In 2022, Visikol launched a new imaging service in partnership with Standard BioTools Inc, this partnership has allowed us to bolster our imaging capabilities with the support of an industry leader in imaging. With new imaging services, our Business Development team is frequently asked about the use case of each imaging modality. The following few paragraphs will delineate the different imaging modalities and the use cases for each.

The process for designing a study plan requires consideration into one’s desired endpoints. Endpoints can be pathology review, discovery data, or clinical insights. To get to each endpoint, there is a different modality.

Standard Imaging

In the case of standard imaging, H&E, DAB, Masson’s Trichrome (and others) are staples of staining and brightfield imaging. These stains are often easy to use, efficient and cost effective. The use case is for projects of scale, which require simple area defining analysis or pathology review. Brightfield projects also are typically fast turnaround, making it a great fit for a library of samples that need digitization.

Multiplex Immunofluorescent

The next imaging method is IF (immunofluorescent) – specifically multiplexing. Multiplex IF refers to the labeling of a tissue section with more than 2 antibodies and fluorescently imaging antibodies to create an image. These projects are often more labor intense, and expensive than standard imaging. The pricing and turnaround time for these projects are based on complexity. In other words, a sample imaged with 3 antibodies or 20 antibodies will have a major impact on materials needed and effort required to produce images. The use case for IF is for preclinical or clinical tissues with a medium to high throughput sample quantity. IF can prove useful in drug discovery research as well as translational research and is a great starting point for understanding the TME (tumor microenvironment).

Imaging Mass Cytometry (IMC)

Similar to IF imaging, is IMC (Imaging Mass Cytometry). At Visikol, IMC imaging is available through the Hyperion+ Imaging System and provides researchers the opportunity to generate very large amounts of data from small sections of tissue. Unlike IF, IMC allows researchers to choose up to 40 markers per tissue and there is no background noise because the process is a metal-binding labeling approach. While IMC is more expensive than IF, it provides a greater amount of data that is best used for clinical samples in which researchers need to derive as much data as possible out of valuable tissue samples.

At Visikol, we help our clients with all three of the aforementioned imaging approaches. Our team has worked on projects of all types and are happy to help with you about your next project!

In histology, staining is the method used to identify regions, counts or other structural features of a cell. Nuclear stains are used for the staining of the nuclei and they work by reacting with negatively charged components such as nucleic acids in the nucleus. A frequently used nuclear stain, hematoxylin, comes from a logwood tree, scientifically known as the Haematoxylum campechianum. The logwood tree is a tall crooked growing tree that is native to Central America and has been scientifically cultivated in Jamaica since the early 1700s. At Visikol, hematoxylin is a crucial component of one of the most widely used histological stains, Hematoxylin and Eosin (H&E). 

Hematoxylin alone is not considered to be the dye; the oxidization of hematoxylin produces the weak anionic dye called hematein. The oxidization process, also known as "ripening," can be achieved by exposing the solution to atmospheric solution or by using oxidizing agents. There are several different aluminum hematoxylin formulas:

• Delafield and Ehrlich are ripened
  • Delafield hematoxylin, a regressive stain, is commonly used to get precise staining in the cellulose cell walls.
  • Ehrlich hematoxylin, also known to be a regressive stain, is one of the few commonly used stains for routine H&E staining.
• Gill, Harris, and Mayer are chemically oxidized
  • Gill hematoxylin, a progressive or regressive stain, is a great H&E stain that is the only hematoxylin that illustrates goblet cells. 
  • Harris hematoxylin, a progressive or regressive stain, is another commonly used stain for H&E and a useful counterstain in immunohistochemical or hybridization procedures. 
  • Mayer hematoxylin, a progressive stain, makes great use in immunoperoxidase techniques when 3-amino-9-ethylcarbazole is used as a chromogen. 
  • There are also non-routine techniques, such as special stains, that use hematoxylin. Weigert's iron hematoxylin, stains the nuclei in tissues and acts as a counterstain that is resistant in the decolorization in acidic solutions.  

At Visikol, the frequently used hematoxylin is Gill's hematoxylin. Gill is widely accepted due to the three different strengths that are used in histology and cytology. Gill 1, the weakest strength stain is better used in cytology. Gill 2, the medium strength stain is often used for histology and cytology. Gill 3 is primarily used for staining tissues and triple the strength of Gill 1. Gill hematoxylin consists of ingredients such as: distilled water, ethylene glycol, hematoxylin (anhydrous), sodium iodate, aluminum sulfate, and glacial acetic acid. Gill has great qualities such as a longer shelf life, greater control over quality and reproducibility, and overstaining is unlikely. Another benefit is whether laboratories prefer progressive (more consistent results) or regressive staining. If laboratories prefer regressive staining it is more likely for overstaining to occur so a differentiation process must be in place so the excess hematoxylin can be properly removed. At Visikol, we prefer the regressive staining of Gill's hematoxylin as it allows our histology team to get satisfactory intensity between different types of tissues and thicknesses. Visikol works closely with clients to help them meet their drug discovery goals, reach out to us today if you have any questions or would like to chat with our team! 

3D cell culture models (e.g. organoids, spheroids, micro-tissues) provide researchers with the ability to better mimic the in vivo environment compared to traditional 2D cell culture models. However, these more complex biological models present several challenges for researchers trying to characterize them via imaging as traditional high content imaging approaches are highly limited. For example, trying to image a 3D cell culture model with wide field or confocal microscopy results in significant characterization bias as light only penetrates a few cell layers into these models and thus the characterization is biased towards the cells on the exterior which are most exposed to nutrients and compound treatments.

This misrepresentation can be clearly seen from confocal imaging wherein the center of individual Z slices are typically dark which is due in many cases to optical attenuation and not a lack of cells or staining. However, some models of larger sizes will form necrotic cores and some researchers can have challenges with getting antibodies to penetrate deep into their models.

Another characterization problem with these 3D cell culture models is that while they are three-dimensional tissue-like constructs, they cannot be treated like traditional pieces of tissue and processed in accordance with traditional histopathology. The reason for this is that they are as small as 200 µm in diameter and are thus very easy to lose during the embedding and sectioning process. Furthermore, due to their size, many 3D cell culture models are typically embedded together to increase the probability that they can be found and subsequently sectioned. This creates several problems as this means that a 96 well plate will only yield eight slides if twelve models are pooled in order to get a single slide. Another problem is that the orientation of the slide is challenging to adjust and thus for 3D cell culture models which are heterogenous such as those that have cell sub-type polarity it is possible to take a section which is not representative of the sample.

At Visikol, we have developed several reagent, imaging and image analysis tools which allow us to side-step these problems through the use of tissue clearing combined with fluorescent labeling, high content confocal imaging and true 3D analysis. However, there are still many researchers who want to see traditional sections from 3D cell culture models and thus there is still a need for embedding, sectioning and traditional processing of these models. Therefore, Visikol has launched 3D cell culture histopathology services through which sections can be routinely collected from 3D cell culture models. These services include H&E, IHC and IF tissue labeling as well as slide scanning and digital pathology analysis.