A p p l i c a t i o n G u i d e Peter Banks, Ph.D. and Peter J. Brescia, Applications Department, BioTek Instruments, Inc., Winooski, VT Table of Contents Introduction ---------------------------------------------------------------------------------------------------------------------- 2 Microscope Slides and Microplates for Imaging --------------------------------------------------------------------- 2 Optical Microscopy with Microplates ----------------------------------------------------------------------------------- 3 Cytation 3 Overview ----------------------------------------------------------------------------------------------------- 4 Microscopy Applications using Cytation 3 Image Analysis Software ---------------------------------------------- 4 Cell Counting ----------------------------------------------------------------------------------------------------------------- 4 Quantifying Transfection Efficiency -------------------------------------------------------------------------------------- 6 Automated Fixation and Staining of Cells in Microplates --------------------------------------------------------- 7 Microscopy Applications using Third Party Software ------------------------------------------------------------------- 8 Montage Image Stitching (ImageJ) -------------------------------------------------------------------------------------- 8 Z-Stacking (CombineZP) ---------------------------------------------------------------------------------------------------- 9 Time Lapse Microscopy (Camtasia Studio) --------------------------------------------------------------------------- 10 Summary ------------------------------------------------------------------------------------------------------------------------- 11 References ----------------------------------------------------------------------------------------------------------------------- 11 BioTek Instruments, Inc. P.O. Box 998, Highland Park, Winooski, Vermont 05404-0998 USA Phone: 888-451-5171 Outside the USA: 802-655-4740 Email: customercare@biotek.com www.biotek.com Copyright 2014
Introduction Microscope Slides and Microplates for Imaging The standard microscope slide is a 1 mm thick flat piece of borosilicate glass, typically 75 by 25 mm, used to hold objects for examination under a microscope. It has been around for about 150 years and is still used widely by microscopists due to the wide range of specimens that can be mounted, its optical clarity and perhaps most importantly, its low cost. A single blank microscope slide costs about ten cents. The principle limitation of the slide is that when experimental variables are to be investigated or many specimens or samples need to be analyzed, the process of preparing and imaging all these experiments becomes laborious as each requires a separate slide. Historically, large sets of slides have been painstakingly prepared for the production and sharing of information across academia, medicine and industry. Microplates are the laboratory sample vessel of choice when an array of experimental conditions need to be investigated, experimental statistics are required and/or many samples need to be analyzed. Microplates allow multiple experiments on a single vessel, increased efficiency, and reagent cost savings to be realized. Typically, 96 experiments can be performed in the wells of one microplate (see Figure 1), but both higher and lower densities are available, such as 6-, 12-, 24-, 48-, 384- and 1536-well microplates. Microplates are produced in a wide range of materials (polystyrene, polypropylene, etc.); suitable for various optical methods (clear, black and white); treated with various coatings (tissue culture-treated, low adhesion, etc.); and black with clear bottom wells suitable for bottom reading. Some clear bottom microplates are suitable for high resolution optical microscopy. A number of vendors provide microplates specifically for this application (Table 1). Figure 1. Black, clear bottom 96-well microplate suitable for optical microscopy. Vendor Product Description Density Catalog #s Corning Greiner Bio-One Black with clear bottom cyclic olefin copolymer film, 127 µm thick with various coatings Black with glass bottom (200 µm thick), well flatness <50 µm with various coatings Table 1. Various microplates designed specifically for optical microscopy. 1536 4560-61; 4563-68; 4570-73 96 half area, 384 CELLSTAR tissue culture treated 96, 394, 1536 Advanved TC for sensitive, stressed and primary cells CELLCOAT protein coated microplates for sensitive, stressed and primary cells 4680-81 4580-87 655087; 655090; 675090; 781090-092; 783092; 788091-092 96, 384 655986; 781986; 788986 96, 384 655936; 655946; 655948; 655956; 781936; 781946; 781948; 781956 Cycloolefin SCREENSTAR (ultralow well base) 96, 384, 1536 655866;789836; 789866 Glass bottom SensoPlate 96, 384, 1536 662892; 655892; 781892; 788892; 782892; 788892; 782892; 783892; 655891; 781855; 781856 2
Optical Microscopy with Microplates The most common form of detection using microplates is PMT-based optics designed to capture as much light at the selected wavelength from the microplate well as possible. These optical paths are typical of conventional microplate readers and if performing cell-based assays, provide a cell population-averaged response. There are microplate readers that are actually automated digital fluorescence microscopes, available either with wide field or confocal optics to provide high resolution fluorescence microscopy of cells. These have been available for almost two decades and are the so-called high content screening (HCS) instruments. These HCS instruments are complex, dedicated units requiring large capital investments and only make economic sense if used for multiparametric phenotypic screening assays based on the use of microscopy. They are not designed for typical laboratory microscopy applications such as cell counting, determination of transfection efficiency and visualization of cell biology processes both as end point and in time lapse. A less costly solution for common laboratory cell imaging needs is provided by digital wide field microscopes using both bright field and fluorescence detection, such as BioTek s Cytation 3 Cell Imaging Multi-Mode Reader. Automation through typical microplate reader mechanics and software algorithms provides for a cost-effective solution for automated wide field fluorescence microscopy using microplates and other vessels, including microscope slides and cell culture flasks. Figure 2 demonstrates the high resolution microscopy available from Cytation 3 using either conventional microscope slides or clear bottomed microplates. A. B. C. D. Figure 2. Optical microscopy using Cytation 3. A: Fixed PtK2 cells in ScreenStar 96-well microplates using 40x microscope objective. Central cell is undergoing mitosis: chromatin is condensing into chromosomes in Prophase. The cells are stained with Hoechst 33342 (blue), a primary antibody against tubulin with secondary antibody labeled with Alexa Fluor 488 (green), and Texas Red phalloidin (red); B: 6x5 montage of images stitched together from a 16 µm cryostat section of mouse intestine on a microscope slide using 4x microscope objective. The tissue slice was fixed and stained with Alexa Fluor 350 wheat germ agglutinin, Alexa Fluor 568 phalloidin and SYTOX Green nucleic acid stain. C: Z-stacked image of an InSphero Liver Microtissue cell aggregate (actual size 200 µm ID) in 96-well GravityTrap microplate under physiological conditions, 10x microscope objective. Red: MitoTracker Red Mitochondrial Dye; Blue: Hoechst 33342 Nuclear Stain; Green: Calcein Mitochondrial Stain. D: Zebrafish embryo development. Image displays bright field microscopy of the embryo in a 96-well microplate with 4x microscope objective. 3
Cytation 3 Overview The Cytation 3 is a Multi-Mode Microplate Reader that provides modular detection options. These modular options include: PMT-based whole well detection using monochromators PMT-based whole well detection using spectral filters and dichroic mirrors CCD-based wide field microscopy Various combinations of these modules are available, including a model that provides only automated digital wide field microscopy. Cytation 3 s microscopy module uses a 16 bit gray scale scientific grade CCD camera, high power LED sources for fluorescence excitation and bright field, a wide variety of filter cube options allowing for the use of fluorophores that cover the visible spectrum and a broad range of microscope objectives including 2.5x, 4x, 10x, 20x, 40x and 60x. The Cytation 3 is also designed to enable kinetic live cell imaging. The detection chamber can be held to any temperature from ambient up to 45 C; or specifically for the health of cells, at 37 C ±0.5 C. The Gas Controller accessory also allows for the control of both CO 2 and O 2 in the detection chamber with a resolution of ± 0.1%. Gen5 software with Cytation 3 is designed for ease of use by providing auto-focus and auto-exposure capability for a variety of sample vessels including petri dishes, T-25 culture flasks, microscope slides and 6- to 384-well microplates. Captured images can be viewed on a monitor, downloaded as a variety of data files including TIFF, PNG, and JPG for data sharing or further processing. Gen5 Image+ Software allows for cell counting and cell sub-population analysis such as the quantification of transfection efficiency and percentage of cells expressing phenotypes in the field of view. Microscopy Applications using Cytation 3 Image+ Software Gen5 Image+ software provides for common image analysis procedures such as those described above using the Cellular Analysis module. Captured images can be analyzed for cell level details, including object size, intensity and circularity measurements. A total cell count is returned for all identified objects in the image which is useful in of itself and for many applications such as transfection efficiency and phenotypic assays. Cell Counting The most common form of cell counting using fluorescence microscopy uses nuclear stains such as DAPI and Hoechst 33342. These fluorogenic stains can be added to live or fixed cells and the dye preferentially binds to dsdna present in the nucleus, whereupon a large increase in fluorescence quantum yield provides a means for identifying nuclei by blue emission. Because most eukaryotic cells contain only one nucleus, automated cell counting using the Cellular Analysis module in Gen5 Image+ is fast and easy. A number of analysis parameters are available to define the nuclei for cell counting, such as minimum and maximum object sizes, split touching objects, but most important is the threshold. A threshold is the pixel intensity value in the image, above which is considered the signal of interest, in this case stained nuclei. The threshold is used to define which pixels comprising the image identify nuclei. Then using minimum and maximum object sizes in conjunction with threshold, the software creates an object mask that defines one typical nuclei. This object mask is then applied to other nuclei-like objects in the field of view allowing for the number of cells to be counted (Figure 3) 1. 4
Figure 3. Cellular Analysis tab of Gen5 Image + : Analysis parameters are located above the 20x image of DAPI-stained nuclei. In this analysis, the stained nuclei are the objects which have been analyzed with all analysis parameters set to default. Counted nuclei (and thus cells) have a gold perimeter associated with them, drawn by Gen5. Note nuclei at the edge of the image are not counted (no gold perimeter) as the Include edge objects analysis parameter is unchecked. The most critical parameter for accurate cell counting is the threshold. If a threshold of 1,000 is applied for example, only pixels from 1,000 to the maximum of 65,534 in intensity value in the image are identified and circled with a mask. If the threshold were increased to 10,000, the size of the object mask will shrink or disappear since only pixels from 10,000-65,534 are included. The images below (Figure 4) illustrate the masking behavior at three different threshold levels. Note the shrinking of the object mask area as the threshold is increased. 5 Figure 4. Effect of Threshold and Object Size Definition on cell count analysis. Cellular analysis of 20x images at various Threshold and Object Size values. White arrows indicate a common cluster of three stained nuclei which becomes better analyzed as a cell nucleus with increasing Threshold intensity levels. Yellow arrows indicate exclusion of small or large artifacts from cell count can be achieved by placing upper and lower limits on the object size. DAPI stained cells were imaged using the Blue filter cube of the Cytation 3 and cellular analysis performed with default settings except for Threshold. Threshold value for each analysis is indicated.
Quantifying Transfection Efficiency Transient transfection has become a common method for the introduction of reporter genes (i.e. the family of GFP photoprotins) into cultured cells. The efficiency of transfection can be dependent on several factors and significantly impact experimental results dependent on the successful introduction and expression of the reporter gene. Factors such as cell type, choice and concentration of transfection agent and reporter gene all influence the extent of transfection. Creating an object mask in Gen5 Image+, similar to that done with Cell Counting, is an effective method of quantifying transfection efficiency. The object mask created with nuclear staining is typically made using the blue emission channel since ordinarily DAPI or Hoechst 33342 dyes are used. Other fluorescence channels can also use this object mask such that the relative numbers of cells expressing blue and different color emission can be assessed. This is demonstrated below (Figure 5) using Bacmam transfection of a Histone H3-GFP fusion protein into U-2 OS cells 2. By staining the cells with a nuclear stain following transfection, transfection efficiency can be determined by a simple ratio of green and blue channels: blue yielding the total number of cells; and green, the number of cells transfected with the GFP fusion protein (Figure 6). Figure 5. Live-cell imaging with BacMam Histone H3 virus infected cells. U-2 OS cells were infected with different concentrations of BacMam Histone H3-GFP (Numbers reported in the bottom left-hand corner of each image are % (v/v)). After a 24 hour incubation cells were stained with 5μg/mL Hoechst 33342 for 15 min @ 37 C. Cells were imaged with the 20x objective using blue and green LED cubes. Scale bar indicates 80 μm. Figure 6. Effect of Virus concentration on Transfection Efficiency. Using a signal threshold of 10,000 and a minimum size of 10 μm (all other analysis parameters at default), object counting for nuclei identified total cell number in blue (Hoechst 33342) and transfected cells in green (GFP) channels, respectively. The ratio of cells positive for Histone H3-GFP transfection can then be assessed and plotted against virus concentration for both 4x and 20x images. 6
Automated Fixation and Staining of Cells in Microplates The advantages of performing fluorescence microscopy in a microplate-based format include the ability to rapidly analyze multiple experimental conditions, obtain statistical information from repetitive experiments and increase sample throughput. This latter attribute is fully enabled by the automation of the experiment workflows. This is certainly true for immunocytochemistry applications, where cells must be fixed, permeabilized and stained which can be laborious, even when using a single slide. By using liquid handling devices specifically for microplate operation, these workflows can be efficiently automated. The MultiFlo FX automates reagent dispensing onto cells in microplates and also washes cells to remove excess reagent. A typical automated workflow is demonstrated in Figure 7. Figure 8 demonstrates the quality of automated staining process using three separate stains. Figure 7. Automated Workflow for Cell Seeding, Fixation, Permeabilization and Three Color Staining Process. An EL406 Combination Washer Dispenser controlled by Liquid Handling Control (LHC) software was used to carry out the process steps for cell fixation, permeabilization and staining with three colors: DAPI nuclear stain, Alexa-Fluor 488 phalloidin actin stain and Texas red labeled secondary antibody. 7
Figure 8. U-2 OS cells stained for mitochondria, F-actin and nucleus. Cells in 96-well plates were fixed and stained for mitochondria (primary antibody to mitochondrial proteins and secondary antibody labeled with Texas Red), F-actin (phalloidin Alexa Fluor 488), and DNA (DAPI). Scale bar indicates 80 μm. Microscopy Applications using Third Party Software The excitation light source for widefield microscopy has historically been a mercury vapor lamp but it is becoming more common to find LEDs and CCD detectors for image capture as instruments move to the digital age. LEDs provide a stable light source without the need to pre-warm while LED detectors allow immediate visualization on a computer screen and, in many cases, real-time analysis via integrated or available software. In addition, digital images, much like the prepared microscope slides of the past, provide a means for development and dissemination of information for analysis and/or teaching purposes. Their portability in any number of acceptable digital formats allow images to be quickly transported and shared to the global community across a wide range of devices including PCs, tablets, and smart phones. Additionally, images can be analyzed using a variety of 3 rd party software with a broad range of assessable analytical algorithms. Montage Image Stitching (ImageJ) Cytation 3 can capture montages of images that cover a large objects such as a tissue slice, organism (i.e. zebrafish, nematode) or a complete microplate well. The montages can then be stitched together to form a complete image of the object of interest using ImageJ (Figure 9). 8
A. B. C. D. E. Figure 9. Image stitching. 10x images of a 16 µm cryostat section of mouse kidney stained with Alexa Fluor 488 wheat germ agglutinin (green), Alexa Fluor 568 phalloidin (red) and DAPI (blue). Images were collected as a 5x5 montage by Cytation 3 and imported to ImageJ where they were stitched for analysis. A: Individual fluorescence channel images (RGB); B: Composite image of RGB images: C: Complete 5x5 montage of composite images (images A & B are in the top left-hand corner of the 5x5 montage); D: Stitched images from the montage using Image J; E: Expanded portion of top left area repe=resenting four images from the montage demonstrating seamless stitching. Z-Stacking (CombineZP) Under the high magnification needed to view sub-cellular objects, the depth of field is typically limited to less than the depth of a eukaryotic cell, thus some cellular structure will not be in focus. Confocal microscopy can alleviate this issue by restricting the field of view and taking a number of images at different focal planes such that a 3D reconstruction of the cell can be made. This method also suffices for more three dimensional structures such as tissue slices or 3D cell culture models. While wide field microscopy cannot provide 3D reconstructions, deconvolution software can provide composite images from a similar workflow where multiple images at different focal planes are captured. The software will then analyze each image and only retain portions of each image that are in focus. Thus the composite image appears to provide focus through the depth of the object. Figure 10 illustrates this with a 3D collagen-based scaffold containing HCT116 tumoroid cell aggregates with a depth of 120 m 4. 9
A. B. C. D. Figure 10. Single plane and z-stacked images of HCT116 tumoroids. (A-C) Multi-color overlaid images captured at multiple z-planes within the 120 μm hydrogel, and (D) final CombineZP stacked image. 22 images captured with each fluorescent probe were combined using the Maximum Contrast algorithm. Time Lapse Microscopy (Camtasia Studio) Cytation 3 is fully equipped for live cell microscopy over extended periods. The detection chamber within the instrument is temperature controlled (37 C ± 0.5 C) and both CO 2 and O 2 controlled with the Gas Control accessory. This allows for time lapse microscopy within the Cytation 3. Gen5 Image+ software captures images at the desired time resolution, then the TIFF files can be ported to the 3 rd party software Camtasia Studio for the creation of a video comprising the images. An example of images captured of live cell RNA expression using RNA probes 5 is illustrated in Figure 11. A time lapse video of the events can be viewed here. 10
Figure 11. Live Cell Imaging Time Course. MCF-7 cells were seeded at 15,000 cells per well and grown overnight. The following day cells were stained with 0.5 μg/ml Hoechst 33342 for 15 min at 37 C, treated with 4 μl CY3- GAPDH reagent and immediately imaged kinetically with the 20x objective using the DAPI and CY3 LED cubes and the images overlaid. Scale bar indicates 100 μm. Summary The advantages to performing automated digital widefield fluorescence microscopy in a microplate format are clear as they allow the examination of an array of experimental conditions to be performed and/or enable multiple replications of experiments to provide statistical data all while lowering reagent costs using the small volume typical of a microplate well. High resolution optical microscopy is attainable with both glass bottomed and thin plastic bottomed microplates in 96-, 384- and even 1536-well densities. The Cytation 3 is specifically designed to work with microplates for automated digital wide field microscopy, although a number of other vessels can be used, such as microscope slides, Petri dishes and T-25 culture flasks. Gen5 Image+ software enables cell counting, quantifying transfection efficiency, phenotypic changes in cells and many other applications. Furthermore, the portability of TIFF files from Gen5 allows the use of multiple 3 rd party software to further extend capabilities such as stitching of montages, z-stacking and time lapse videos. References 1. Analysis of Nuclear Stained Cells: http://www.biotek.com/assets/tech_resources/cytation3_nuclear_staining_app_note.pdf 2. Imaging of BacMam Transfected U-2 OS Cells: http://www.biotek.com/assets/tech_resources/bac_mam_transfection_app_note.pdf 3. Automated Tissue Culture Cell Fixation and Staining in Microplates: http://www.biotek.com/assets/tech_resources/automated_cell_fixation_and_staining_app_note.pdf 4. Z-Stacking of Single Plane Digital Widefield Fluorescent Images: http://www.biotek.com/assets/tech_resources/z-stacking_app_note.pdf 5. Live Cell Imaging of RNA Expression: http://www.biotek.com/assets/tech_resources/smartflare_app_note.pdf 11 Rev. 05/13/14