Applications - Technical Notes

Dual-Wavelength Measurements Compensate for Optical Interference

07-May-04

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Related Products: ELx808

 

An experiment with plate lids and induced condensation using the BioTek ELx808IU Microplate Absorbance Reader

 

Dual wavelength is used in many microplate-based applications to reduce optical interference caused by scratches, fingerprints or other matter that absorb light equally at both wavelengths. For example, many investigators prefer to read microplate-based assays with lids or membrane seals in place to reduce biohazards, as well as evaporation. As a result of using lids, condensation may collect on the lid during the assay process. To illustrate the benefits of dual wavelength in these types of applications, BioTek performed experiments showing what effect on results a plate lid, with or without condensation, can have when readings are taken at single vs. dual wavelength. The experiments demonstrated that when the plate is read at two wavelengths and the difference in optical densities is computed, this technique adequately compensated for these effects.

 

Experimental Results

 

The experiment performed compared single and dual wavelength measurement techniques using a BioTek ELx808IU Absorbance Microplate Reader and a filled microplate with and without lid and condensation present. A NUNC MaxiSorp 12-1x8 removable strip microplate was used. Two different plate covers were tested: a NUNC 96-well hard cover, and a generic adhesive cover common in many assay kits on the market. An 8-channel micropipette was used to dispense 200μl of deionized water into wells A1-A12, and a 3:5 dilution of QC Dye #2 and QC Dye #3 (P/Ns 7120783 and 7120784 respectively, BioTek Instruments, Winooski, VT) into remaining wells. Plates were then read at single and dual wavelength. Plate lids were applied to the plates and they were read again at single and dual wavelength. This was followed by a 30-minute incubation at 5oC to induce condensation, and the plates were re-measured at single and dual wavelength with visible condensation on the plate lid. Table 1 shows a chart of the experimental process including the mean plate optical density (OD) and mean plate CV% for each reading.

 

 

Adhesive soft plate cover

 

Plate, Read

Wavelength

Lid Present

Visible Condensation

Mean Plate OD

Mean Plate CV%

P1,R1

Single (450)

No

No

1.107

0.646

P1,R2

Dual (450,630)

No

No

1.07

0.501

P1,R3

Single (450)

Yes

No

1.229

0.721

P1,R4

Dual (450,630)

Yes

No

1.085

0.604

Refrigeration

 

 

 

 

 

P1,R5

Single (450)

Yes

Yes

1.566

0.670

P1,R6

Dual (450,630)

Yes

Yes

1.086

0.730

 

 NUNC hard plate cover

 

Plate, Read

Wavelength

Lid Present

Visible Condensation

Mean Plate OD

Mean Plate CV%

P2,R1

Single (450)

No

No

1.109

0.726

P2,R2

Dual (450,630)

No

No

1.071

0.734

P2,R3

Single (450)

Yes

No

1.15

0.669

P2,R4

Dual (450,630)

Yes

No

1.069

0.725

Refrigeration

 

 

 

 

 

P2,R5

Single (450)

Yes

Yes

1.37

1.640

P2,R6

Dual (450,630)

Yes

Yes

1.064

0.697

 Table 1. Plate readings with mean plate ODs and mean plate CV% for comparison of single and dual-wavelength techniques.

 

The presence of an adhesive plate cover at single wavelength adds just over 10% to the mean OD (P1,R1 vs. P1,R3). At dual wavelength, however, the difference in mean OD with and without the adhesive plate cover is only 1.5% (P1,R2 vs. P1,R4). Following the 5°C incubation used to induce condensation, the results are even more noticeable. At single wavelength there is an almost 25% increase in mean OD (P1,R3 vs. P1,R5) caused by the presence of condensation on the plate lid, while the dual wavelength mean OD compensates as shown by a less than a 0.5% increase (P1,R4 vs. P1,R6).

The results for the NUNC cover are similar, but less pronounced. Using single wavelength measurements it adds just over 3.7% to the mean OD (P2,R1 vs. P2,R3), while with dual wavelength the difference is almost non detectable at 0.02% (P2,R2 vs. P2,R4). Following the 5oC incubation the compensation of dual wavelength clearly makes a difference. At single wavelength there is an almost 20% increase in mean OD (P2,R3 vs. P2,R5) caused by the presence of condensation on the plate lid, while the dual wavelength mean OD compensates as shown by a less than 0.046% increase (P2,R4 vs. P2,R6). The change in CV% for the NUNC cover also illustrates the compensatory properties of dual wavelength. At single wavelength there is over a 100% change in CV% with and without condensation (P2R3 + P2R5), but only about a 4% change when condensation is present at dual wavelength (P2R4 + P2R6).

 

Conclusions

 

Condensation on plate lids can be significant whenever a temperature differential exists between the plate and the fluid at the time of pipetting, or whenever a filled plate experiences an appreciable change in temperature. If condensation does form on the plate lid, light scattering effects can significantly change absorbance readings and CVs. These detrimental effects of condensation are compensated by taking advantage of readings at two wavelengths, and subtracting absorbance at the reference wavelength from those at the reading wavelength.

It is strongly advisable for any investigator contemplating performing assay readings with plate lids in place acquire a microplate reader that permits dual-wavelength readings over a broad light spectrum and that physically accommodates the covered plate. These criteria are met by the BioTek ELx808IU and ELx800UV absorbance microplate readers, which feature a wavelength range of 340 to 900nm and 340 to 750nm respectively, while also accommodating a variety of plate lids within the reading chamber.



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