# Multimode Plate Reader ## expand\_more [**CMI Spark resources and user guide**](/node/1876901#spark-resources)**⬇︎** Multimode plate readers are used for measuring fluorescence, absorbance or luminescence in a microplate and support a wide array of assays. The CMI has a [Spark Multimode Plate Reader](https://www.tecan.com/spark-overview) from [Tecan](https://www.tecan.com/), configured with Absorbance mode, Luminescence mode, and enhanced Fusion Optics Fluorescence mode. ![Tecan Spark Plate Reader](/sites/g/files/omnuum4016/files/styles/hwp_1_1__360x360_scale/public/2025-11/tecanspark_nobackground.png?itok=ajiKSG0K) Fusion Optics supports flexible set-up of fluorescence experiments using monochromator-based optics, filter-based optics, or a combination. Filters allow for higher sensitivity and speed and monochromators allow for greater flexibility and specificity in wavelength selection. **Key Features and Applications** - Fluorescence (enhanced) - Top and bottom read – End point and spectra - Fusion Optics - Monochromator spectral range: Ex. 230-900 nm; Em. 280 – 900 nm - Excitation and Emission Filters: 12 common filters (see [specifications](/node/1876901#spark-resources)) - [Fluorescence Polarization (FP)](/node/1876901#FP_assays) - [Fluorescence Resonance Energy Transfer (FRET)](/node/1876901#FRET_assays) - Time-resolved fluorescence (TRF) - [Time-Resolved Förster Resonance Energy Transfer (TR-FRET)](/node/1876901#FRET_assays) - Absorbance - Top read – Endpoint and UV/Vis spectra - spectral range: 200 – 1000 nm - Luminescence (enhanced) - Top read – glow, flash, multi-color, spectra - spectral range: 370 – 700 nm - NOT configured for AlphaScreen, Fluorescence Imaging or Cell-based assays. ## Fluorescence Polarization (FP) ### About FP expand\_more **Fluorescence Polarization (FP), also called Fluorescence Anisotropy,** measures the rotational mobility of fluorescently labeled molecules in solution. When a sample is excited with plane-polarized light, only those fluorophores aligned with the light’s electric vector become excited. If the labeled molecule rotates slowly (typically because it's bound to a larger partner), the emitted light retains much of the original polarization. If the molecule is small or unbound and rotates rapidly, the polarization decreases as the emission becomes depolarized. FP is widely used to monitor binding interactions, such as protein–protein, protein–DNA, or small molecule binding assays. High polarization values indicate binding (reduced rotational mobility), while low values indicate free, unbound molecules. The method is homogeneous (no separation/washing required), rapid, and suitable for high-throughput screening. ![Fluorescence polarization assay](/sites/g/files/omnuum4016/files/2025-12/Fluorescence%20Polarization%20Assay.png) SparkControl will measure both parallel and perpendicular fluorescence intensities for each well. **Polarization (mP or P):** \\\[P=\\frac{I\_{\\mid\\mid}-I\_\\bot}{I\_{\\mid\\mid}+I\_\\bot}\\\]**Anisotropy (r):** \\\[r=\\frac{I\_{\\mid\\mid}-I\_\\bot}{I\_{\\mid\\mid}+2I\_\\bot}\\\]Where I|| is intensity of parallel and I⟂ is perpendicular emission. ### FP Fluorophores expand\_more SortFluorescence Polarization Fluorophores \*The optimal mass range for FP using a given fluorophore is when the rotational correlation time (τr) of the labeled molecule is close to the fluorescence lifetime (τf): **τr≈τf** Rotational correlation time for a roughly spherical protein in water at 20°C: τr(ns)=0.012×MW2/3; MW = molecular weight in Daltons (Da). Adapted from the Molecular Probes Handbook (ThermoFisher).Fluorophore**τf** (ns)Optimal Mass\*(kDa)Usable Mass Range (kDa)Ex (nm)Em (nm)Cy3 0.5 0.35 <1–10 550 570 Cy5 1 0.98 <1–15 650 670 Tetramethyl- rhodamine 2.5 3 <10–50 555 580 **Fluorescein** **FITC** **4** **6** **<10–75** **495** **520** AlexaFluor 488 4.1 6.5 <10–75 495 519 BODIPY TMR 5.4 9.8 <10–100 544 574 BODIPY FL 5.5 10 <10–100 503 512 Dansyl ~20 54 <50-300 340 520 ## Förster Resonance Energy Transfer (FRET) ### About FRET expand\_more **Förster Resonance Energy Transfer (FRET)** is a mechanism describing the transfer of energy between two fluorophores. The **donor molecule**, absorbs light energy and, if the **acceptor molecule** is close enough, can transfer this energy without the emission of light (non-radiatively). This energy transfer, which occurs through a dipole-dipole coupling, then causes the acceptor to emit its own fluorescence. The efficiency of this energy transfer is very sensitive to the distance between the donor and acceptor, decreasing proportionally to the sixth power of the distance separating them. This makes FRET an extremely precise "spectroscopic ruler" for measuring nm distances. For FRET to occur, several conditions must be met: - **Proximity:** The donor and acceptor must be very close, typically within 1-10 nanometers of each other. - **Spectral Overlap:** The emission spectrum of the donor fluorophore must overlap with the absorption spectrum of the acceptor. - **Dipole Orientation:** The orientation of the donor's emission dipole and the acceptor's absorption dipole must be roughly parallel. A key parameter in FRET is the **Förster radius (R₀)**, which is the distance at which the energy transfer efficiency is 50%. This value helps determine the feasible range of distances that can be measured with a specific donor-acceptor pair. **Applications of FRET**: - Detecting and tracking interactions between proteins. - Measuring distances between different parts of a single protein to understand its conformation and folding. - Studying DNA and RNA structures. **Limitations of FRET:** - Standard FRET is its highly susceptibility to background noise from scattered excitation light and natural fluorescence from other molecules in the sample (autofluorescence). ![FRET](/sites/g/files/omnuum4016/files/2025-12/FRET.png) ### FRET Fluorophores expand\_more SortFRET Pairs \* Förster Radius (R₀): This value represents the distance at which energy transfer efficiency is 50%. It is specific to each donor-acceptor pair and is influenced by factors such as the spectral overlap between donor emission and acceptor absorption, quantum yield of the donor, and the relative orientation of the dipoles. Adapted from Molecular Probes Handbook (ThermoFisher).**Donor****Ex (nm)** **Em (nm)** **Acceptor****Ex (nm)** **Em (nm)** CFP (Cyan FP) 433 475 YFP (Yellow FP) 514 527 GFP (Green FP) 488 509 YFP (Yellow FP) 514 527 FITC (Fluorescein) 495 519 TRITC (Tetramethylrhodamine) 557 576 Cy3 550 570 Cy5 649 670 Alexa Fluor 488 495 519 Alexa Fluor 555 555 565 GFP (Green FP) 488 509 mCherry 587 610 Cy3 550 570 Cy3.5 581 596 FAM (Carboxyfluorescein) 495 520 Texas Red 595 615 Cy3.5 581 596 Cy5.5 675 694 Alexa Fluor 546 556 573 Alexa Fluor 647 650 668 B-Phycoerythrin 545 575 Cy5 649 670 ### Time-Resolved Förster Resonance Energy Transfer (TR-FRET) expand\_more **Time-Resolved Förster Energy Transfer (TR-FRET)**, also called Time-Resolved Fluorescence Resonance Energy Transfer, is a highly sensitive and robust biochemical technique used to study molecular interactions. It combines the principles of **FRET** (measuring energy transfer between two light-sensitive molecules, or fluorophores) with **Time-Resolved Fluorescence** (measuring fluorescence signals after a specific delay). This unique combination dramatically reduces background noise and interference, making it a standard for high-throughput screening (HTS) in drug discovery and for quantifying biomolecular interactions. **FRET** occurs when a **donor** (excited by a light source) transfers energy non-radiatively to a nearby **acceptor**, but only if they are within a 1–10 nm proximity. This results in quenching of donor fluorescence and emission of light from the acceptor. **Time-Resolved**-FRET, uses a **long-lifetime fluorophore** as the donor. After excitation, a delay is introduced (~50–150 μs) before the emission signal is detected. This delay allows short-lived background fluorescence from the sample matrix or autofluorescence to decay, resulting in a *much lower background and higher assay sensitivity*. - Donor Fluorophore - a lanthanide chelate (Europium or Terbium). Lanthanides have an very long fluorescence lifetime (milliseconds), whereas typical background fluorescence lasts only for nanoseconds. - Acceptor Fluorophore - a conventional fluorophore (e.g., Alexa Fluor, Cy5, or specialized proprietary dyes) that can accept energy from the donor. **Advantages** - Low Background Signal. Due to time-resolved measurement, native fluorescence and other short-lived signals are excluded. - Sensitivity. Capable of detecting low-affinity or transient interactions. - Versatility. Suitable for a wide range of applications including protein-protein, protein-peptide, and small molecule binding studies. ![TR-FRET](/sites/g/files/omnuum4016/files/2025-12/TR-FRET.png) ### TR-FRET Fluorophores expand\_more SortTR-FRET Donor Fluorophores**Fluorophore****CoraFluor 1****LanthaScreen****LANCE****Supplier** Tocris Bioscience Thermo Fisher Scientific Revvity **Donor Chemistry** Terbium chelate Terbium (Tb³⁺) chelate Europium (Eu³⁺) chelate/cryptate **Excitation Wavelength** 337 nm 340 nm (typical) 320–340 nm **Emission Bands** 490, 545, 590, 620 nm (Tb³⁺) 495, 545, 570, 620 nm (Tb³⁺) 615 nm (Eu³⁺); long-lived **Compatible Acceptors** FAM (fluorescein), TMR, Cy5, GFP, mCherry, Alexa Fluor™ 488 Alexa Fluor 488, 546, 594, 647, FAM, GFP, Cy5, others ULight (Revvity proprietary), Cy5, APC, Alexa Fluor™ 647 **Reactive Chemistries** Amine-reactive (PFP ester), Thiol-reactive (maleimide), Haloalkane Amine-reactive (NHS ester), protein labeling kits Amine-reactive (ITC chelate), protein labeling kits **Key Features** High brightness/stability Multiple reactive forms Best for in vitro/cell-free assays Widely compatible, robust kits multiple acceptor choices optimized for screening Sensitive, robust long-lived fluorescence low background **Product Page** [CoraFluor™ 1](https://www.tocris.com/products/corafluor-1-amine-reactive_7920) see also [CoraFluor™ 2](https://www.tocris.com/products/corafluor-2-amine-reactive_7950) [LanthaScreen™ Amine Reactive Tb Chelate](https://www.thermofisher.com/order/catalog/product/PV3583) [LANCE® Europium Labeling Reagent (W1024-ITC)](https://www.revvity.com/product/lance-eu-w1024-itc-chelate-eustd-200ug-ad0096#product-overview) Spark Plate Reader Resources Instrument Specifications ## Spark Plate Reader Resources [CMI Tecan Spark Multimode Plate Reader User Guide](/sites/g/files/omnuum4016/files/2026-04/CMI%20Tecan%20Spark%20Multimode%20Plate%20Reader%20Getting%20Started%20Guide%20.pdf "CMI Tecan Spark Multimode Plate Reader Getting Started Guide") - NEW *Please let us know if you find errors.* [Spark Multimode Plate Reader](https://www.tecan.com/spark-overview) from Tecan. ## Instrument Specifications Tecan Spark Temperature Control: Ambient +3C – 42C Plate Formats: 96-well, 384-well, 1536-well ### Fluorescence expand\_more SortFluorescenced - EnhancedSpecificationLight source High energy xenon flash lamp Spectral range (monochromator) Ex: 230-900 nm Em: 280-900 nm Wavelength accuracy Ex: <0.5 nm; Em: <0.5 nm Wavelength reproducibility <0.5 nm Bandwidth Adjustable from 5-50 nm Optical mirrors 50%, 510, 560, 625 nm built-in Well scanning Up to 100 x 100 data points Fastest read time 384-well plate (FI), ≤22 sec 1,536-well plate (FI), ≤34 sec Filters, wavelength/bandwidth (nm) Ex. 320/25, 340/35, 360/35, 465/35, 485/20, 495/10 Em. 520/10, 535/25, 540/25, 620/10, 635/35, 665/8.5 SortFI (fluorescence intensity) Specification Limit of detection1 Filter – top ≤8 amol/well (10 μl; 1,536 wells). Fusion – top ≤15 amol/well (10 μl; 1,536 wells) Mono – top ≤20 amol/well (10 μl; 1,536 wells) Filter – bottom ≤180 amol/well (10 μl; 1,536 wells) Fusion – bottom ≤200 amol/well (10 μl; 1,536 wells) Mono – bottom ≤220 amol/well (10 μl; 1,536 wells) SortFP (fluorescence polarization)2Specification Spectral range 300-850 nm Precision Filter ≤1.25 mP2 Precision Fusion ≤2.0 mP Precision Mono ≤2.5 mP SortTRF (time-resolved fluorescence)3SpecificationLimit of detection Filter ≤0.5 amol/well (20 μl; 384 sv wells)3 Limit of detection Fusion ≤0.6 amol/well (20 μl; 384 sv wells) Limit of detection Mono ≤0.7 amol/well (20 μl; 384 well) 1 Detection limit for fluorescein 2 FP detection limit @ 1 nM fluorescein 3 Detection limit for europium ### Absorbance expand\_more SortAbsorbanceSpecificationLight source Dedicated xenon flash lamp Spectral range 200-1,000 nm OD range 0–4 OD Scan speed (200–1,000 nm) ≤5 sec Wavelength accuracy <0.3 nm Wavelength reproducibility ≤0.3 nm λ ratio accuracy (260/230) <0.08 λ ratio accuracy (260/280) <0.07 Precision @ 260 nm <0.2 % Accuracy @ 260 nm <0.5 % Limit of detection (nucleic acids) <1 ng/μl ### Luminescence expand\_more SortLuminescenceSpecificationSpectral range 370-700 nm Luminescence (glow) – Limit of detection4 ≤225 amol/well, (25 μl; 384 sv wells)4 Luminescence (flash) – Limit of detection5 ≤12 amol/well, (55 μl; 384 wells)5 Dynamic range >9 orders of magnitude Multi-color luminescence 38 spectral filters Attenuation OD1,2,3 attenuation filters 4 Detection limit for ATP (144-041 ATP kit SL (BioThema)) 5 Detection limit for ATP (ENLITEN. Kit)