Factors to Consider When Choosing a High Content Plate

Interviews By Steve Titus:  CHARLES POWELL OF AURORA MICROPLATES

I recently had the pleasure of attending a seminar given by Charles Powell of Aurora Microplates. Charles highlighted several important features of imaging plates that I had not considered previously. Charles did a great job of highlighting the importance of the microenvironment of a well, particularly with plates dedicated to high content imaging assays. I recently spoke with Charles about microplate design and function in a high content imaging context.

 

*NOTE - SBI2 does not endorse any products, nor is this interview an endorsement for Aurora Microplates*

 

ST: Can you explain what makes a good imaging plate?

 

CP: High performing imaging plates need to have consistent well bottom thickness, they need to be flat across the entire well bottom surface area, as well as across the entire range of the plate- typically 100 microns or less “lack of flat”. The plate polymer material needs to be 100% biocompatible to allow for long incubation work with cells. The film material used should be heat-fused or over-molded to the plate frame to avoid the use of adhesives or other bonding agents which can leach into the well over time and contribute to assay variability. Finally, the film material should have a refractive index similar to borosilicate glass (approx. 1.523) and exhibit very low birefringence- essentially being optically isotropic.

 

Finally, the microenvironment of the well is not to be taken for granted. Dust and lint will ruin images and many detergents (in lint from clothing) will fluoresce brightly. Plates should not be exposed to or be kept near cardboard boxes after being opened for use. Static electricity is also a “ghost” to be dealt with- plates should be packaged in anti-static bags for delivery and on-site storage. Managing to keep dust and static out of the equation is vital to achieving consistent quality images with microplates.

 

 

ST: You mentioned thin. How thick it too thick and how thin is too thin? Most microscope objectives are calibrated for cover glass thickness (~170 microns) which is what I prefer. Do you see this feedback in the field?

 

CP: While it might be true that the thinner film bottom materials for imaging plates can potentially let more light intensity through to the samples in question, the thicker films (approx. 180 micron) actually are preferred by most users. I think the main reason is the auto-focus times on most scopes- which can take considerable time with thinner films, and in high-density (384 and 1536 well) imaging, these extra times certainly add up fast.

 

ST: What about properties of clear plastic and light transmission. I’ve heard of cyclic olefin co-polymers; are these the best for imaging and if so, why?

 

CP: Cyclo-Olefin Polymer (COP) is an excellent choice of material for imaging plates. It is a material that is optically isotropic- so it doesn’t polarize or de-polarize light, and has a broad transmission window, from approx.. 230nm to over 800nm. Other popular polymers for microplate construction (such as Polystyrene) have very poor optical properties when compared to COP or Glass.

 

ST: Anything you see or would like to see in a new polymer design?

 

CP: We always keep our eyes open for new materials, or for newer levels of purity of existing materials. So far we are extremely pleased with COP as a plate material for imaging applications. We have used the material for over 20 years with outstanding performance feedback from our users. If a new material came along that matched all of the critical attributes of COP we would certainly investigate its’ potential as a plate material.

 

ST: We are examining a wide variety of plate coatings (vitronectin, Poly D- Lysine, Collagen, BMT etc). What have you seen and can you comment on trends in coatings?

 

CP: Coatings serve an important role in working with challenging cells lines, or challenging assay conditions. For work with primary and stem cells, there are many cases where a coating of some type helps maintain cell viability- especially in long incubation assays. We developed a Multi-Coat plates years ago that puts up to 6 different coatings regionally into one plate- to enable a researcher to test a variety of coatings at one time. We have also seen many instances where a slightly different TC-treatment can provide an environment where cells can perform- without the need for coatings.

 

ST: We are doing a lot of 3D spheroid work, particularly in  ultra-low attachment (ULA) round bottom plates. Do you have any experience with these, any comments?

 

CP: It seems to be a new technique that is becoming quite popular. We are working to make a version of the ULA plate-which has a dimpled (rounded) well bottom- versus a flat well bottom. This application puts even more importance on the choice of plate material, as many polymers will reflect or refract light when it hits a surface at other than exactly 90 deg. COP will make a great choice of materials for these plates as it passes light through very well even when the light hits it at an angle.

 

ST: As we approach near IR imaging in HCA (both from a reagent and hardware perspective)  can we expect that the plastics will follow suit in terms of light transmission. Is this an easy task?

 

CP: As I mentioned previously, this is strictly a materials question and answer. High quality polymers that have excellent optical properties will enable imaging at higher wavelengths of light. COP certainly meets these criteria.

 

ST: Where do you see plates in 2018 or 2020? What’s coming that we can look forward to? Any other comments you would like to share?

 

CP: We think there are opportunities that are related to a combination of microfluidics and microplates. There are opportunities in signal generation from surfaces that can receive deposits of fluorescent or luminescent materials into the plate material. We generally focus on Better, Cheaper, Faster performance criteria for our designs- as it relates to the value proposition for users. There are many possibilities to extend the performance utility of current microplates- with the need for much exotic invention. We listen to our users, and brainstorm with them as to how we can help make the microplate more invisible in their research.

*NOTE - SBI2 does not endorse any products, nor is this interview an endorsement for Aurora Microplates*

titus

 

Steve Titus; Staff Scientist

High Content Imaging & Discovery

NCATS|NIH