Imaging Flow Cytometry

The ImageStream X mk II captures images of objects in flow, therefore is a powerful technique able to combine the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy. It produces multiple high resolution images of every cell directly in flow, including brightfield, darkfield (SSC) and up to 10 fluorescent markers. Applications include the enumeration of rare cells or objects, the characterization of circulating tumour cells, cell cycle analysis, analysis of intracellular colocalisation and translocation, as well as many others.

the internal optics of the image stream

In the laboratory the ImageStream is equipped with five lasers for fluorescence (405nm, 488nm, 561nm, 592nm, 642nm) and one for side scatter (785nm) in addition to an LED light source for brightfield imaging. It features 20x, 40x and 60x objectives with the option of extended depth of field (EDF) to improve detection of small objects. Images are captured in 12 channels across two cameras. Passing through the flow cell, around 5,000 cells are procesed and scored in a second, 10mls of processed blood from a patient takes around 10 minutes to quantify.

imaging biomarkers

Image: Karen Morris & Caroline Dive, Clinical and Experimental Pharmacology

 

In the above example EpCAM (epithelial cell adhesion molecule) positive circulating cells have been imaged and assessed. These cells have been donated by a small cell lung cancer patient and the sample enriched by the Cell Search platform. The labels used are DAPI (blue), side scatter for cell granularity (pink) and antibodies against CD45 (red) and cytokeratin (yellow). Examples of circulating tumour cells (CTCs) are in the top four image sets, these cells are DAPI positive, CK positive and CD45 negative. Contaminating white blood cells (WBC) were defined as DAPI positive and CD45 positive.

 

image stream analysis

Image: Karen Morris & Caroline Dive, Clinical and Experimental Pharmacology

 

ImageStream image data files were analysed by selecting single cells of the correct size on a plot of object area against object aspect ratio (width/length), focused cells using the Gradient RMS feature and DAPI positive cells on a histogram of channel 7 intensity. Finally WBCs and CTCs were identified on a scatter plot of channel 3 (CK) against channel 11 (CD45) intensity.

Institute equipment

The facility has one ImageStream X Mark II for the imaging of cells under flow cytometry. The equipment is based in a GCP(L) laboratory and the equipment is operated under those standards. This includes active monitoring of illumination, outputs and strict control over consumables. Three members of the facility aid researchers in making use of the equipment