1. Sample PreparationSample preparation is critical to ensure high quality images. Cells should be at an adequate density when harvested from the tissue culture flask, and removed with an appropriate dissociation solution to retain essential cellular function. Use aseptic technique to prevent contamination during the experiment and avoid unnecessary introduction of foreign particles or cellular debris that can negatively impact image quality.
For live cell assays, fluorescent probes need to be cell membrane permeable to assess structure and function within the cell. Optimize concentrations and incubation times prior to performing the actual assay to maintain cell viability while still ensuring sufficient fluorescence. Be aware of potential background fluorescence - it may be necessary to incorporate wash steps before imaging. Together, these will help to provide images of live cells with good signal to background ratios.
For fixed cell assays such as immunofluorescence, always use a proven fixing/permeabilizing/staining protocol, including, addition of a blocking agent to prevent non-specific binding, and optimization of antibody concentrations when necessary.
2. Sample Vessel ConsiderationsThe bottom thickness of sample vessels used for microscopy is important, since inverted microscopes view samples through this thickness. Different vessels have different bottom thicknesses. Some common vessel bottom thicknesses include:
- microscope slide cover glass: 0.17 mm
- common plastic microplates: 0.5 mm
- low density microplates: 1.0 mm
3. Cell Number OptimizationTypically cell-based assays are more robust when there are more cells. Cellular imaging is a bit trickier; plating too many cells can cause cells to grow on top of each other, confusing proper segmentation of cells for cell counting or other assessments. Plating too few cells can be statistically insignificant and skew cell sub-population analysis. Post-plating and post-treatment incubation times should also be factored into cell number determination, as cell propagation can continue during the preparation and execution of the experiment.
4. Cell TypeA wide variety of cell types can be used for cellular imaging, including immortalized cell lines, primary cells, and stem cells. Most cell types used in microscopy applications are adherent in nature; with the right treatment, the cells will adhere and form a two-dimensional (2D) layer of cells across the bottom of the well. 2D is the most straightforward format for imaging, as the cells are in a single plane which can be found using the auto-focus capability of the imager.
Suspension cells, such as erythrocytes and leukocytes, can also be imaged. These cell types lack the ability to adhere to a surface and require additional manipulation to restrict them to a single focal plane at the surface of the vessel used for microscopy. Suspension cells can be transferred onto a microscope slide with coverslip or hemocytometer with cover glass which restricts the cells in the axial direction and provides better image clarity. With microplates, a centrifugation step can also be used to induce cells to the bottom of the well.
More complicated biology, such as tissues and three-dimensional (3D) cellular structures, have also been incorporated into image-based experimental processes. The difference in size and shape compared to individual 2D-plated cells, make more advanced imaging procedures necessary. Tissue samples can often be much larger than the field of view provided by the microscope objective, particularly if high resolution is desired with high numerical aperture objectives. An image montage procedure captures numerous images across the entire tissue which can then be stitched together into a single composite image which can then be used for analysis. 3D cellular structures contain hundreds to thousands of cells which extend not only in the x- and y-axes, but also in the z-axis. Capturing a z-stacked set of images across multiple z-planes, coupled with z-projection image processing allows for a composite image with better focus than any one of the individual z-stack images. This z-projected composite image can increase the accuracy of any subsequent analysis.
5. Fluorophores and Imaging Filter SetsIt’s essential to use the best fluorophore for successful imaging. Excitation and emission spectra of the fluorescent probe or protein should be matched with LED light sources, excitation and emission filters, and dichroic mirrors available to assure satisfactory fluorescent signal. The fluorophore's Stokes shift is an important variable to consider as narrow Stokes shift can lead to excessive background fluorescence and poor signal to background. Additional optimization is necessary if multiple fluorophores are used together in a multiplexed format. Molecular spectra tend to be broad and overlap in both excitation and emission spectra can occur, resulting in bleed-through of one fluorophore into the fluorescent channel of another. This is particularly important should both fluorophores be colocalized in the same area within a cell.
By: BioTek Instruments