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Cell imaging multi-mode microplate reader

11th December 2014

Posted By Paul Boughton


Top is an image of the Cytation 5 showing the open microplate tray. Below is an artistic impression highlighting the phase contrast and fluorescence microscopy optics
A: Brightfield microscopy using a 2.5x microscope objective of 1,000 MCF-7 cells forming a spheroid in a hanging drop of media; B: H&E stained section of human intestinal wall mounted on a microscope slide and imaged with a 20x microscope objective as a 10x8 montage (80 separate images) which have been stitched together with Gen5 software to provide a composite image of wide field of view; C: High resolution microscopy of a PtK2 cell undergoing mitosis (metaphase as the chromosomes line up along the metaphase plate) using a 60x microscope objective (blue depicts the chromosomes; green the spindle fibres and red the cytoskeleton); D: Phase contrast image of lillium anther using a 4x microscope objective; E: Phenotypic assay in 384-well microplates to quantify the number of autophagosomes in the RFP channel (number of red spots) relative to the number of cells identified in the GFP channel using a 40x microscope objective; F: Dose response curves for the inhibition of AKT phosphorylation (Ser 493) by inhibitors

Enabling optical microscopy and cell-based assays with a flexible new solution.

The cell is life at its most basic. It is used as a model system for the study of organism structure, function and dysfunction. Myriad cell lines and primary cells isolated from organisms are used throughout the life sciences. 

These cell lines are probed in many ways, but two laboratory methods are critical to most research programmes involving the use of cells: optical microscopy and cell-based assays.

Optical microscopy is used to perform routine laboratory tasks such as cell counting and determination of transfection efficiency. When used with stains, such as hematoxylin and eosin, optical microscopes can be used for histological applications useful for the diagnosis of cancer.

Equipped for fluorescence microscopy, a broad palette of fluorescent probes can be used, which include intercalating stains, reactive fluorescent dyes, fluorescently-labelled antibodies and the family of GFP variants. 

These probes allow for a number of experiments including localisation of quantification of DNA and proteins within the cell; conducting phenotypic assays based on protein translocation and cell morphology changes; and performing sub-population analysis of the cells in the field of view based on a phenotype expression (i.e. cell cycle). Furthermore, optical microscopy with phase contrast optics provides adequate contrast in cell monolayers and thin tissue sections such that label-free, live cell analysis is possible.

Cell-based assays typically quantify attributes of populations of cells. These assays are most often conducted in 96- or 384-well microplates with microplate readers. These readers use PMT detection and optical paths designed to capture as much light from the well as possible. Cell-based assays can quantify ligand binding to receptors; the expression of second messengers; post-translational modifications of proteins, such as phosphorylation and ubiquitinylation; cellular viability, toxicity and apoptosis; and many other cellular attributes.

Multi-mode microplate readers provide many detection modes including the most common based on absorbance, fluorescence and luminescence detection.

Multiple instruments to conduct these tasks

In some laboratories, perhaps as many as half a dozen different instruments are used to perform all the applications described above with optical microscopy and cell-based assays. These include: bright field microscope with oculars; colour-based digital microscope for histological staining; fluorescence microscope with oculars or monochrome CCD digital microscope; phase contrast microscope for label-free, live cell analysis; automated fluorescence digital microscope for high content analysis using phenotypic assays; and multi-mode microplate reader.

A new solution

The Cytation 5 is a new cell imaging multi-mode microplate reader that is equipped to perform all the applications described for optical microscopy and cell-based assays.

Furthermore it provides equivalent performance to the six instruments listed above in one bench-top instrument. The microscopy components are digitally based, consisting of LEDs for illumination, filter cubes, microscope objectives (2.5x – 60x) and a CCD camera.

Most microscopy components are located in the lower half of the Cytation 5, except for phase contrast illumination, which resides in the same upper space as the PMT-based optics of the conventional microplate reader.

The design is also modular, allowing selection of microscopy and/or microplate reader optics as desired. Various sample vessels can be used with the microscopy modules, including microscope slides, a broad range in microplate densities (6- to 384-well), Petri dishes and tissue culture flasks (T-25). 

Image acquisition, processing and analysis is performed by Gen5 software available with the Cytation 5. Fig. 1 illustrates the actual laboratory instrument and a cut-away impression of its modularity as part microscope/part microplate reader.

As shown in Fig. 2, Cytation 5 is capable of capturing images using a variety of methods. These include fluorescence, brightfield, H&E and phase contrast, along with data from a cell-based assay performed with the multi-mode detection function in the system. 

The images and data highlight the many practical applications of Cytation 5 that traditionally would have required using two or more of the instruments described above. 

Hybrid approach for maximum flexibility

The multi-mode detection module features BioTek’s Hybrid Technology, which incorporates variable bandwidth monochromator optics and high sensitivity filter-based detection optics for excellent versatility and performance. Temperature control to 65 °C and shaking, plus available CO2/O2 control and dual reagent injectors optimize conditions for cell-based imaging and detection.

The fluorescence monochromators offer a variable bandwidth from 9nm to 50nm in 1nm increments for ultimate flexibility. Another key feature is laser-based excitation for AlphaScreen/AlphaLISA assays.Finally, the powerful yet easy to use Gen5 software enables efficient plate reading, image capture and analysis

For more information, visit www.scientistlive.com/eurolab

Peter Banks is scientific director of BioTek Instruments, in Vermont, USA.






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