University of Pittsburgh Cancer Institute (UPCI)

Imaging Cytometry

The Imaging Cytometry platform houses the Cellomics ArrayScan VTI and Amnis ImageStream cytometers, which enable the collection of quantitative data on many cellular processes, including:

Advantages of Imaging Flow Cytometry over Conventional Flow Cytometry

• Ability to localize fluorescence within cells and quantitatively determine fluorescence in defined cellular features (e.g., nucleus versus cytoplasm, endosomes versus lysosomes)
• Ability to co-localize fluorescent signals in the entire cell or within selected cellular features (e.g., quantifying tagged proteins within organelles)
• Ability to localize fluorescent signals during cellular interactions (e.g., adhesion molecules in the immune synapse)
• Availability of 35 intensity-based and morphologic parameters per channel with ability to create user-defined parameters based on Boolean and algebraic calculations (e.g., nucleus-to-cytoplasm ratio)
• Ability to view images (single or composite fluorescent images, or brightfield) associated with a single point or a region on a bivariate scatter plot
• Morphologic parameters can serve as surrogates for one or more fluorescent parameters, freeing up fluorescent channels (e.g., detection of apoptosis cells by comparing nuclear area and peak nuclear pixel intensity, ability to distinguish prophase, metaphase and anaphase on the basis of nuclear texture, size and aspect ratio)

Advantages over Fluorescence Microscopy-Based Image Analysis

• Higher throughput by 2-3 orders of magnitude gives better statistical analysis
• Choice of objects and subpopulations for analysis is objective rather than subjective

Advantages over Fluorescence Activated Cell Sorting Followed by Image Analysis

• Can “sort” on calculated morphologic features and by location of fluorescence within a cell
• Does not require two complex instruments which are usually not in the same location (1.8 miles apart in our case)
• No loss of cells or viability issues
• Can perform kinetic experiments

Advantages over Image Scanning Cytometry

• Better suited to cells in suspension (simplified sample handling)
• Gives light scatter measurements
• Findings more easily translated to flow cytometry (e.g., for polychromatic multiparameter analysis or cell sorting)
• Cells in suspension are not altered in function or appearance by adherence to a solid substrate

Instrumentation

Cellomics ArrayScan VTI Imaging Cytometer

The Imaging Cytometry platform centers on a Cellomics ArrayScan VTI imaging cytometer with up to 40X magnification and a 120W mercury-halide white light source capable of excitation from 300nm-700nm and up to 5-color capability. Related equipment also includes a Twister robotic plate loader, a Zeiss Apotome for pseudo-confocal capability (7uM sectioning), a 1T server for data storage and a dedicated data analysis terminal.

The ArrayScan VTI is an automated fluorescence image analysis system with integrated image analysis and data management that together allow the user to perform High Content Screening analysis (HCS). Conventional inverted fluorescence microscopy is applied with robotics to enable fully automated imaging of cells in multi-well cell culture plates. The VTI brings three common technologies--a fluorescent plate reader, fluorescent microscope, and flow cytometer--into a single instrument. The focus of the VTI is to establish event dynamics across populations and subpopulations like a flow cytometer, while incorporating sophisticated image processing to allow for spatial discrimination of fluorescent dyes within the cell. Sophisticated image analysis enables quantification of cellular fluorescence on a large scale to include highly complex phenomenon like morphological changes involving sub-cellular constituents, whole cell morphology, and multi-cellular structures, for example. Biological content is further increased through spatial analyses of targets labeled with fluorescent dyes. The integrated database allows for remote sharing of data and images and features logical in-line operations like automated dose response curve reporting and relationships within reams of cellular data.

More information about the ArrayScan VTI

Some commonly used fluorophores compatible with the ArrayScan VTI:

Amnis ImageStream© 100

The Amnis ImageStream 100 is the first commercially available imaging flow cytometer. It combines advantages of flow cytometry (ability to interrogate large numbers of cells in suspension, well-developed color compensation, analysis and display methods) with those of image analysis (digital imagery of each individual cell, calculation of morphological features from digital images, localization of fluorescence to morphologic features, co-localization of fluorescent probes).

In the simplest terms, the ImageStream may be thought of as a flow cytometer in which the photomultiplier tubes have been replaced by an array of sensitive charge coupled device (CCD) cameras. Cells in the sample are hydrodynamically focused within a quartz cuvette, where they are interrogated by a 480 nm solid-state laser. Scattered incident light and emitted fluorescence are collected at 90 degrees to the excitation beam and broken into 6 images of distinct bandwidth by a dichroic filter stack that directs the resulting spectral bands at different angles. These images are captured by adjacent segments of the camera chip. The images include brightfield (transmitted light), 488nm side-scatter, and 4 emitted fluorescence channels corresponding to those used on many 4-color flow cytometers. Individual objects are identified and distinguished from background in real time in a process analogous to live gating, and the images are stored to a raw image file (RIF). In post-acquisition processing, the 6 images of each object are put into register, color is compensated pixel by pixel, and features such as area and intensity are calculated for each parameter and each image (see table below). The results are stored in a compensated image file (CIF) for analysis.

The parameters include those familiar to flow cytometry, such as intensity, defined as the total pixel intensity of an object in a given fluorescence channel (minus background), and side scatter, the intensity of the 488nm image. Cell size can be measured directly as the brightfield area. A wealth of additional parameters (see table below), drawn from the repertoire of image analysis but foreign to most flow cytometrists, allows characterization of cellular features and localization of fluorescent probes.

*Calculated Image Features and Definitions. These features are calculated individually for all 6 image channels. Additional parameters can be defined by the user and calculated for each object. For example, if the nuclear stain Draq5 (collected in channel 6) is used, the nucleus-to-cytoplasm ratio can be calculated as the channel 6 area, divided by channel 2 (violet brightfield) area. Table courtesy of Amnis.

Special Guidelines for the Amnis ImageStream 100 Imaging Flow Cytometer

• Samples must be at a very high concentration. A minimum of 20 x 10⁶ cells/mL is permissible, but acquisition will be slow. The optimal concentration is 50 x 10⁶ cells/mL. The minimum cell number is 2 x 10⁶ per sample.
• Samples must be filtered through a 70 micron cell strainer (Falcon 35-2350 or equivalent). Undoing a sample clog can take up to 2 hours on the ImageStream; you will be charged for this time if you did not filter your sample. Samples should be prepared in 0.5 mL flat top microcentrifuge tubes (Fisherbrand 05-408-128 or equivalent). Use these tubes from the start to avoid sample loss during transfer.
• Remember to prepare single-stained tubes for each fluorochrome, for color compensation. Draq-5 (Alexis Biochemicals, BOS-889-001-R200) is very useful for visualizing the nucleus. This dye occupies the FL6 channel (similar to PE-Cy5). Cells do not have to be permeabilized for Draq-5 uptake. It is not washed out and can be added 10 minutes prior to sample acquisition. Use the Amnis worksheet to design your experiment and record cytometer settings.