Whole Slide Imaging

What is Whole Slide Imaging?

Whole slide imaging, also known as virtual microscopy, refers to scanning a complete microscope slide and creating a single high-resolution digital file. This is commonly achieved by capturing many small high-resolution image tiles or strips and then montaging them to create a full image of a histological section. 

Whole slide imaging is changing the workflows of many laboratories. Specimens on glass slides can now be transformed into high-resolution digital files that can be efficiently stored, accessed, analyzed, and shared with scientists from across the web using slide management technologies like Biolucida

The transformative technology of whole slide imaging has had a dramatic impact on many fields of research, including:

  • pathology
  • connectomics
  • genomics
  • proteomics 
  • neuroanatomy

Whole slide imaging has also had a significant impact on the fields of:

  • medical education
  • archival libraries
  • medical research publishing
The digital nature of whole slide images allows for microscopic specimens to be easily be shared with others. Unlike the actual specimens, whole slide images do not deteriorate over time like specimens on glass slides. Whole slide images contain complete histological sections and enable the viewer to zoom to see high levels of detail, e.g. individual cells and subcellular structures. Put in another way, whole slide images enable the examination of histological sections at magnifications that range from a “bird’s eye view” to the highest level of detail provided at the time of image acquisition.  

Advanced technology solutions for whole slide imaging allows:

  • Fast image acquisition
  • Efficient data storage
  • Repeatable data analyses across experiments
  • Novel, automated analyses
  • Easy and instantaneous data sharing


Data Storage and File Formats

Whole slide imaging produces data sets that require careful plans for data storage, organization, and access. There are currently few solutions that meet this requirement. 

File formats used in whole slide imaging have evolved with little or no standardization across the industry. Many commercial whole slide imaging scanners have adopted proprietary file formats that are dependent on unique software solutions to read and analyze the files. Other standards, such as OME TIFF, are loose standards that have many variations, many of which are not efficient for viewing, analyzing or web streaming.

The JPEG2000 (JP2) file format is the most effective one for all aspects of whole slide imaging. JP2s include a streaming format, multiresolution capabilities, and efficient data compression. Only the most advanced software uses JP2 files format effectively.  MBF Bioscience products enable the utilization of formats from all major microscope and scanner manufacturers to ensure that scientific workflows produce data that and be efficiently accessed, analyzed, and shared.

JPEG 20000 extended part-2 specifications (JPX) files are able to store multiple JP2 files and MBF Bioscience software uses them to create three-dimensional image sets. By combining many whole slide images of serial sections into a JPX file, users can create massive image data files of whole organ reconstructions that are highly compressed for data sharing a storage.  


 Whole Slide Imaging Technology Falls into Three Main Categories:

⦿ Acquisition

⦿ Analysis

⦿ Management and Sharing


Acquisition of whole slide images is achieved using a combination of microscope-based hardware and software. 

Whole Slide Scanners

Whole slide image scanners are specialized devices that are dedicated to acquiring high resolution images of entire slides. There are a wide variety of these products currently on the market. These scanners are typically designed for a single mode of illumination, either brightfield or fluorescent microscopy. The primary distinguishing factors (in addition to cost) in these products include:

1. imaging modality (brightfield or fluorescence)
2. quality of the images
3. speed of acquisition
4. size of the glass slides that can be scanned
5. optical resolution
6. 2D or 3D imaging capabilities.
Secondary distinguishing factors include ease of use, reliability, and file format support.


MBF Bioscience has partnered with Huron Digital Pathology to offer a line of brightfield slide scanners, TissueScope, that is available from MBF Bioscience which have been specially designed for neuroscience and other biomedical research applications. These scanners can create 2D and 3D whole slide images using a variety of glass slides ranging in size from standard 1”x3” (25mm x 75mm) to ultra large 6”x8” (150mm x 200mm).


MBF Bioscience has partnered with Caliber ID to create Vesalius a confocal fluorescent slide scanner. Vesalius is specially designed for cutting-edge neuroscience and other biomedical research applications. Vesalius scanners can create 2D and 3D whole slide images using a variety of glass slides ranging in size from standard 1”x3” (25mm x 75mm) to 3”x2” (50mm x 75mm). Vesalius can rapidly scan slides with up to 4 fluorescent wavelengths using objective lenses up to 100x in magnification.


Microscope-Based Slide Scanners

Advanced research microscopes may be configured to create whole slide images. These microscopes can be multi-purpose laboratory devices that, with the proper ancillary hardware, i.e., motorized stages, cameras, etc., will allow you to capture high-resolution, 2D or 3D compatible whole slide images. The advantages to using these microscope-based scanners, (in addition to them being multi-purpose devices) are primarily related to the wide flexibility for scanning specimens. These devices can be configured to acquire with both brightfield and fluorescence. They can even be configured to use structured illumination microscopy (SIM) techniques, such as the Zeiss Apotome. Other advantages include being able to use a wide variety of objective lenses, e.g., from 5x to 100x oil, and custom motorized stages to handle extremely large slides.

MBF Bioscience offers a variety of microscope systems to meet specific lab needs, including solutions for brightfield and fluorescence. Neurolucida or Stereo Investigator systems can also be upgraded with the 2D Slide Scanning and 3D Slide Scanning modules to achieve this functionality.



The analysis of whole slide images is a field that is rapidly evolving. There are numerous quantitative measurements that can be performed on these images. Specialized software is often required, as the large size of the images requires software techniques designed to work with these images without requiring long wait time by users.

Types of quantitative analysis include:

  • measuring sizes of objects and regions,
  • counting objects such as cells, axon terminals, etc.
  • measuring lengths of objects such as dendrites, capillaries, etc.
  • measuring density objects, such as protein based biomarkers, etc.

There are two main categories of analysis techniques:

  • Image processing and machine learning, including high-throughput and user-assisted Stereology.


MBF Bioscience has a Diverse Range of Software Designed to Analyze Whole Slide Images. These Products Include:

Management and Sharing

Effective management of whole slide images is an important, but often-forgotten aspect of whole slide imaging.  Since the files are often very large and in non-standard file formats, specialized software is required to manage the files.  Typically, laboratories generate terabytes of image data and don’t have a good solution to categorize and view this data.  We designed Biolucida to solve this problem. After whole slide images are acquired, these images can be viewed, categorized and stored on either local networks or using cloud-storage. Whole slide images can be instantly shared with colleagues for collaboration in your lab, down the hall, or around the world.  Biolucida supports file formats from virtually all commercial whole slide scanners.

Archival Libraries

Digital libraries containing collections of important microscopic specimens are increasingly being used in medical research and education. Important uses for whole slide libraries and web resources include:  digital histology collections, brain atlases, connectivity maps, and gene databases. Biolucida is designed to manage large collections of on-line whole slide images. It includes special features for collections, tags and sophisticated searches.


Specific Applications

Counting Cells and Measuring Objects

The locations and distribution of cell types in the whole slide images, can be analyzed with sophisticated automatic detection and image processing techniques in Neurolucida 360, NeuroInfo, TissueMaker, Tissue Mapper and BrainMaker. Each of these products provide cutting-edge solutions for specific classes of analyses. For example, researchers can map the distributions of a particular cell type, like those producing parvalbumin, and obtain analyses such as the nearest neighbor analysis to determine the distance from one cell to another or the distance from the cells to an anatomical boundary.

3D Serial Section Reconstruction

BrainMaker, TissueMaker and NeuroInfo can generate 3D image reconstructions from whole slide images of serial sections so that you can easily view cells, structures and lesions. With the integrated 3D visualization, you can see the reconstruction of the entire organ. For example, you can see the location of all neurons expressing a particular gene, visualize the axonal projections of specific neurons with full anatomical context, or automatically detect cells throughout the brain. Neurolucida and Stereo Investigator with the Serial Section Reconstruction module can be used to produce 3D solid modeling graphics representations from serial sections.

Whole-Brain Reconstruction, Annotation, and Registration

Recent advances in molecular neuroanatomical tools have expanded the ability to map connections of specific neuron subtypes in the context of behaviorally driven patterns of neuronal activity. Analysis of such data across the whole mouse brain, registered to a reference atlas, aids in understanding the functional organization of brain circuits related to behavior. NeuroInfo works with whole slide images of mouse brain sections labeled with standard histochemical techniques, reconstructs those images into a whole brain image volume and registers those images to the Allen Mouse Brain Atlas. Image detection of cells and quantification of axon density labeling are then combined with the structures in the annotated reference atlas. Examples of these analyses include mapping the axonal projections of layer‐specific cortical neurons using Cre‐dependent AAV vectors and for mapping inputs to such neurons using retrograde transsynaptic tracing with modified rabies viral vectors.


Design-based stereology is a rigorous set of data analysis methods to ensure integrity in the quantitative analysis of the size, shape, and number of objects (usually cells) in a tissue sample. Stereology is considered to be the gold standard in quantification. It is essential to validating and rejecting experimental hypotheses and produces results that are unbiased, efficient, and more reliable than non-stereological quantitative analyses. Non-stereological cell and object detection methods are highly susceptible to sampling bias and often fail to account for differences in object size and distribution within a region of interest. Stereo Investigator - Whole Slide Edition is a tool for analyzing whole slide image data accurately and efficiently.

Medical Education

Many medical educators now use whole slide images, also known as virtual slides, instead of microscopes to teach histology and histopathology. Virtual slides allow instructors to present the very best specimens ranging from traditional H & E staining to immuno-labeled confocal slides. Biolucida is in use worldwide in leading universities and medical schools an works with virtual slides in 2D or focusable 3D formats. To date over 300,000 students have learned histology and histopathology using Biolucida. 


Professional Technical Support

When you call us you will speak with a person - not an automated system. Talk to us about your hardware, software, or experimental design questions. Our team includes Ph.D. neuroscientists and experts in microscopy, stereology, neuron tracing, and image processing; ready to help you over the phone or online.

Contract Slide Scanning and Imaging Services

MBF Lab's Whole Slide Scanning services provides you with high-quality, high-resolution, 2D and 3D images of micropscope specimen slides. Scanning option include brightfield, fluorescence, or confocal microscopy, magnifactions can range from 10x to 100x. Our Slide Scanning services can be used for a standalone project or as a valuable complement to our stereology or morphology services.

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