3D Imaging of Tissues and Specimens

Switching from widefield to confocal without losing focus

Mica images of an intestine tissue section – specimen: 20x magnification widefield images were processed with THUNDER (Computational Clearing) and 63x confocal images with LIGHTNING. Nuclei are labeled blue, mitochondria green, and detyrosinated tubulin red. Mica allowed the progression from overview to segmentation of the tubulin network and identification of detyrosinated-tubulin-positive cells. Intestine_tissue_section_Teaser.jpg

3D imaging enables thick tissue specimens and 3D cell cultures to be characterized intact, enabling scientists to visualize natural biological interactions. However, it is challenging to screen thick tissues and 3D cell cultures efficiently to find areas of interest. It becomes even more difficult when you need to transfer your specimen between instruments, e.g., from a widefield to a confocal microscope to get a more detailed view.

Discover how Mica can be used to obtain 3D images using widefield and then seamlessly move to higher resolution confocal imaging without transferring your sample. Here, it is shown that 3D imaging of intestinal tissue with Mica enables a rapid switch from a specimen overview to a more detailed segmentation of the tubulin network with the aim of identifying efficiently detyrosinated-tubulin positive cells.

Image: Mica images of an intestine tissue section – specimen: 20x magnification widefield images were processed with THUNDER (Computational Clearing) and 63x confocal images with LIGHTNING. Nuclei are labeled blue, mitochondria green, and detyrosinated tubulin red. Mica allowed the progression from overview to segmentation of the tubulin network and identification of detyrosinated-tubulin-positive cells.

3D imaging of tissues and specimens

3D imaging of tissues refers to visualizing clearly in real time and analyzing details even deep inside large volume, thick specimens of biologically relevant models [1], like model organisms, tissue sections, and 3D cell cultures, like organoids and spheroids [2,3].

The 3D imaging of a thick tissue specimen and 3D cell culture could take from minutes to many hours depending on how large the specimen’s volume is. It is ideal for many biomedical applications where these specimens are required, including regenerative medicine, cancer, and stem cell research.

Thick tissues and 3D cell cultures

The cells of native tissues in an organism interact with each other in three dimensions. For this reason, thick, large-volume tissue specimens or 3D cell cultures, e.g., organoids or spheroids, mimic more realistically the physiological functions of living tissues [3,4].

These 3D specimens can be used by researchers to obtain a better understanding of how tissues and cells function in an actual organism. Applications for thick specimens include neuroscience, developmental biology, cancer, biomedical, and translational research.

Challenges

When doing 3D imaging of tissues, certain challenges have to be overcome. For example, the specimen may not remain in focus when the objective is changed, so when this occurs, users will need to spend time refocusing on the area of interest within the specimen. Whenever magnification is increased, it can be difficult to keep a specimen overview. Also, increasing magnification can make it cumbersome to relocate the same area of interest in the specimen.

When using confocal microscopy, searching for the right area in the specimen can be very time consuming. Also, when first viewing a specimen with a widefield microscope and then transferring it to a confocal system, it can be difficult to find the same location of interest on the specimen. When analyzing heterogeneous tissue with global-thresholded parameters, there is a tendency that interesting specimen features and structures are missed.

A significant number of research labs may not have available all the instrumentation and microscopes needed for 3D imaging. This problem is usually resolved with access to shared facilities utilized by multiple users, but that can mean long waiting times before the needed instruments are available. These challenges can lead to delays in acquiring the important, quantifiable results that lead to fundamental breakthroughs and the gaining of new insights.

Introducing Mica

Mica, the world's first imaging Microhub, unifies everything a researcher needs in one completely controlled, highly flexible environment, supercharging microscopy workflows to get meaningful scientific results faster.

Mica offers the benefits of:

  • Access for all: Mica provides a fast sample overview and allows users to easily change observation conditions with just a few clicks
  • No constraints: Mica seamlessly combines transmitted and fluorescence Widefield with Confocal imaging
  • Radically simplified workflows: Mica allows users to effortlessly transit from a large sample overview to highly resolved images and provides AI based analysis within the same workflow

Methods

Mica was used to 3D image an intestinal tissue section with the aim of demonstrating that users can seamlessly move from fast overview to high resolution as required. A large overview of the intestine slide was obtained followed by higher magnification viewing to identify appropriate areas of the intestine where confocal z-stack images could be acquired.

Results

The 3D imaging results show how an intestine tissue overview is rapidly attained in widefield and a more detailed view of areas of interest is acquired at 10x and 20x magnification. Then, a high magnification widefield view at 63x was obtained followed by acquisition of a confocal image. The acquired confocal image was then segmented to quantify sizes.

Mica allowed the progression from intestinal tissue overview to the segmentation of the tubulin network in order to identify detyrosinated-tubulin-positive cells.

With Mica, widefield and confocal microscope modalities are seamlessly connected . Without an integrated system like the Microhub , when performing widefield and confocal imaging, then users have to either carry samples between microscopes or compromise on efficiency. On top of that burden, the alignment of image data after moving from one system to another means extra work.

Mica provides users with seamless widefield and confocal imaging in a single system,  so they can use the most appropriate and optimal imaging modality whenever it is needed.

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