Emission Spectra of Luminescent Signals

Technical Note

Emission spectra of luminescent signals

Luminescence scans on the Infinite® M1000 multimode reader

Introduction

This technical note describes the successful implementation In addition, the Infinite M1000’s ability to perform of the new luminescence scan feature on the Infinite M1000 simultaneous excitation and emission scans (3D scanning) premium Quad4 Monochromators™-based multimode reader. has been successfully used in research. The Center for Integrated Protein Science Munich (CIPSM), for example, is The Infinite M1000 multimode reader and its predecessor, the using the Infinite M1000 to identify nanobody-induced Safire2TM, are well known for their superb absorbance and changes to the spectral properties of green fluorescent protein fluorescence intensity scanning capabilities. Premium Quad4 (GFP)4, 5. Monochromators technology enables high speed absorbance scanning and has been used for a variety of research This comprehensive range of scanning capabilities has now applications, such as development of a novel DNA detection been extended to include luminescent signals. The new platform based on a hairpin DNA switch, nanoparticles and luminescence scan mode allows emission spectra of enzyme signal amplification for ultrasensitive detection of DNA luminescent signals to be recorded with 1 nm resolution, hybridization1. thanks to the high performance of Tecan’s premium Quad4 Monochromators. Basic fluorescence excitation and emission scans performed on the Infinite M1000 have previously been described in Luciferase-encoding genes are commonly used as reporters several publications, for applications including the study of in a variety of cell-based studies and animal disease models, genetically encoded fluorescent sensor proteins2 and and a broad range of recombinant luciferases and recording the emission spectra of fluorescent molecular rotors corresponding substrates are commercially available. to characterize polysorbate-containing IgG formulations3.

Technical Note

Therefore, the development of new, genetically enhanced Luciferases luciferases is of high importance, and the Infinite M1000 now Stock solutions (protein concentration approximately 5 µg/µl) provides the functionality to easily characterize these proteins. of firefly and Renilla luciferases were prepared using 1 x PLB/G. All further dilutions were prepared in 1 x PLB/G, from The luciferase protein from the firefly beetle (Photinus pyralis) the stock solutions. is a monomeric enzyme that catalyzes the oxidation of luciferin while emitting light at approximately 550 – 570 nm. For recording the firefly luciferase emission spectrum, 20 µl of The smaller Renilla luciferase from the sea pansy (Renilla the appropriate dilution of firefly luciferase was mixed with reniformis) is also monomeric, oxidizing coelenterazine and 100 µl LAR II. To induce the Renilla luminescence signal, emitting light at about 480 – 500 nm. Usually the 100 µl LAR II and 100 µl Stop & Glo reagent, plus 20 µl of the Dual-Luciferase® Reporter (DLR™) assay is used to appropriate Renilla dilution, were dispensed into a white determine the activity of firefly and Renilla luciferase in cell 96-well microplate. Emission scans were performed using lysates in a sequential manner. In the present experiment, different measurement parameters (integration time, independent emission spectra of firefly and Renilla luciferases bandwidth, z-position), with LAR II (firefly), or LAR II mixed were recorded. The substrates and buffers provided with the with Stop & Glo reagent (Renilla), used as blanks. DLR assay, plus firefly / Renilla luciferase, were used to generate luminescent signals, and the influence of the Infinite Measurement parameters M1000 measurement settings (integration time, bandwidth, Tecan’s i-control™ software now includes a luminescence z-position) on the luminescence emission spectra were scan option and pane (Figure 1). evaluated.

Material and methods

Instrument

Tecan Infinite M1000 premium Quad4 Monochromators-based multimode microplate detection system

Microplates 96-well, flat bottom, white, polystyrol microplates (Greiner

Bio-One) Figure 1 The Luminescence Scan pane in i-control software (V1.8). 96-well, flat bottom, white, transparent base, polystyrol microplates (Greiner Bio-One)

Reagents Dual-Luciferase Reporter assay (Promega, E1960) Renilla reniformis luciferase, recombinant, 1 mg (LUX biotechnology Inc., 20207) QuantiLum® recombinant luciferase, 1 mg (Promega, E1701)

Reagent preparation Assay buffer (Dual-Luciferase Reporter assay) Luciferase assay reagent II (LAR II), Stop & Glo® reagent and passive lysis buffer containing 1 mg/ml gelatine (1 x PLB/G) were prepared according to the manufacturers’ instructions6.

Technical Note

Table 1 gives an overview of the parameters that can be high temperatures – above 30 °C – and decreases at selected. The measurement parameters used for the temperatures below 30 °C. It is recommended that luminescence scans summarized in this technical note are measurement parameters are optimized according to the shown in Table 2. Modifications of these settings are environmental conditions and the sample concentrations described separately. The instrument temperature was used. maintained at 26 °C for all measurements. Results Measurement Instrument settings parameter The luciferase stock solutions were diluted from 1:100 to Wavelength range 280 – 850 nm 1:10,000 using 1 x PLB/G, and the emission spectra were Step size 1 nm, up to selected recorded in top and bottom mode. The results of Renilla wavelength range luciferase diluted 1:10,000 and measured in top mode, and Bandwidth 5 – 20 nm the results of firefly luciferase diluted 1:5000 and measured in Top / bottom Top or bottom optics bottom mode, are shown in Figure 2. Luminescence scan Gain – default Recommended results are given in relative luminescence units (RLU). Gain – manual Selectable (1 – 255) Integration time 1– 1000 ms Top and bottom emission spectra z-position – default 22,000 µm 50000 20000 45000 18000 z-position – manual Selectable 40000 16000 Table 1 Available measurement parameters for luminescence scans. 35000 14000 30000 12000 RR Top optics 25000 10000 FF Bottom optics 20000 8000

Measurement Instrument Instrument Top [RLU] 15000 6000 Bottom [RLU] Bottom parameter settings – FF settings – RR 10000 4000 5000 2000 Mode Luminescence Luminescence 0 0 350 400 450 500 550 600 650 700 scan top / scan top / bottom Wavelength [nm] bottom Figure 2 Emission spectra of luciferases recorded in top and bottom Wavelength 500 – 650 nm 400 – 600 nm mode. Luminescence top mode: Renilla luciferase (RR) diluted 1:10,000. Step 5 nm 5 nm Luminescence bottom mode: firefly luciferase (FF) diluted 1:5000. Bandwidth 20 nm 20 nm Gain manual manual The integration time is definable between 1 and 1000 ms, with Integration 1000 ms 1000 ms longer integration times resulting in higher luminescence z-position 22,000 µm 22,000 µm signal intensities. A strongly emitting firefly luciferase sample Table 2 Measurement parameters for the Infinite M1000 with Tecan (dilution 1:100) was measured using integration times i-control software; firefly luciferase (FF) and Renilla luciferase (RR). between 200 ms and 1000 ms (Figure 3). All other measurement parameters were as stated in Table 2. The Infinite M1000 reader records emission spectra of luminescent signals using the top or bottom fluorescence emission optics. In comparison to fluorescence intensity scans, longer integration times are necessary to detect luminescent signals. Long signal integration times, in the millisecond range, combined with high gains, may result in unwanted detector noise. Each instrument offers a preset gain value, keeping the noise level low at maximum signal integration. However, the sensitivity of the detector is also influenced by temperature; detector sensitivity increases at

Technical Note

Impact of integration times on luminescence emission spectra z-position [µm] Maximum signal [RLU]

100000 16,000 30,378 90000

80000 18,000 39,549 70000 IT = 1000 ms 20,000 52,488 60000 IT = 800 ms 50000 IT = 600 ms 22,000 64,278 RLU 40000 IT = 400 ms 30000 IT = 200 ms 24,000 66,379 20000

10000 26,000 65,603 0 Table 3 Optimization of z-position for a 96-well microplate with a fill 475 500 525 550 575 600 625 650 675 Wavelength [nm] volume of 120 µl.

Figure 3 Influence of the integration time on the signal intensity of firefly Conclusion luciferase diluted 1:100.

This technical note describes the successful recording of the The bandwidth defines the amount of light passing through the emission spectra of different luciferases on the Tecan Infinite monochromator. A large bandwidth implies more light, M1000 premium Quad4 Monochromators-based multimode whereas a small bandwidth implies less light. When applied to detection system with regard to the influence of the main luminescence scans, this means that higher bandwidths result emission spectrum measurement settings on signal detection. in higher signal intensities (Figure 4). The emission spectrum of firefly luciferase was recorded with different bandwidth The new luminescence scan functionality enables settings. All other measurement parameters were as stated in characterization of new or genetically enhanced luciferases in Table 2. high throughput formats. With the ability to run absorbance,

fluorescence – excitation, emission and 3D – and Impact of bandwidth on luminescence emission spectra luminescence scans, the Infinite M1000 provides great 80000 scanning flexibility in all detection modes, providing a great 70000

60000 tool for academic research and assay development.

50000 Bandwidth = 20 nm 40000 Bandwidth = 15 nm RLU 30000 Bandwidth = 10 nm

20000

10000

0 475 500 525 550 575 600 625 650 675 Wavelength [nm]

Figure 4 Influence of bandwidth on signal intensity of firefly luciferase diluted 1:100.

The position of the measurement head (z-position) can be manually adjusted to optimize the signal intensity in luminescence scan top mode (Table 3), and this option is especially useful if low sample volumes are used. By changing the z-position using the manual z-position option (Figure 1), the maximum signal intensity can be determined. In addition to manual adjustment, a default z-position value is offered (z = 22,000 µm).

Technical Note

Literature List of abbreviations

1. Youyu Zhang et. al. (2010). Hairpin DNA switch for LAR II Luciferase assay reagent ultrasensitive spectrophotometric detection of DNA PLB/G Passive lysis buffer / gelatine hybridization based on gold nanoparticles and enzyme RLU Relative luminescence unit FF Firefly luciferase signal amplification. Anal Chem 82(15), 6440–6446. RR Renilla luciferase 2. Matthias A. Brun et. al. (2009). Semisynthetic fluorescent

sensor proteins based on self-labeling protein tags. J Am

Chem Soc 131(16), 5873-84. Epub 2009 Apr 06.

3. Andrea Hawe et. al. (2010). Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations. Pharm Research 27(2), 314–326. 4. Seeing is believing. Tecan Journal, Edition 1/2011, p18-19. 5. Axel Kirchhofer et.al. (2010). Modulation of proteins properties in living cells using nanobodies. Nat Struct Mol Biol Jan, 17(1), 133-138. Epub 2009 Dec 13.

6. Dual-Luciferase Reporter assay system, technical manual, part# TM040 (Promega Corporation).

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Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative.

Tecan and Infinite are registered trademarks, and Safire2, i-control and premium Quad4 Monochromators are trademarks, of Tecan Group Ltd., Männedorf, Switzerland. Dual-Luciferase Reporter assay, QuantiLum recombinant luciferase and Stop & Glo reagent are registered trademarks, and DLR a trademark, of Promega Corporation, Madison, WI, USA. Greiner is a trademark of Greiner Bio-One, Germany. 396660 V1.0, 05-2011 05-2011 V1.0, 396660