Dual-illumination holographic microscopy visualizing blood flow in zebrafish embryos.

Blood Flow Visualization: A New Dimension in 3D Imaging

Kai Mendoza in Science & Nature August 2025 • 3 min read.

"Dual-Illumination Holographic Microscopy Offers High-Speed, Quantitative Insights into Microcirculation"

Visualizing blood flow, especially in the microcirculation, is crucial for understanding various biomedical processes and detecting early signs of diseases. Traditional blood flow imaging (BFI) techniques often require contrast agents, making them invasive. While methods like scanning Doppler imaging exist, they can be time-consuming.

Holography, also known as digital holography (DH), provides a non-invasive alternative. By reconstructing wavefronts, DH captures 3D information effectively. Recent advances combine DH and microscopy with dual illumination, offering detailed 3D images of red blood cells (RBCs) in living organisms, such as zebrafish embryos.

A new study introduces an improved dual-illumination holographic microscopy technique. This method uses two microscope objective lenses at a wider separation angle, enhancing resolution and allowing easier sample manipulation. The technique's effectiveness is demonstrated through experiments on zebrafish larvae, paving the way for live 3D holographic imaging.

Dual-Illumination Holographic Microscopy: How It Works

Dual-illumination holographic microscopy visualizing blood flow in zebrafish embryos.

The core of this innovation lies in its unique setup. The system uses two microscope objective lenses positioned at a 90-degree angle. This wide separation enhances angular rotation and improves z-resolution. The setup also simplifies the displacement of the sample in multiple directions, making it more versatile for live imaging.

Key components and processes include:

Laser Light Source: A laser diode splits light into two beams.
Acousto-Optic Modulators (AOMs): These modulate the reference beam.
Microscope Objectives: Water-based objectives image the sample.
CCD Camera: Captures the interference pattern created by the beams.
Holographic Reconstruction: Processes the captured data to create 3D images.

Zebrafish larvae are used as a model organism. They are embedded in a low melting point agarose and mounted in a transparent FEP tube. The tube is positioned vertically, allowing for easy rotation and displacement of the sample. The chamber is filled with tricaine to maintain the zebrafish during imaging.

Potential and Future Directions

This new technique addresses limitations of previous methods by providing a less invasive and more efficient way to visualize blood flow in three dimensions. The dual-illumination approach enhances angular rotation and z-resolution, offering a significant improvement over traditional methods.

The study successfully captured moving RBCs and demonstrated the technique's ability to perform phase-shifting reconstruction for both beams simultaneously. This is crucial for implementing live 3D holography.

Future work will focus on identifying the coincidence plane to refine the reconstruction process. The researchers believe that this technique, combined with advanced cleaning algorithms, will further improve z-resolution and extend the angle diversities of 3D reconstruction, potentially reaching a full 360-degree view.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1002/jemt.23076, Alternate LINK

Title: High-Speed Quantitative 3D Imaging By Dual-Illumination Holographic Microscopy

Subject: Medical Laboratory Technology

Journal: Microscopy Research and Technique

Publisher: Wiley

Authors: Dario Donnarumma, Nitin Rawat, Alexey Brodoline

Published: 2018-11-15

Everything You Need To Know

What is Dual-Illumination Holographic Microscopy?

The core concept is **Dual-Illumination Holographic Microscopy**, a novel method for visualizing blood flow. It utilizes two microscope objective lenses set at a 90-degree angle to enhance the angular rotation and improve the z-resolution. This setup allows for the creation of detailed 3D images of blood flow, especially of **red blood cells (RBCs)**, without the need for invasive contrast agents. The **Holographic Reconstruction** processes the captured data to generate these 3D images, offering a significant advancement over traditional methods.

Why is this new microscopy technique important?

The significance of the technology lies in its ability to non-invasively and efficiently image blood flow, particularly within the **microcirculation**. Understanding blood flow is crucial for biomedical research and early disease detection. Traditional methods are often invasive and time-consuming. **Dual-Illumination Holographic Microscopy** overcomes these limitations by providing high-speed, quantitative 3D insights. The improved resolution allows for more detailed examination of **RBCs** and their movement, offering new possibilities in diagnosing and studying diseases affecting the microcirculation.

What are the implications of using this technology?

The implications are far-reaching. By using this technique, scientists can gain a better understanding of various biomedical processes. The ability to visualize blood flow in three dimensions and in real-time offers potential for earlier and more accurate disease detection. This is especially important for diseases affecting the **microcirculation**. Furthermore, the use of **zebrafish larvae** as a model organism allows for live imaging experiments. This approach reduces the need for invasive procedures and facilitates the study of physiological processes in living organisms.

What are the main components and processes involved in this technique?

The basic components of **Dual-Illumination Holographic Microscopy** include a **Laser Light Source**, **Acousto-Optic Modulators (AOMs)**, two **Microscope Objectives**, a **CCD Camera**, and the **Holographic Reconstruction** process. The laser diode splits light into two beams which are then modulated by the AOMs. The **Microscope Objectives** image the sample, while the CCD camera captures the interference pattern. Finally, the Holographic Reconstruction transforms the captured data into 3D images.

Why are zebrafish larvae used in the experiments?

The use of **zebrafish larvae** is key because they are transparent and provide a suitable model for studying blood flow. The larvae are embedded in a low melting point agarose and mounted in a transparent FEP tube, facilitating easy sample manipulation, rotation, and displacement. The chamber is also filled with tricaine to maintain the zebrafish during imaging. This experimental setup allows researchers to observe the movement of **RBCs** within the zebrafish's microcirculation in a non-invasive and efficient manner.