Fluorophores, also known as fluorescent probes, fluorochromes, fluorescent dyes or simply dyes, are molecules that absorb light at a specific wavelength and emit light at a longer wavelength. Designing an immunohistochemistry study begins with the crucial decision of selecting antibodies and fluorochromes, which can be challenging. Base your decision on the available filter sets and practical testing to identify combinations that yield bright and clear images.
Some fluorophores are generic names, referring to a particular chemical structure, while others are brand names, which are usually proprietary and trademarked by specific companies.
The number that follows the name of a dye, such as Alexa Fluor 488 or DyLight 488, generally represents the dye’s peak excitation wavelength in nanometers (nm). This value indicates the wavelength at which the fluorophore absorbs light most efficiently, leading to excitation.
Optimal specificity by pairing the right filter sets with the appropriate fluorochromes
The filter sets, the fluorescent channel, needs to be optimized for the fluorochrome you aim to detect. Filter sets are designed to block wavelengths outside the excitation peak and collect peak emission wavelengths. Since the incoming light from the light source is much more intense than the emission signal, it must be blocked to even detect the emission signal.
If the excitation filter allows a broad range of wavelengths to pass through, it may inadvertently excite other fluorochromes in your multiplex. Conversely, if the wavelength window is too narrow, the excitation peak of the fluorochrome might be missed. Furthermore, if the emission filter transmit a wide range of wavelengths, other fluorochromes may be detected. Keep in mind that detectors capture photons, not wavelengths, meaning they cannot distinguish between different colors; the specificity lies in the filter sets.
Multiplex immunohistochemistry images
When conducting a multiplex immunohistochemistry assay, it’s crucial to ensure that filters block and transmit each fluorochrome’s excitation and emission without any spillover between them. To achieve this, examine the full absorption and emission spectra, rather than just the peaks, as partial excitation and emission can cause unspecific signals in multiplex IHC images. Compare these spectra to the wavelengths transmitted by each excitation and emission filter. Perform this procedure for all fluorophores in your assay. If potential spillover is observed, consider switching fluorochromes or filters. To detect bleed-through, use single-stained slides, which will reveal any presence of other fluorochromes in the channel. If this process seems tedious, we have a solution for you.
Our patented SpectraSplit® 7filter set allows for 7-plex IHC without spillover, eliminating the need for spectral unmixing. Compatible with most fluorescent microscopes and commercially available fluorochromes, you can select one fluorochrome from each class in the fluorochrome chart to produce sharp, clean 7-plex images without bleed-through.
Figure: Red areas indicate overlapping wavelengths. Fluorochrome B will be excited from the excitiation filter for fluorochrome A. In addition, emission from fluorochrome B will be detected with using the emission filter of fluorochrome A. This is called bleed-through or spillover between channels.
Fluorophores for Immunohistochemistry and Immunofluorescence
Numerous fluorophores are available for use in research, and scientists continue to create new dyes with enhanced brightness, photostability, and spectral properties to address the expanding needs of diverse research applications.
Here is a list of common fluorochromes:
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein that was first described in 1942 and has been commercially available for quite some time. With excitation and emission peak wavelengths of around 495 nm and 519 nm, FITC produces a green color. Although photobleaching is an issue with FITC, other fluorescein derivatives, such as Alexa 488 and DyLight 488, have been developed for applications requiring greater photostability, higher fluorescence intensity, or different attachment groups. Nonetheless, FITC remains popular due to well-established protocols, widespread availability, and relatively low cost.
Green Fluorescent Protein (GFP) is a bright green, genetically-encoded fluorophore originally derived from the jellyfish Aequorea victoria. GFP has also been found in other marine organisms, such as corals, sea anemones, zoanthids, copepods, and lancelets. Variants with different colors have been developed, including Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), and Red Fluorescent Protein (RFP). In recognition of their discovery and development of GFP, scientists Roger Y. Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry 2008.
Alexa Fluor is a brand name created by Molecular Probes, which is now part of Thermo Fisher Scientific. It represents a series of highly photostable and bright dyes that come in a wide range of colors, such as Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647. Alexa Fluor dyes are synthesized from other fluorophores like fluorescein and rhodamine. These dyes are designed to be less pH-sensitive and more photostable compared to the original fluorophores.
Cyanines, sometimes called tetramethylindo(di)-carbocyanines, belong to a family of synthetic dyes known for their high extinction coefficients (which measure how strongly a substance absorbs light at a specific wavelength) and good photostability. The most frequently used dyes are Cy3, which has a orange fluorescence, and Cy5, which fluoresces in the far-red region. Although patent protection for the standard Cy series of dyes has expired, the trademarked Cy naming remains. As a result, dyes identical to Cy dyes but bearing different names are now available on the market.
Unlike the previous mentioned fluorescent dyes that label primary or secondary antibodies, DAPI (4′,6-diamidino-2-phenylindole) strongly binds to adenine-thymine-rich regions in DNA, effectively staining cell nuclei. This blue-emitting dye can pass through an intact cell membrane, making it suitable for staining both live and fixed cells. However, DAPI penetrates the membrane less efficiently in live cells, providing a marker for membrane viability.
When bound to double-stranded DNA, DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and an emission maximum at 461 nm (blue). Its emission peak is relatively broad. DAPI can also bind to RNA, though its fluorescence is weaker. When bound to RNA, its emission shifts to around 500 nm.
Hoechst stains are similar to DAPI, as they are also blue-fluorescent DNA stains compatible with both live and fixed cell applications. They can be visualized using the same equipment filter settings as DAPI. Three related Hoechst stains are available: Hoechst 33258, Hoechst 33342, and Hoechst 34580. Hoechst 33258 and Hoechst 33342 are the most widely used, and they have nearly identical excitation-emission spectra. The number 33342 for example, refers to the 33342nd compound developed by the company Hoechst AG.
SYBR Green, a green-emitting dye, is primarily used in molecular biology for real-time PCR due to its ability to selectively bind to double-stranded DNA. Though occasionally employed in fluorescent microscopy, SYBR Green’s main application is in PCR. Developed by Molecular Probes and now part of Thermo Fisher Scientific, SYBR Green is a well-known brand name in the field.
The Rhodamine family
The Rhodamine family consists of a collection of red and orange-emitting dyes, featuring compounds such as Tetramethylrhodamine (TRITC) and Rhodamine B. Additional rhodamine derivatives used for imaging include branded fluorophores like Alexa 546, Alexa 633, DyLight 550, and DyLight 633, as well as other examples like Carboxytetramethylrhodamine (TAMRA) and Texas Red.
The mFruits family
mOrange, mCerulean, mPlum, and mCherry are genetically-encoded fluorescent proteins derived from coral species. They emit light in the orange, cyan, far-red, and red regions of the spectrum, respectively. As monomeric red fluorescent proteins (mRFPs), they are advantageous due to their lower molecular weight and faster folding compared to tetramers. Second-generation mRFPs, such as mStrawberry, mOrange, and dTomato, offer enhanced brightness and photostability compared to the first-generation mRFP1.
Contrary to its brand-like name, Texas Red is actually a generic term for a red-emitting dye that fluoresces around 615 nm, with an absorption spectrum peak at 589 nm. Newer rhodamine derivatives, such as Alexa 594 and DyLight 594, have been developed to match Texas Red’s excitation and emission spectra.
Opal dyes are a series of fluorescent dyes developed by Akoya Biosciences. The Opal dyes themselves are considered brand names, as they are proprietary and developed specifically by Akoya Biosciences for their multiplex immunofluorescence platform. The Opal dyes are available in a range of excitation and emission wavelengths, which enables the detection of multiple targets with limited spectral overlap.
Quantum dots (QDs) are tiny semiconductor nanocrystals, usually ranging from 2 to 10 nanometers in size. When exposed to light, they can absorb and emit photons with high brightness and photostability. QDs offer several advantages over traditional organic fluorophores, including broad excitation spectra and narrow emission spectra, which minimize spectral overlap and allow for the simultaneous detection of multiple fluorophores in multiplexed assays. Furthermore, the emission wavelength of QDs can be precisely controlled by altering their size and composition. Smaller QDs emit light at shorter wavelengths (e.g., blue), while larger ones emit light at longer wavelengths (e.g., red). QDs are known for their high brightness and photostability, but it’s important to note that they have shown varying levels of toxicity.
DyLight fluorochromes, a series of fluorescent dyes developed by Thermo Fisher Scientific, are renowned for their brightness, photostability, and water solubility. Available in various excitation and emission wavelengths, these dyes support multiplexing in experiments that necessitate the simultaneous detection of multiple targets. DyLight Fluors exhibit excitation and emission spectra similar to those of fluorescein, rhodamine, Cy3, and Cy5 but are said to offer greater photostability, brightness, and reduced pH sensitivity.
AZDye is a collection of trademarked fluorochromes known for their high brightness and photostability. After evaluating numerous fluorophores, we chose to incorporate AZDyes in our StreptaClick® kits. The StreptaClick®-color kit enables the labeling of any biotinylated antibody with AZDye™ 488 (Green), AZDye™ 594 (Red), or AZDye™ 647 (Far red). We also offer Atto™ 542 (Yellow) for simplified four-color multiplex IHC. For our StreptaClick®-HRP kit, we recommend and provide various AZDyes based on extensive testing to achieve the brightest 7-plex results on both FFPE and frozen tissue.
Selected fluorochromes available with StreptaClick®
Atto fluorescent dyes represent a category of synthetic fluorescent dyes characterized by high absorption, high fluorescence emission, and high photostability. In our StreptaClick®-color kit, we use the yellow Atto™ 542 due to its exceptional performance, as demonstrated through our extensive testing.
CF dyes, Cyanine-based fluorescent dyes produced by Biotium, encompass a variety of dyes with different excitation and emission wavelengths, such as CF488A, CF568, and CF647. We recommend and offer CF®430 as a tested and highly efficient tyramide dye for use with ourStreptaClick®-HRP kit.
Selected Tyramide dyes available with StreptaClick®-HRP kit