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Krystal Courtney D. Belmonte, PhD | Field Applications Scientist | Molecular Devices
Christina Nadolny, PhD | Field Applications Scientist II | Molecular Devices

Introduction

Different fluorophores exhibit unique excitation and emission spectra, which can impact the sensitivity and accuracy of the measurements. SpectraMax® microplate readers vary in instrument configurations and settings such as variations in wavelength selection, bandwidth, and excitation and emission wavelength pairs were considered in their performance across a range of fluorophores. The lower limit of detection (LLD) is a critical parameter in these comparisons, representing the smallest concentration of a fluorophore that can be reliably distinguished from background noise.

Here, we provide a comparison of SpectraMax microplate readers across a total of seven fluorophores spanning the blue to red range, focusing on key parameters such as linear range and the LLD.

Fluorophore standards ranging from 100 µM to 0.001 pM were prepared as a serial dilution series in tris buffered saline (TBS). 100 µL of standard was pipetted into wells of a black 96-well microplate (Greiner cat. #655076; n=4 for each standard). Blank wells containing 100 µL of TBS buffer only (n=8) were also prepared. Fluorescence was then measured using the optimized settings for each microplate reader (Table 9). Standard curves were plotted using SoftMax Pro Software. To assess the sensitivity of each microplate reader, the LLD for each fluorophore was estimated using the following formula:

LLD=3 SD_blankConcentration/(RLU_sample-RLU_blank).

Instruments compared

• SpectraMax® Paradigm® Multi-Mode Microplate Reader with TUNE module (Molecular Devices P/N 0200-7050)
• SpectraMax® iD5 Multi-Mode Microplate Readers
• SpectraMax® i3x Multi-Mode Microplate Readers
• SpectraMax® Mini Multi-Mode Microplate Readers with Filter Cubes (see Table 10)
• SpectraMax® M5e Multi-Mode Microplate Readers
• SpectraMax® Gemini™ EM Microplate Reader

Fluorophores compared

• Alexa Fluor 350 (Molecular Probes)
• Coumarin B (Thermo Fisher)
• Fluorescein (Invitrogen)
• Alexa Fluor 555 (Molecular Probes)
• Alexa Fluor 594 (Molecular Probes)
• Alexa Fluor 647 (Invitrogen)
• Alexa Fluor 750 (Molecular Probes)

Figure 1. Fluorescence SpectraViewer¹ excitation and emission spectra of fluorophores compared.

Table 1. Lower limits of detection (pM) summary of all fluorophores compared across SpectraMax microplate readers.

Alexa Fluor 350

Figure 2. Alexa Fluor 350 fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 2. Lower limits of detection for Alexa Fluor 350 (AF350) calculated and excitation/emission wavelength parings used for SpectraMax microplate readers.

Coumarin B

Figure 3. Coumarin B fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 3. Lower limits of detection for coumarin B calculated and excitation/emission wavelength parings used for SpectraMax microplate readers.

Fluorescein

Figure 4. Fluorescein fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 4. Lower limits of detection for fluorescein calculated and excitation/emission wavelength parings used for SpectraMax microplate readers.

Alexa Fluor 555

Figure 5. Alexa Fluor 555 fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 5. Lower limits of detection for Alexa Fluor 555 (AF555) calculated and excitation/emission wavelength parings used for

Alexa Fluor 594

Figure 6. Alexa Fluor 594 fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 6. Lower limits of detection for Alexa Fluor 594 (AF594) calculated and excitation/emission wavelength parings used for SpectraMax microplate readers.

Alexa Fluor 647

Figure 7. Alexa Fluor 647 fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations. Table 7. Lower limits of detection for Alexa Fluor 647 (AF647) calculated and excitation/emission wavelength parings used for SpectraMax microplate readers.

Alexa Fluor 750

Figure 8. Alexa Fluor 750 fluorophore standard curves read on SpectraMax microplate readers.

*See Table 10 for SpectraMax Mini Filter Cube configurations.Table 8. Lower limits of detection for Alexa Fluor 750 (AF 750) calculated and excitation/emission wavelength parings used for

Instrument Acquisition Settings

Table 9. Instrument settings used for SpectraMax microplate readers.

SpectraMax Mini Filter Cubes

Table 10. Filter cube wavelength configurations used for SpectraMax Mini microplate reader.

Reference

1. ThermoFisher. Fluorescence SpectraViewer. https://www.thermofisher.com/order/fluorescence-spectraviewer/#!/

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