Purpose

These references are designed for the routine qualification of fluorescence-based plate readers for fluorescence intensity and well-to-well reading reproducibility.

Description and Discussion

These references were formerly marketed by Matech.

96-well microplates containing inorganic fluorescent reference materials, available in both top- and bottom-reading configurations. Plate dimensions conform to international standards for biomedical screening. 

 

The inorganic fluorophores used are inherently stable and do not suffer from photodecomposition or “bleaching”. Solvent evaporation is not an issue as these references are solid state. Four different fluorophores, identified by the wavelength of their principal emission peak, provide a range of excitation and emission wavelengths to match the user’s application:

Identification PEAK EXCITATION WAVELENGTH (NM)  Excitation Range (nm) Peak Emission Wavelength (nm) Emission Range (nm)
Violet - 395 335  250 - 375 395 340 - 460    
Blue - 455 325  250 - 410 455 410 - 550    
Green - 517 280  250 - 490 530 480 - 600    
Red - 615 280  250 - 530 525, 590, 615, 655, 705 470 – 715    
Infrared - 800  440  380 - 680 702 620 - 820    

Typical spectra

Note that spectra obtained on individual instruments will vary, especially when cut-off filters are used to distinguish between excitation and emission energies. Instrument resolution will also be a factor. The spectra above should be representative of what is achievable with modern plate reader instruments. 

The Calibration Certificate supplied with each reference plate gives normalized emission spectrum data over the working range, plus relative intensity values for all the wells in the plate and the calculated coefficient of variation for those readings. Typical data (for a bottom reading BR615 plate) is shown below:

  1 2 3 4 5 6 7 8 9 10 11 12
A 0.941 0.957 0.953 0.957 0.952 0.954 0.95 0.949 0.927 0.925 0.918 0.913
B 0.952 0.951 0.953 0.96 0.956 0.957 0.946 0.932 0.938 0.908 0.918 0.931
C 0.956 0.957 0.96 0.96 0.951 0.949 0.946 0.948 0.929 0.926 0.918 0.916
D 0.951 0.962 0.961 0.965 0.961 0.952 0.952 0.945 0.941 0.938 0.928 0.921
E 0.965 0.962 0.96 0.966 0.96 0.961 0.957 0.957 0.945 0.953 0.931 0.939
F 0.972 0.969 0.948 0.966 0.963 0.958 0.963 0.958 0.942 0.935 0.932 0.965
G 0.987 0.968 0.964 0.958 0.965 0.967 0.97 0.982 0.971 0.945 0.963 0.96
H 1 0.971 0.967 0.971 0.977 0.983 0.982 0.973 0.944 0.942 0.938 0.947

CV: 1.83 %

The maximum coefficient of variation (CV) in fluorescence intensity across all wells is warranted to not exceed five percent.

  

Catalogue Number

Identification  Emission Peak Wavelengths (nm) No. of wells Top/Bottom Reading CATALOGUE NUMBER
Violet 395 395 96 Top TR-395        
    96 Bottom BR-395        
Blue 455 455 96 Top TR-455        
    96 Bottom BR-455        
Green 517 530 96 Top TR-517        
    96 Bottom BR-517        
Red 615 525, 590, 615, 655, 705 96 Top TR-615        
    96 Bottom BR-615        
Infrared 800 702 96 Top TR-800        
    96 Bottom BR-800