The selection of a microtiter plate is often an iterative process that must balance competing factors such as automation compatibility, cost, availability, and performance.
We have created a checklist for you to quickly overview the factors that could affect the quality of imaging in high throughput and high-content screening.
Microtiter plate properties
Microtiter plate properties that affect the quality of imaging in high-throughput and high-content screening (HCS) include color, well shape, well volume, well density, polymers and surface treatments.[1]
Microplate color
Black pigmented microplates are commonly used for fluorescence applications, whereas white pigmented microplates typically support luminescence measurements, and are sometimes used to enhance fluorescence signal intensity. Both pigmentations help overcome critical issues for these techniques, such as background, autofluorescence, and well-to-well crosstalk.
- Clear – for colorimetric-based readouts
- White – for luminescence-based readouts
- Black – for fluorescence-based readouts
- Black with clear bottom – for cell visualization in normal and confocal microscopic assays and fluorescence-based bottom read-outs
- White with clear bottom – for luminescence-based bottom read-outs
Well design
The well bottom design is also a critical aspect when choosing microtiter plates for high throughput applications:
Well shape
- Round – for minimizing reaction volumes
- Square-shaped with flat bottoms – for maximizing the area for light transmission
Well bottom
- Flat (F-bottom) – for maximum light transmission for bottom-reading applications
- Rounded (U-bottom) – to facilitate mixing, washing, coating and retrieval of solutions
- Conical (V-bottom) – for maximal retrieval of small sample volumes
- Flat with a rounded edge (C-bottom) – to combine features of flat and round bottom
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Bottom type
New applications in high-throughput and high-content screening, as well as high-resolution and confocal microscopy, have increased the demand for microtiter plates with pigmented walls and clear bottom. The product portfolio of Greiner Bio-One contains clear bottom plates either with glass or a high-quality film bottom.
Film bottom microtiter plates (µClear®) combine a pigmented frame with a transparent bottom, a prerequisite for luminescence and fluorescence applications where bottom-reading or microscopy are involved. Due to the optimized thickness of the film, the intrinsic autofluorescence of polystyrene is minimized.
Microtiter plates with cycloolefin film bottom (SCREENSTAR) are optimised for the specialized requirements of high-content screening and high-resolution microscopy. The 190 µm cycloolefin film bottom guarantees maximum resolution, even at high microscopic magnification. SCREENSTAR microtiter plates provide a recessed bottom to allow full use of high-magnification oil or water immersion objectives with access to all microplate peripheral wells, including perimeter and corner positions.
Microtiter plates with glass bottom (CELLview) are designed for demanding and high-resolution microscopic applications. They consist of a cycloolefin-based black frame with a 170 µm thin borosilicate glass bottom providing superior images of in-vitro cultures. The geometry is also optimized for high-resolution microscopy that allows imaging with short working distance. The round and conical well design reduces the meniscus effect in order to assure equal cellular distribution and constant imaging results.
- Solid clear – for top and bottom reading (bottom only for wavelengths > 400 nm)
- Solid black or white – for top reading
- Polystyrene film bottom (µClear®) – for microscopy and spectroscopic measurements down to 340 nm (340 - 400 nm)
- Cyclic olefin film bottom (UV-Star®) - for UV spectroscopy (230 - 340 nm)
- Cyclic olefin bottom (SCREENSTAR) – for high magnification microscopy
- Glass bottom (CELLview) – for high magnification microscopy
Surfaces and treatments
At the well surface, interaction between the sample and the microplate takes place. Therefore surface properties play an important role for the functionality of a vessel. Surface properties can be modified in many ways, whether by physical, chemical or coating methods, to fulfill various demands.
- Non-treated – for homogeneous assays
- Non-binding – for sensitive homogeneous assays
- Medium-binding – for immunoassays
- High-binding – for immunoassays
- Streptavidin – for immunoassays
- Tissue culture (TC)-treated – for general adherent cell culture
- Suspension – for non-adherent cell culture
- Advanced TC – for cultivation of fastidious cell lines
- Protein coating (Poly-(D ± L)-lysine (PDL, PLL), ECM coatings – for cell type-specific needs for growth and adhesion
- Cell Repellent - for spheroid and organoid formation
Microtiter plates for biological assays
Microtiter plates for biological assays should meet the following criteria: [1]
- Dimensional stability under multiple temperature and humidity conditions
- Flatness
- Chemical and biological compatibility with assay reagents
- Low adsorption of chemicals or biologicals
- Low autofluorescence
- Supports cell viability, attachment, and growth
- Supports relevant optical detection modes
- No leaching of solvents, metals, or chemicals
HCS imaging instrument optics and microtiter plate specifications
The optics of the HCS imaging device and the microtiter plate specifications must be matched. Experiments need to consider all aspects of the microtiter plate specifications along with the following key features of the objective lens:
- Magnification power
- Numerical aperture
- Working distance
- Depth of field
- Minimal Z-plane interval spacing
Other factors to consider
- Optical isotropy – important for imaging-based assays and assays involving polarized light because anisotropic materials can produce light-based interferences.
In order to select the best plate for your imaging-based assays, download our checklist and use it for your daily work!
Find the right microplate with our checklist:
References
[1] Auld DS Ph.D., Coassin PA B.S., Coussens NP Ph.D., et al. Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology. In: Markossian S, Grossman A, Brimacombe K, et al., eds. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; June 1, 2020. https://pubmed.ncbi.nlm.nih.gov/32520474/