Black Microplates for Fluorescence Assay

Fluorescence-based assays are extensively employed in high-throughput analysis due to their elevated responsiveness, varied array of fluorescent molecules, simplicity of usage, and multiple reading modalities.

What is fluorescence?

Fluorescence refers to the characteristic of certain chemicals or dyes to absorb light at a specific wavelength, known as excitation (Ex), and subsequently emit light at a longer wavelength, known as emission (Em). This phenomenon allows for the detection and study of various substances in scientific research and other applications. In fluorescence assays, the difference in wavelength between the excitation and emission peaks is called the Stokes shift. This shift varies depending on the type of fluorophore used, as different chemicals or dyes can exhibit different fluorescent properties. The term “fluorophore” is commonly used to describe the specific chemical or dye that emits fluorescence in these assays.

Fluorophores, which are used in various scientific and imaging applications, possess distinct spectral properties. To activate a fluorophore, it needs to be excited at a specific wavelength. Once excited, it emits light at a particular wavelength. It’s important to note that the excitation and emission wavelengths are not limited to single values but rather encompass a range of wavelengths that are unique to each fluorophore.

During a fluorescence assay, a common method used in scientific research and diagnostics, the fluorophore is stimulated by light of a specific wavelength. This excitation leads to the emission of light at a different wavelength, which can be measured using a plate reader. This technique allows scientists to detect and quantify certain substances or analyze biological processes accurately and efficiently.

Typical fluorescence assays

Fluorescence assays commonly used in scientific research include fluorescence intensity (FI), fluorescence polarization (FP), and Förster resonance energy transfer (FRET) assays.

Fluorescence assays are widely used in scientific research to study various biological and chemical processes. These assays utilize the phenomenon of fluorescence, where molecules absorb light at a specific wavelength and emit light at a longer wavelength.

One commonly used fluorescence assay is the fluorescence intensity (FI) assay. In this assay, the intensity of emitted light is measured to quantify the concentration or activity of a target molecule. FI assays are versatile and can be used to measure various parameters such as enzyme activity, protein-protein interactions, and DNA/RNA quantification.

Another popular fluorescence assay is the fluorescence polarization (FP) assay. FP measures the degree of rotational movement of fluorescent molecules in solution. By monitoring changes in polarization, researchers can study molecular interactions, ligand binding events, and enzymatic activities. FP assays are particularly useful for high-throughput screening applications due to their simplicity and sensitivity.

Förster resonance energy transfer (FRET) assays are another important tool in fluorescence-based research. FRET occurs when two fluorophores are in close proximity and energy is transferred from an excited donor molecule to an acceptor molecule without emitting photons. This phenomenon allows researchers to investigate molecular interactions, conformational changes, and protein-protein interactions with high spatial resolution.

Overall, these typical fluorescence assays – FI, FP, and FRET – offer valuable insights into various biological processes by harnessing the power of fluorescent molecules like fluorescein, cyanine 3, cyanine 5, green fluorescent protein (GFP), rhodamine, Texas Red, coumarin, and various others. They enable scientists to study molecular dynamics, protein function, drug discovery, and much more with precision and sensitivity.

Fluorophores used in microplate-based assays often have short half-lives, lasting less than a microsecond. This is the reason why, to minimize background autofluorescence, it is recommended to conduct assays using black plates for these short half-life fluorophores.

Typical excitation and fluorescence emission spectra of fluorophores with relatively short half-lives

What is autofluorescence?

Autofluorescence refers to the phenomenon of fluorescence emitted by substances other than the specific fluorophore being studied and is initiated by the same excitation light that is employed to stimulate the fluorophore in a fluorescence assay. It can have a negative impact on assays by increasing the background signal, leading to inaccurate results and reduced sensitivity.

In various laboratory settings, autofluorescence can arise from components present in assay buffers and biological samples. These components may include proteins, lipids, pigments, or other molecules that possess intrinsic fluorescent properties. Autofluorescent substances have the ability to absorb light in the UV-blue range (355-488 nm) and emit light in the blue-green range (350-550 nm). As a result, this autofluorescence leads to a decrease in signal sensitivity and loss of signal resolution within these specific light ranges since when excited by an appropriate light source, these substances emit fluorescence signals that can interfere with the detection of the desired fluorophore.

The level of background autofluorescence depends on the excitation wavelength used in an assay. Generally, higher excitation wavelengths (above 650 nm) result in lower autofluorescence compared to wavelengths in the UV-Vis range.

The choice of plate color can have a significant impact on the results of fluorescence assays. White plates reflect light, leading to higher background fluorescence compared to black plates. On the other hand, black plates tend to absorb light, reducing background interference. Consequently, black plates are generally recommended for fluorescence assays involving fluorophores with a short half-life to minimize background noise and optimize signal detection.

In addition to the right plate color selection, to minimize the effects of autofluorescence, researchers employ various strategies such as spectral unmixing techniques or using blocking agents that reduce non-specific binding. Additionally, careful selection of fluorophores with minimal overlap in emission spectra can help mitigate interference caused by autofluorescence.

Understanding and addressing autofluorescence is crucial for accurate data interpretation in fluorescence-based assays. By identifying and mitigating sources of autofluorescence, researchers can enhance assay sensitivity and specificity while obtaining reliable experimental results.

Time-resolved fluorescence

Time-resolved fluorescence (TRF) is a specialized technique that diverges from conventional fluorometric detection primarily in the timing of the excitation and emission processes. In traditional fluorometric detection, the excitation and emission occur simultaneously. This means that as the sample is being excited, its emitted light is simultaneously being measured. However, TRF takes a different approach. This method employs specific fluorescent molecules known as lanthanide chelate labels, such as the Europium ion (Eu3+), which is the most prevalent. These labels have a unique characteristic: their half-lives span from microseconds to milliseconds, especially in microplate-based assays.

Due to the extended half-life of these fluorescent molecules, the instrumentation can introduce a “delay time” or “lag time” between the moment of excitation and the point when the emission signal is read. This time-resolved mode proves beneficial as it allows any background autofluorescence to diminish before the main emission signals are recorded, enhancing the clarity of the results. The extended half-life of fluorescent molecules in this context refers to the length of time these molecules remain in an excited state before emitting light. When using instrumentation to measure fluorescence, there can be a delay or lag time between the moment of excitation and when the emission signal is actually read.This delay time can be advantageous because it allows any background autofluorescence to dissipate before recording the main emission signals. Background autofluorescence refers to any unwanted fluorescence from sources other than the target molecules being studied.By allowing this delay, researchers can enhance the clarity of their results by reducing interference from background autofluorescence. This time-resolved mode helps ensure that only the desired fluorescence signals are captured and analyzed, leading to more accurate and reliable data interpretation.

When it comes to the practicality of running time-resolved fluorescence assays, both black and white plates can be utilized. White plates tend to produce amplified raw signals since the white hue maximally reflects the light. On the other hand, black plates yield subdued raw signals due to their light-quenching property. Yet, black plates can be advantageous in certain scenarios, particularly when reducing cross-talk is essential, thereby improving sensitivity.

For assays demanding higher accuracy and those run in denser settings, such as in 384-well and 1536-well plates or when a fainter signal is anticipated, white plates are generally the preferred choice.

Typical excitation and fluorescence emission spectra of fluorophores with relatively long half-lives like Europium. Europium has a large Stokes shift, a wide excitation spectrum, and a narrow emission spectrum, typical of lanthanide chelates.

Biomat black microplates for biochemical immunofluorescence assays

Microplate Surface	Description
Medium-Binding	These plates are designed with a hydrophobic surface that is capable of passively adsorbing proteins with varying degrees of hydrophobicity. This feature makes them well-suited for protein adsorption in various applications. – Available black, white and clear – Available in the 96-well breakable, strip and solid format
High-Binding	These plates have a hydrophilic surface that is specifically designed to passively adsorb proteins with varying degrees of hydrophilicity. This means that the proteins can easily bind to the surface without any additional steps or active involvement.. – Available black, white and clear – Available in the 96-well breakable, strip and solid format
Carboxylated	These plates are activated with carboxylic groups which can promote the covalent immobilization of biomolecules containing reactive free amino groups using EDC mediated amination – Available black, white and clear – Available in the 96-well breakable, strip and solid format
Aminated	These plates are activated with primary amino groups which can promote the covalent immobilization of biomolecules containing reactive groups such as carboxyl, thiol, or amino via well-known homo/hetero-bifunctional linkers, e.g. N-Hydroxysuccinimide (NHS) or Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) – Available black, white and clear – Available in the 96-well breakable, strip and solid format
Streptavidin	These plates are activated with Streptavidin, a powerful and universal instrument for binding any biotinylated molecules (antibodies; antigens; proteins; peptides; polysaccharides; oligonucleotides; DNA fragments; etc.). They are used especially for molecules which do not offer reliable bonding by passive adsorption, or that adsorb in an unfavorable orientation. – Available black, white and clear – Available in the 96-well breakable, strip and solid format
Streptavidin High-Binding	These plates are activated with a dedicated form of Streptavidin that, besides having the same properties of basic Streptavidin, is particularly useful to set up competitive tests to measure biotinylated low molecular weight molecules – Available black, white and clear – Available in the 96-well breakable, strip and solid format
A, G, and A/G proteins	These plates are activated with Protein A, Protein G, or mixed A/G that provide alternatives to direct, passive adsorption methods for immobilizing antibodies for immunofluorescence plate-based assay techniques. They specifically bind to the Fc region of immunoglobulins of many mammalian species with an orientation that allows the F(ab)2 binding sites to be freely available for efficient binding to epitopes. – Available black, white and clear – Available in the 96-well breakable, strip and solid format

 

Biomat black microplates for cell-based fluorescence assays

Microplate Surface	Description
Tissue Culture Treated (polystyrene)	TC-treated plates guarantee that the surface chemistry offers a uniform surface with both hydrophilic and negative-charge properties; this treatment will lead to increased cell attachment – The bottom can be either opaque or transparent – Sterilized with lid – Available in 96- and 384-well formats
Poly-D-lysine and Poly-L-lysine	In certain cases, for optimal cell attachment, growth, and differentiation, it is necessary to have a thin layer of polymer polycationic on the plastic support. This layer helps cells or tissues adhere to the surface more effectively. This enhanced surface is obtained by coating it with Poly-lysine, a synthetic, positively charged polymer. – The bottom can be either opaque or transparent – Sterilized with lid – Available in 96- and 384-well formats
Glass Bottom Plates	Glass Bottom Plates are suitable for high-resolution imaging, where low autofluorescence and optical clarity are required. The high-quality glass provides lower fluorescent background and higher signal-to-noise ratio, essential in all fluorescence applications. – Produced in clean rooms – Available in 96-, 384- and 1536-well formats