Concurrent Scientific Session (Imaging): Imaging Flow
Fluorescence in situ hybridization and nuclear translocation events quantified by imaging flow cytometry
Imaging flow cytometry with its ability to perform image analysis on up to 10 spectrally separated but spatially correlated images of the same cell in statistically robust cell population sizes has steadily seen an increase in the number of applications and the use of this platform in various research settings to a point where it is now commonly found in flow and/or image cytometry shared resource centers. One of the originally anticipated applications of imaging flow cytometry was in the area of fluorescent in situ hybridization (FISH), a technique which, due to its manual evaluation of slide-based sample preparations, suffers from a relatively low sensitivity of detection. A second early application was in the area of quantifying nuclear translocation events of transcription factors such as NFkB and NFAT which conventionally had been analyzed by microscopy or molecular approaches such as western blot or gel shift assays of nuclear protein extractions. Practical examples and considerations of using imaging flow cytometry to assess FISH and nuclear translocation of NFkB and NFAT will be presented with a discussion of the challenges of sample preparation and data analysis faced with each of these applications and the experience at Roswell Park with incorporating this analysis platform in a shared resource setting. Supported by NIH 1S10OD018048, R50CA211108, and NCI Cancer Center Support Grant to Roswell Park P30-CA016056
Imaging Flow Cytometry for Phytoplankton Analysis: Instrumentation and Applications
Imaging flow cytometry was first introduced into algal research in the late 1990’s. The ability to enumerate, classify and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function, carbon density, and the role phytoplankton play in both freshwater and marine microbial food webs. Traditional micro-phytoplankton quantification methods use microscopic techniques that require preservation and are slow, tedious and very laborious. The availability of automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and phytoplankton biomass. Currently many imaging cytometry tools are available for use in aquatic sciences and provide a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments. Imaging flow cytometry, combines the speed and statistical capabilities of flow cytometry with imaging features of microscopy, and has contributed significantly to the advancement of phytoplankton analysis in recent years. This presentation discusses various instrumentation and applications relevant to phytoplankton communities.
Imaging in flow in the 70’s—The “Correlation System”
The vast majority of flow cytometers in use since the inception of this technology operate by making cellular measurements derived from a pulse of fluorescence or laser-light scatter generated as a particle intercepts a focused laser in a flow system. In the case of fluorescence, these “zero resolution” systems accurately quantify the amount of light without knowledge of the location or distribution of the source of this light within the cell, since the laser focal point typically is larger than the cell being measured.
There are situations where the spatial distribution of fluorescence in a cell can provide important additional information, but this information is not available in most contemporary zero resolution flow cytometers.
In the early 70’s, there was interest in developing rapid automated diagnostic systems for the fields of hematology and cancer diagnostics based on either flow cytometry or microscopic image analysis. In the lab of Leon Wheeless at University of Rochester, scientists and engineers set forth to utilize Stanley Patten’s diagnostic criteria in cytopathology towards the development of an automated pre-screening system for detection of atypical or malignant cells in cytologic samples derived from cervical sampling or urine.
The principle of concept of “slit-scanning,” or restricting the detection of light from a cell by sampling through a slit mechanically moved over the image of the cell, was initially tested with a custom fluorescence microscope. This proof of concept led to the development of a slit-scan flow cytometer, where the laser illumination field was narrowed to a plane of laser light, oriented orthogonal to the direction of cell flow, with a thickness much smaller than that of the cell. By evaluating the fluorescence intensity pulse shape in real time, this technique proved highly effective at detecting atypical or malignant cells in a flow cytometer. It was noted with this system, however, that there was an unacceptable false positive rate, although only slightly higher than that obtained using microscopic screening by trained cytotechnologists.
In order to evaluate cells in flow and their correlated real-time classification as normal or abnormal, a “correlation system” was built which provided images in flow of cells correlated with their pulse shape analysis and classification. This presentation will describe the characteristics and operation of this original imaging flow cytometer.