressing needs in translational and clinical research, including oncology, immunology, and immunotherapy.  Learn more at www.standardbio.com or connect with us on Twitter®, Facebook®, LinkedIn, and YouTube™. Standard BioTools, the Standard BioTools logo, Fluidigm, the Fluidigm logo, “Unleashing tools to accelerate breakthroughs in human health,” CyTOF, Hyperion, Hyperion+, Imaging Mass Cytometry and IMC are trademarks and/or registered trademarks of Standard BioTools Inc. or its affiliates in the United States and/or other countries. All other trademarks are the sole property of their respective owners. Standard BioTools products are provided for Research Use Only. Not for use in diagnostic procedures.

About Visikol
Visikol is a contract research services company that is focused on accelerating the drug discovery and development process through providing its clients with advanced tissue imaging and advanced cell culture services. Today, Visikol counts the top 20 pharmaceutical companies as clients and has been instrumental in dozens of drug discovery programs. The company provides end-to-end services that include 3D tissue imaging, multiplex tissue imaging, digital pathology, high-content imaging, 2D cell culture assays, 3D cell culture assays, and ex vivo tissue slice assays. Visikol’s expertise lies in both transforming tissues into actionable insights and bridging the gap between in vitro assays and in vivo results through the use of best in-class cell culture models. In addition to its services, Visikol sells a suite of tissue clearing reagents and kits as well as the HUREL® Micro Liver portfolio of primary hepatocyte liver models. Learn more at www.visikol.com.

Available Information
Standard BioTools uses its website (standardbio.com), investor site (investors.standardbio.com), corporate Twitter account (@Standard_BioT), Facebook page (facebook.com/StandardBioT), and LinkedIn page (linkedin.com/company/standard-biotools) as channels of distribution of information about its products, its planned financial and other announcements, its attendance at upcoming investor and industry conferences, and other matters. Such information may be deemed material information, and Standard BioTools may use these channels to comply with its disclosure obligations under Regulation FD. Therefore, investors should monitor Standard BioTools’ website and its social media accounts in addition to following its press releases, SEC filings, public conference calls, and webcasts.

Investors:
Peter DeNardo
415 389 6400
ir@standardbio.com

Source: Standard BioTools Inc.

Download our Multiplex Tissue Imaging eBook Today!

With the recent advances in the field of tumor immunology and cancer immunotherapy, there is a growing need for multiplex imaging methods that can simultaneously label multiple tumor and immune cell biomarkers providing users valuable information about the complex tumor microenvironment.

This eBook will examine the pros and cons of using:

• Tissue-Based Cyclic Immunofluorescence (t-CyCIF) Method
• Co-Detection by Indexing or Fluorescent Immunohisto-PCR
• Fluorescent Tyramide mediated amplification
• Visikol Multiplex Immunofluorescence (VMIF)
• Imaging Mass Cytometry

…and more!

One of Visikol’s main areas of focus is histology and multiplex imaging. Visikol’s multiplex imaging is driven by patented Histo clearing reagents that work to make images of cells clearer and fit for scientific research. A problem that often occurs while prepping and clearing tissues for imaging is something known as Autofluorescence. Autofluorescence is the fluorescence of the naturally occurring substances in a cell, and these substances usually gain fluorescence through accidental excitation. Autofluorescence can happen for multiple reasons, including issues during fixation, heat and dehydration, and fluorescence of heme. Autofluorescence can complicate the analysis of a section and skew the results of an experiment. However, Autofluorescence can be helpful if it labels something that would otherwise require labeling.

Fixation-Induced Autofluorescence

Since there are several causes of Autofluorescence, there are many things to consider when attempting to mitigate the effects of Autofluorescence. In fixation-induced Autofluorescence, the cause of the fluorescence is the cross-linking of tyrosine and tryptophan residues in proteins that contain formaldehyde. This cross-linking forms a fluorescent formaldehyde adduct that can cause this autofluorescence to appear. The best way to avoid this is to ensure that the tissues have been fixed for the minimum amount of time required for the size and type of tissue. In Visikol’s Tissue Clearing eBook, the best protocol for slide fixations is outlined.

Heat and Dehydration-Induced Autofluorescence

The next cause of Autofluorescence to discuss is heat and dehydration-induced Autofluorescence. Treatment of fixed tissues at too high a temperature can cause background fluorescence. When dehydrating, staining, and clearing your tissues, be sure to do so with tissues incubated at room temperature. The effect of heat-induced Autofluorescence is much more significant in the 530-600 nm (red) region.

Endogenous Pigments

The last type of Autofluorescence to discuss is Autofluorescence by endogenous pigments. Heme Autofluorescence is a type of fluorescence caused by the pigment in blood cells. To control this, you should perfuse tissues with phosphate buffer solution before fixation. This will remove the blood cells from tissues and eliminate problems pre-processing. Other types of endogenous pigment Autofluorescence include fluorescence through lipofuscin, collagen, and other lipophilic pigments. The fluorescent spectra of these pigments vary along the spectrum, and the protocol to get rid of their fluorescence can vary.

Visikol has found that background Autofluorescence can be managed if you follow methods correctly. In Visikol’s Tissue Clearing eBook, there are many tips and strategies for keeping Autofluorescence down, including tables and charts documenting the wavelengths at which common pigments tend to be excited. Visikol offers high-quality clearing reagents and kits that are great for your next clearing or imaging project. If you are interested in working with Visikol, don’t hesitate to get in touch with us.