How to Use Imaging Flow Cytometry

How to Use Imaging Flow Cytometry

Introduction of Imaging Flow Cytometry

Imaging Flow Cytometry has emerged as a powerful tool in the realm of cell analysis, offering researchers the ability to simultaneously capture high-resolution images and gather quantitative data on thousands of individual cells. This cutting-edge technology combines the strengths of traditional flow cytometry with microscopy, providing a deeper understanding of cellular morphology, spatial distribution, and functional characteristics. In this article, we will delve into the fundamentals of Imaging Flow Cytometry and guide you through the steps to effectively utilize this technique in your research.

Imaging Flow Cytometry integrates the principles of flow cytometry and microscopy, allowing researchers to analyze cells based on multiple parameters while capturing detailed images of each individual cell. Unlike conventional flow cytometry, which provides quantitative data on cellular characteristics, Imaging Flow Cytometry adds a visual dimension to the analysis. This enables the examination of cellular structures, subcellular localization of proteins, and the dynamics of cellular processes.

Key Components of Imaging Flow Cytometry

Optics and Imaging System: Imaging Flow Cytometers are equipped with sophisticated optics and imaging systems. These systems use multiple lasers and detectors to excite and capture fluorescent signals from labeled cellular components. The high-resolution camera records detailed images of cells as they pass through the flow cell.

Imaging flow cytometry.Figure 1. Imaging flow cytometry.

Fluidics System: Similar to traditional flow cytometry, Imaging Flow Cytometry employs a fluidics system to ensure a single-cell suspension. The cells are hydrodynamically focused to pass through the imaging area one at a time, allowing for precise imaging and data collection.

Image Analysis Software: The images acquired during the analysis are processed using specialized software. This software facilitates the identification and quantification of various cellular features, such as cell morphology, size, and fluorescence intensity. Users can define specific regions of interest and customize analysis parameters to extract meaningful data.

Steps to Use Imaging Flow Cytometry

Sample Preparation:

  • Prepare a single-cell suspension: Ensure that your cell sample is free of clumps or aggregates to facilitate accurate analysis.
  • Labeling: Use fluorescent dyes or antibodies to label specific cellular components of interest. This step is crucial for capturing fluorescence signals during imaging.

Instrument Setup:

  • Calibrate the instrument: Perform instrument calibration using calibration beads or particles to ensure accurate and reproducible results.
  • Choose appropriate lasers and filters: Select lasers and filters based on the fluorophores used for labeling. This step is essential for optimal signal detection.


  • Load your prepared sample into the Imaging Flow Cytometer.
  • Set acquisition parameters: Adjust settings such as laser power, exposure time, and acquisition speed to optimize image quality and data collection.

Image Analysis:

  • Import acquired images into the analysis software.
  • Define analysis parameters: Specify parameters for cell identification, including size, shape, and fluorescence intensity.
  • Analyze and visualize data: Utilize the software tools to generate visual representations of cellular features and export quantitative data for further analysis.

Applications of Imaging Flow Cytometry

Cellular Morphology:

  • Study cell shape and size variations.
  • Identify and quantify cellular abnormalities.

Subcellular Localization:

  • Investigate the distribution of proteins within cells.
  • Examine organelle localization and dynamics.

Functional Analysis:

  • Assess cellular functions such as proliferation, apoptosis, and autophagy.
  • Monitor intracellular signaling pathways.

Rare Event Analysis:

  • Detect and analyze rare cell populations within heterogeneous samples.
  • Enhance the sensitivity of detection for rare events.


Imaging Flow Cytometry represents a groundbreaking advancement in cell analysis, providing researchers with a holistic view of cellular characteristics. By combining the strengths of flow cytometry and microscopy, this technique enables the comprehensive study of cellular morphology, spatial distribution, and functional attributes. As technology continues to evolve, Imaging Flow Cytometry is poised to play a pivotal role in advancing our understanding of complex biological processes at the single-cell level. Integrating this powerful tool into your research workflow can unlock new insights and contribute to breakthroughs in various fields, from immunology to cancer research.

  1. Barteneva NS, et al.; Imaging flow cytometry: coping with heterogeneity in biological systems. J Histochem Cytochem. 2012, 60(10):723-33.

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