Applications - App Guides
Automated Digital Microscopy
Authors: Peter Banks, Ph.D. and Peter J. Brescia, Applications Department, BioTek Instruments, Inc., Winooski, VT
Microscope Slides and Microplates for Imaging
The standard microscope slide is a 1 mm thick flat piece of borosilicate glass, typically 75 mm 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 to be performed in a single vessel, increased efficiency, and reagent costs 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 come 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.
Table 1. Various microplates designed specifically for optical microscopy.
Optical Microscopy with Microplates
The most common form of detection using microplates is PMT-based optics designed to capture as much light as possible at the selected wavelength from the microplate well. These optical paths are typical of conventional microplate readers and if performing a cell-based assay, provide a cell population-averaged response. There are microplate readers that are actually automated digital fluorescence microscopes, available with either widefield or confocal optics that 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 much less costly solution for common laboratory cell imaging needs is provided by digital widefield microscopes utilizing both brightfield and fluorescence detection such, as BioTek’s Cytation 5 Cell Imaging Multi-Mode Reader. Automation through typical microplate reader mechanics and software algorithms provides a cost-effective solution for automated widefield fluorescence microscopy using microplates and other vessels, including microscope slides and cell culture flasks. Figure 2 demonstrates the high resolution microscopy available from Cytation 5 using either conventional microscope slides or clear bottomed microplates.
Figure 2. Optical microscopy using Cytation 5.
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: H & E fixed and stained tissue slice of human intestinal wall imaged with a 60x objective generated a 12 x 8 montage. Montage images were then stitched using linear blend method with registration on the red channel.
C: Label-free monitoring of apoptosis induction using phase contrast imaging. Apoptotic cells were imaged using a 20x objective following a seven hour incubation period with 40 μM oridonin.
D: Zebrafish embryo development. Image displays brightfield microscopy of the embryo in a 96-well microplate with 4x microscope objective.
Cytation 5 Overview
The Cytation 5 Cell Imaging Multi-Mode Reader 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
- Laser-based excitation for AlphaScreen®/AlphaLISA® assays
- CCD-based fluorescence, brightfield, color brightfield and phase contrast widefield microscopy
Various combinations of these modules are available; including a model that provides only automated digital widefield microscopy.
Cytation 5’s microscopy module uses a 16 bit monochrome scientific grade CCD camera and a broad range of microscope objectives from 1.25x to 60x. Fluorescence microscopy is enabled using an inverted geometry with high power LED sources situated beneath the specimen for fluorescence excitation and a wide variety of filter cube options allowing for the use of fluorophores that cover the visible spectrum. The transmission microscopy modes, brightfield, color brightfield and phase contrast, have their light sources situated above the specimen.
The Cytation 5 is also designed to enable kinetic live-cell imaging. The detection chamber can be held to any temperature from ambient up to 65 °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 CO2 and O2 in the detection chamber with a resolution ± 0.1%.
Gen5 software with Cytation 5 has been 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 microplates ranging from 6- to 384-well densities. Captured images can be viewed on a monitor, downloaded as a variety of data files including TIF, PNG, and JPG for data sharing or further processing. Gen5 Image+ Software allows for cell counting and cell subpopulation analysis such as the quantification of transfection efficiency and percentage of cells expressing phenotypes in the field of view.
Contrast Enhancement with Cytation 5
With its high quality microscopy optics and automated focus, exposure and LED intensity settings, Cytation can acquire amazingly detailed images from whole organisms to sub-cellular details.
Cytation fluorescence imaging features include:
- 6-position turret for user-exchangeable objectives
- 4 channels available for multi-color imaging
- 15 imaging filter cubes and 9 imaging LED cubes are available to work with a very wide range of dyes
Cytation uses its high quality monochrome camera and sequential red-green-blue LED illumination to capture color images, which are then precisely combined to provide RGB sampled color without interpolation common to the use of color filter arrays. Cytation’s color brightfield imaging mode is particularly suited to H&E staining, immunohistochemistry with color deposition and samples with intrinsic color. Color brightfield imaging with Cytation offers the following benefits:
- Lower cost and maintenance than a system using both monochrome and color cameras
- Higher resolution in final image compared to color cameras using a color filter array
- Automated slide scanning with 20-slide stacker
Phase Contrast Imaging
Typically, cells are difficult to discern with brightfield imaging as their structures provide poor contrast due to sparse natural pigmentation and a relatively uniform transparency across the sample. Phase contrast is an important mode for label-free imaging of live-cells as it relies on differences in refractive index across the sample rather than absorption. This provides significantly enhanced contrast relative to brightfield.
Phase contrast imaging with Cytation 5 provides:
- 4x, 10x, 20x and 40x magnification
- Live-cell, label-free imaging
- Low intensity illumination for reduced phototoxicity
Microscopy Applications using Gen5 Image Software
Gen5 Image+ software provides for common image analysis procedures such as counting the total number of cells in the field of view or a sub-population based on analysis parameters defined in the Cellular Analysis module. These parameters include object size, intensity and circularity measurements, among others. A 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 is one of the most common laboratory tasks in the life sciences. It is required for routine cell culture propagation and as a starting point for any cell-based assay. While there are many instruments available for automated cell counting, the reusable hemocytometer remains the inexpensive tool of choice. It is used with another common tool of the laboratory, a brightfield optical microscope, such that cells can be visualized and counted on a grid of squares. The counting grid has several regions ruled to varying degrees to accommodate cells of differing sizes. The complete ruled grid area is covered by a coverslip that provides a perpendicular dimension of 100 μm which serves to keep cells roughly in the depth of field of the microscope used and to define the volume of the squares used for counting. The defined volume allows the cell density of the original culture to be easily calculated. Often an exclusion dye such as trypan blue is used to differentiate between live and dead cells such that the cell count reflects only viable cells.
Phase Contrast and Color Brightfield
The hemocytometer cell counting process can be automated using the Cytation 5 equipped with contrast enhancement technologies1. For total cell counts, automated phase contrast microscopy can be used to count the total number of cells in the field of view which represents an area approximately 2.9 times larger than the total ruled grid area. This serves to improve the counting statistics relative to manual counting. Cell viability can be assessed by the use of automated color imaging of the trypan blue stained cells in the same field of view.
Live MCF-7 cells appear as structures with a mostly round morphology when viewed on a hemocytometer, due to the fact that they are added as a suspension and have not attached to the bottom surface. Being more compact, the cells cause a relatively uniform change in phase, allowing them to appear as bright white objects on the dark background (Figure 3A). The lower threshold value in the Gen5 Cellular Analysis tool can be set at an appropriate value to correctly identify cellular structures, while ignoring other areas of the image, including trypan blue stained dead cells. Minimum and maximum object sizes can also be optimized to ensure that single cells are identified (Figure 3B). However, dead cell remnants and other non-cellular debris can also be added to the counting area. These items typically do not have the same level of signal, and are not as circular as live-cells.
Therefore two additional sub-population parameters are applied to the objects that are primarily identified, circularity and minimum mean signal. Items that do not meet the circularity and minimum mean phase contrast signal requirements are then eliminated from the final live-cell determination (Figure 3C).
Figure 3. Determination of live MCF-7 Cell Numbers. (A.) 4x phase contrast image of hemocytometer containing MCF-7 cells. (B.) Total object count using primary cellular analysis parameters. (C.) Live-cell count through incorporated sub-population criteria. Included objects highlighted in red.
Color brightfield imaging is used to identify dead cells within the counting regions. Trypan blue stained dead cells, which appeared as dark areas when using the phase contrast imaging channel, appear as easily identified blue objects with the incorporation of color brightfield (Figure 4). The final dead cell count, coupled with the phase contrast live-cell count, can then be used to calculate a % cell viability value to determine the viability of the cell culture1.
Figure 4. Color Brightfield Image of Live and Dead MCF-7 Cells. Trypan blue stained dead cells identified by red oval.
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 nucleus. 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 5)2.
Figure 5. 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 mask associated with them, drawn by Gen5. Note nuclei at the edge of the image are not counted (no gold mask) 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 6) illustrate the masking behavior at three different threshold levels. Note the shrinking of the object mask area as the threshold is increased.
Figure 6. 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™ 5 and cellular analysis performed with default settings except for Threshold. Threshold value for each analysis is indicated.
Hematoxylin and Eosin (H&E) Staining
Fixed and stained tissue preserved on a microscope slide is a common method of data storage. Pathological samples to be viewed are routinely fixed and embedded in paraffin for long term storage. Thin slices can then be made from the paraffin samples, immobilized on slides, dewaxed and subsequently stained, usually with hematoxylin and eosin, and sealed with a cover slip. Examination of the pathological slide requires that examiners utilize a staged multi-objective microscope to make observations. H&E staining remains the most common staining protocol for applications in histology.
More recently the use of automated digital microscopy has resulted in a simplified workflow and increased portability of the captured data. The capability of the Cytation 5 to image fixed and H&E stained tissue slices in color brightfield is demonstrated in Figure 7, where normal and diseased kidney tissues are compared3. Real time images can be viewed on a computer monitor prior to capture and the desired images can then be saved and downloaded in a number of different formats including TIF and PNG. The Cytation 5 and Gen5 Image+ software allow a range of microscope objectives to be selected as well as automated focus and exposure via software control (Figure 8).
Figure 7. Comparison of normal and diseased human kidney. Hematoxylin and eosin stained tissue from (A) normal kidney and (B) Chronic nephritis kidney. Images represent a stitched 12 x 8 montage made using a 60x objective. Scale bar represent s 200 μm.
Figure 8. Macroscopic and microscopic structure of human chronic nephritic kidney tissue. A series of montage (12 x 8) images at increasing magnification were recorded and stitched from the same tissue slice. Boxed area represents the area imaged in the next higher magnification image. Objective magnification and scale bar are present for each montage.
Quantifying Transfection Efficiency
Transient transfection has become a common method for the introduction of reporter genes (i.e. the family of GFP photoproteins) 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+ Cellular Analysis, 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 emissions can be assessed. This is demonstrated below (Figure 9) using BacMam transfection of a Histone H3-GFP fusion protein into U-2 OS cells4. 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 10).
Figure 9. 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 10. 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.
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 MultiFloTM FX automates reagent dispensing onto cells in microplates and also washes cells to remove excess reagent. A typical automated workflow is demonstrated in Figure 11. Figure 12 demonstrates the quality of the automated staining process using three separate stains5.
Figure 11. 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.
Figure 12. U-2 OS cells stained for mitochondria, F-actin and nuclei. 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.
The capture of multiple images is sometimes required to illustrate the biology of a cellular specimen. These include:
- Capture of multiple images in the same focal plane, but different areas of the specimen (montages) to increase field of view while maintaining magnification;
- Capture of multiple images in different focal planes (z-stack) to provide a 3D representation of specimens thicker than the microscope objective depth of field;
- Capture of multiple images in the same focal plane and area over time to create time-lapse videos of live-cell biology.
Each of these requires image processing steps which are described below for use with Cytation 5.
Montage Image Stitching
Cytation™ 5 can capture montages of images that cover 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 automated image processing capabilities embedded in the Gen5 software (Figure 13).
Figure 13. 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 5 and 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; E: Expanded portion of top left area representing four images from the montage demonstrating seamless stitching.
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 three dimensional structures such as tissue slices or 3D cell culture models. While widefield microscopy cannot provide 3D reconstructions, z-projection 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 14 illustrates this with a 3D co-cultured tumoroid structure6.
Figure 14. Single Plane and Z-Projected Images of Co-cultured Tumoroid Structure. (A. - C.) Images of tumoroid captured at individual z-planes using 4x objective. (D.) Final z-projected image. Image overlay shown using the following channels: Brightfield; GFP for identification of MDA-MB-231 cells; RFP for identification of fibroblasts; DAPI for identification of Hoechst 33342 stained nuclei.
Time-lapse Microscopy (Camtasia)
Cytation 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 CO2 and O2 controlled with the Gas Control accessory. This allows for time-lapse microscopy within the Cytation instrument. Gen5 Image+ software captures images at the desired time resolution, and then the 3rd party software Camtasia Studio can be used for the creation of a video comprising the images. An example of images captured of live-cell RNA expression using RNA probes7 is illustrated in Figure 15. A time-lapse video of the events can be viewed here: www.biotek.com.
Figure 15. 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.
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 5 is specifically designed to work with microplates for automated digital widefield microscopy, although a number of other vessels can be utilized, such as microscope slides, Petri dishes and T-25 culture flasks. Contrast enhancement methods such as color brightfield, phase contrast and fluorescence offer several options to generate rich phenotypic data. Gen5 Image+ software enables multiple applications, such as cell counting and quantifying transfection efficiency and phenotypic changes in cells. Embedded automated image processing further extend Gen5 Image+ capabilities including stitching of montages and z-projection. Furthermore, the portability of TIFF files from Gen5™ allows the use of 3rd party software such as those used for the generation of timelapse videos.
- Automated Hemocytometer based Live/Dead Cell counting
- Analysis of Nuclear Stained Cells
- Color Brightfield Imaging of H&E Staining
- Imaging of BacMam Transfected U-2 OS Cells
- Automated Tissue Culture Cell Fixation and Staining in Microplates
- Z-Stacking of Single Plane Digital Widefield Fluorescent Images
- Live-cell Imaging of RNA Expression