Microscope to Microplate Reader - Increasing the Throughput of Live Fluorescent Protein FRET Assays
Filters vs. Monochromators - When using a microplate reader, these are the two options to filter light into monochromatic wavelengths. Filters offered higher performance because of greater light transmission and wider bandwidths; monochromators offered greater flexibility, no new filters were needed for each new assay.
However, researchers state that monochromators cannot perform many assays (1). Fluorescent protein assays like GFP, mCherry, or CFP-YFP, do not perform well on monochromator-based microplate readers. The same holds true for FRET and BRET assays. One main reason is that wider bandwidths are needed. Some FRET or BRET assays require an emission bandpass up to 100 nm and current monochromators only have fixed or limited bandwidths up to 30 nm.
New LVF MonochromatorsTM Have Filter-like Performance
Realizing the need for a more sensitive, broader bandwidth monochromator in a microplate reader, BMG LABTECH’s German engineers created the CLARIOstar® multimode microplate reader with new innovative LVF MonochromatorsTM. Consisting of linear variable filters separated by a linear variable dichroic mirror, LVF MonochromatorsTM filter light into definable wavelengths and bandwidths up to 100 nm wide.
Confocal Microscope to Microplate Reader – No Filters Needed
Researchers routinely try to adapt their fluorescent protein assay from a confocal microscope to a microplate format. Now with LVF MonochromatorsTM no filters are needed. The figure shows an mTFP1-YFP fluorescent protein FRET response in HEK293 cells as measured on a confocal microscope and on the CLARIOstar®. This FRET assay requires the measurement of two emission signals with bandwidths of 30 nm and 45 nm for the microscope (2), which were the starting points for the CLARIOstar®’s LVF MonochromatorsTM. With further optimization, the same percent change is seen (figures table), making it an almost seamless transition to a higher throughput method without buying new filters.
- Comley, J. Monochromator vs Filter-based Plate Readers; horses for courses, or a winning combination? Drug Discovery World. Fall 2007. http://ddw.net-genie.co.uk/enabling_technologies/347427/monochromator_vs_filterbased_plate_readers_horses_for_courses_or_a_winning_combination.html
- Padilla-Parra S, et al. Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. Biophys J. 2009 Oct;97(8):2368-76.
E.J. Dell, PhD
BMG LABTECH, GmbH