Synopsis
Digital pathology is changing the way you monitor and analyze digital specimens; however, it also has its drawbacks. It creates issues when you must analyze the whole specimen. Z-stacking technology is used to overcome challenges arising from this. The technique creates a three-dimensional (3D) representation of the specimen by combining a series of images taken at different depths. It takes multiple images of a sample in different focal planes and then combines them to create a single image. Z-stacking allows you to observe and analyze sensitive samples, even those with unusual thicknesses. In this article we will discuss Z-stacking and why it is so useful in digital pathology.
Z-Stacking in Modern Digital Pathology
Z-stacking is a digital pathology technique used to create a three-dimensional (3D) representation of a specimen by combining a series of images taken at different depths. In practice, Z-stacking involves taking multiple images of a specimen at different focal planes and then combining these images to create a single image in which the entire depth of the specimen is seen in sharp focus.
Limitation to be Addressed
It is the depth of field that determines the focal point of a digital image. When looking at a sample in a microscope, only a small part of the sample is in focus at any given time, while the rest of the sample is out of focus. The limited depth of field of the microscope means that only a small part of the specimen is in sharp focus at any one time, making it difficult to try to image thicker specimens. The Z-stacking concept is based on the principle of the depth of field.
Z-Stacking Principles
To overcome the challenge, multiple images of the same sample must be taken at different focal points, from the top to the bottom of the sample. In each of these areas, only the area corresponding to the focal plane is in sharp focus. When these images are combined, a composite image is produced in which the entire depth of the sample is in sharp focus. This is often called a “stacking” image.
Z-Stacking in Whole Slide Imaging
The whole slide scanner is a computer-controlled microscope. It is connected to a highly specialized camera containing advanced optical sensors. The main components of an all-slide scanner are:
- Microscope with objective.
- Brightfield or fluorescent light source.
- A robot that moves or repositions the slide.
- One or more digital cameras for taking images.
- A computer.
- Software for processing, managing and displaying digital slides.
Some devices also have a dynamic refocus function that uses one camera to focus and another to scan. This helps to speed up the scanning process.
Tasks Performed
Slides can be scanned manually or automatically. In addition, many WSI devices can perform batch scanning and continuous or random processing. A slide can be loaded while another slide is being scanned. Most devices can read 1D and 2D barcodes on glass. Scanning speeds range from 1 to 3 minutes per slide, depending on the magnification of the object and the number of Z-stacks obtained.
Scanning Flexibility
You can choose to scan the whole slide or, if you prefer, a predefined area of interest on the slide to be observed. Some WSI scanners can digitize slides in different Z-axes (vertical focal planes), resulting in a multiplanar image that mimics the precise focus control of a conventional microscope. In the field of cytological slides, the Z-stacking alignment is an invaluable advantage.
Use of Z-Stacking
Z-stacking is particularly useful in situations where the specimen thickness is greater than the depth of focus of the imaging system. This is often the case with tissue, where the structures of interest may be located at different tissue depths. With Z-stacking, these structures can be imaged with a sharp focal length, providing a more accurate representation of the tissue structure.
Numerous Applications
Z-stacking has many applications in digital pathology other than helping to analyzing tissue structures. It can be used to improve the resolution of images obtained using low magnification targets. These targets have a higher depth of field but also a lower resolution than higher magnification targets. Z-stacking can be used to increase the effective resolution of an image, thus allowing tissue structures to be observed more easily.
Used in Digital Reconstruction
Z-stacking can also be used for the digital reconstruction of 3D structures such as neurons or blood vessels. Z-stacking technique is particularly useful in neuroscience to study the shape of neurons and in vascular biology to study the structure of blood vessels.
Conclusion
Z-stacking is a useful technique in today’s digital pathology practice because it overcomes the challenge associated with viewing the entire specimen. It is also used in various fields such as neuroscience and vascular biology. Z-stacking has significant advantages, especially when working with sensitive tissues, so it is an important feature that facilitates such work. Digital microscopes such as the iO:M8 in–vitro digital microscope are useful in achieving high quality output. In addition to the microscope, tools such as suitable light sources, robots, digital cameras, computers and software can also improve the working environment. Z-stacking is an important part of today’s digital pathology workflow, offering advantages that can have a significant impact on diagnostic and research work.
