Author: kromnigon

How to choose fluorochromes?

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® 7 filter 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.

Absorb (nm)Emit (nm)
AlexaFluor 350346442
AlexaFluor 405401421
AlexaFluor 430434541
AlexaFluor 488495519
AlexaFluor 500502525
AlexaFluor 514517542
AlexaFluor 532532554
AlexaFluor 546556573
AlexaFluor 555555565
AlexaFluor 568578603
AlexaFluor 594590617
AlexaFluor 610612628
AlexaFluor 633632647
AlexaFluor 635633647
AlexaFluor 647650665
AlexaFluor 660663690
AlexaFluor 680679702
AlexaFluor 700702723
AlexaFluor 750749775
AlexaFluor 790782805

Cyanine Dyes

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.

Absorb (nm)Emit (nm)
Cy3(512); 550570; (615)
Cy3B558572; (620)
Cy3.5581594; (640)
Cy5(625); 650670


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 dyes

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

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

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

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.

Absorb (nm)Emit (nm)
DyLight 350353432
DyLight 405400420
DyLight 488493518
DyLight 550562576
DyLight 594593618
DyLight 633638658
DyLight 650654673
DyLight 680692712
DyLight 755754776
DyLight 800777794


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®
Absorb (nm)Emit (nm)
AZDye™ 488494 nm517 nm
Atto™ 542542 nm562 nm
AZDye™ 594590 nm617 nm
AZDye™ 647649 nm671 nm


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 our StreptaClick®-HRP kit.

Selected Tyramide dyes available with StreptaClick®-HRP kit
Absorb (nm)Emit (nm)
AZDye™ 405402 nm424 nm
CF®430426 nm498 nm
AZDye™ 488494 nm517 nm
AZDye™ 555555 nm572 nm
AZDye™ 594590 nm617 nm
AZDye™ 647649 nm671 nm
Cyanine 3555 nm572 nm
Cyanine 5648 nm671 nm

We are My Green Lab certified!

My Green Lab – sustainability in science

My Green Lab is an initiative to build a global culture of sustainability in science. Recognized as the gold standard for laboratory sustainability practices worldwide, My Green Lab Certification equips scientists and laboratory support teams with practical solutions to effect significant change. The Race to Zero Campaign sets ambitious goals for achieving net-zero carbon footprints. As a proven and scalable program, My Green Lab Certification assists organizations in attaining their sustainability objectives.

Aiming for a net-zero carbon footprint in line with AstraZeneca’s sustainability efforts

The My Green Lab certification guarantees our proactive efforts in adopting best practices to minimize waste, conserve energy, and reduce water consumption. Furthermore, the program seeks to transform mindsets in order to establish a culture of sustainability in everything we do.

Our sustainability work at Kromnigon

This certification highlights our commitment to enhancing sustainability in our laboratory. It encourages us to make changes in both our research and development, as well as production practices. By participating in discussions and reviewing our current processes, we pinpointed areas for potential improvement. We then implemented innovative solutions, practices, and behaviors to support us in reaching our sustainability goals.

12 areas for a sustainable lab

Plug Load

In a laboratory, 20-25% of energy consumption comes from electrical devices connected to power outlets. We have assessed all our equipment and ensured that only necessary instruments are plugged in.

Fume Hoods

Fume hoods consume a significant amount of energy. Leaving them open also adds to the ventilation system’s energy load. Therefore, it is essential to develop a habit of consistently closing the hood.

Large Equipment

For larger equipment such as incubators, vacuum pumps, computers, and tissue culture hoods, it is crucial to take extra energy considerations depending on the specific device.

Cold Storage

After fume hoods the refrigerators, freezers and cold rooms are the most energy consumptive equipment category. Routine maintenance can boost energy efficiency by 10%, while maintaining ultra-low temperature freezers at -70 degrees, rather than lower, can result in a 30% energy savings.

Infrastructure Energy

In a typical lab, air handling, air quality, temperature regulation, and lighting account for half of the energy consumption. We strive to be aware of our energy usage and explore ways to reduce it.


Water consumption in laboratories is naturally higher than in office spaces. We are conscious of our freshwater usage and actively seek methods to minimize it.

Waste Reduction and Recycling

Laboratories generate a considerable amount of waste, with plastic waste from labs estimated to constitute nearly 2% of global plastic production. We consistently explore alternatives to plastics and replaces it with better alternatives when possible.

Resource Management

Pooling resources, sharing inventories, and exchanging materials among laboratories saves resources and time, and reduce costs.


We aim to make more intelligent purchasing decisions, focusing on resource efficiency, supporting eco-friendly manufacturers, and reducing the environmental footprint in collaboration with our suppliers.

Green Chemistry

The 12 Principles of Green Chemistry are a set of guidelines for designing environmentally friendly products and processes. We strive to minimize, replace, and design the utilization of chemicals in processes and manufacturing whenever feasible.

Community and Engagement

The green lab movement grows by exchanging experiences, techniques, advice, and ideas. Together, we are building a robust culture of sustainability within the laboratory.


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Learn more about My Green Lab at: Find additional information on the collaborative sustainability efforts between the BioVentureHub at AstraZeneca and My Green Lab at:

How do fluorochromes work?

Fluorochrome excitation spectrum 

When a fluorochrome is exposed to light, a photon with the appropriate amount of energy can excite an electron in the fluorochrome. In this excitation process, the electron absorbs the energy from the photon and moves from a ground state with low energy into an excited state with higher energy. The energy that photons carry is determined by the wavelengths. The absorbed light has shorter wavelengths than the emission wavelengths (the emitted photons). Consequently, the wavelength intervals that that contains photons with the appropriate amount of energy make up the excitation spectrum of the fluorescent dye.

Fluorochrome emission spectrum

In the excited state, the electron loses some energy as heat. The remaining energy is released as a photon of light when the electron returns to the ground state. This causes the fluorochrome to emit light. The emitted light has less energy than the excitation light since some energy was lost as heat. This is why the emitted light has longer wavelengths than the excitation light. Typically, each fluorochrome has a wavelength peak interval wherein most light is emitted. This peak interval determines the color of the fluorochrome. For example, green fluorochromes emit most light at a wavelength around 500-540 nanometer. 

Both the excitation and emission are rapid reactions that occur within nanoseconds. Thereby fluorochromes continuously are excited and emit light when illuminated the excitation light source.

Filter sets for fluorescence microscopy

In fluorescence microscopy, optical filters are used to excite (excitation filter) and collect (emission filter) light from fluorochromes. The microscopy light source provides the excitation light, and the excitation filter determines the exact wavelength interval that will be passed on to the sample. The emission filter determines which interval of the emitted light from the fluorochromes in the sample that will be passed on to the camera/eyes for detection. When samples are immunostained with more than one fluorochrome, it is crucial to have filter sets with high specificity for each fluorochrome in the sample to avoid bleed-through signals.

Filter sets for multiplex IHC

Multiplex immunohistochemistry (IHC) that involves more than four colors is a challenge for the microscope since the excitation and emission spectra of fluorochromes will significantly overlap. Thereby the risk of bleed-through signals from the other fluorochromes rapidly increases. Especially if the staining intensity varies between fluorochromes in the sample. Hence, each filter set must be carefully designed to ignore signals from all other fluorochromes and still efficiently collect signals from its specific fluorochrome. 

SpectraSplit® 7 is patented filter sets optimized for 7-color multiplex IHC. The filter sets effectively separate seven classes of common fluorochromes, with less than 0.5% bleed through between any channels. Hence, you have the freedom to combine any fluorochromes from the SpectraSplit® 7 fluorochrome chart in immunostainings, all the way up to seven colors. This means that you can custom make your own 7-plex IHC with your favorite fluorochromes without spectral unmixing corrections. 

The 7 classes are:

S-Split DAPI

DAPI, AZDye™405 (StreptaClick®-Color and StreptaClick®-HRP), Hoecht

S-Split 440

CF®430 (StreptaClick®-Color and StreptaClick®-HRP), Opal™p.480, Atto®425, cCFP

S-Split 488

FITC, AZDye™488 (StreptaClick®-Color and StreptaClick®-HRP), Opal™520, CF®488, Alexa Fluor®488, DyLight®488, Atto®488, eGFP

S-Split Cy3

Cy®3, TRITC, AZDye™555 and Cyanine 3 (StreptaClick®-Color and StreptaClick®-HRP), Opal™570, CF®555, Alexa Fluor®546/555, DyLight®549, Atto®542, mOrange/mRFP

S-Split 594

Texas Red, AZDye™594 (StreptaClick®-Color and StreptaClick®-HRP), Opal™620, CF®594, Alexa Fluor®594, DyLight®594, Atto®590/594, mCherry/mRaspberry

S-Split 647

Cy®5/Cy®5.5, AZDye™647 and Cyanine 5 (StreptaClick®-Color and StreptaClick®-HRP), Opal™690, CF®647/680, Alexa Fluor®647, DyLight®649, Atto®647/665, miRFP703

S-Split 750

Cy®7, Opal™p.780, CF®750, Alexa Fluor®750, DyLight®750, Atto®740

Almi Invest invests in to Kromnigon – 2022

Almi Invest is investing SEK 2.5 million in the biotechnology company Kromnigon, which is developing new methods for multi-staining of cells and tissues, which is of great importance in research and cancer treatment. The West Coast Business Angels also participate in the issue of a total of just over SEK 5 million. The money will be used for product development, sales and to establish the organization.

Antibody-based staining of cells and tissues (immunostaining) is a common method in research, drug development and healthcare, and an important piece of the puzzle in the development of immunotherapy, precision medicine and diagnostics. Immunostaining allows researchers to label certain cells/proteins of interest. Examples of uses are to distinguish cancer cells from healthy cells in a tumor sample, or to ensure that the correct protein has been identified in research. Tissue staining is also used in cancer care, where it enables an individual and targeted treatment, which can be tailored to each patient to give the best effect.

Gothenburg-based Kromnigon has developed a patented system for multi-staining of cells and tissues, which provides a simpler and faster staining process. With Kromnigon’s product FlexiStain, it is possible to stain different cells in different colors in a single common staining step, instead of doing several color steps sequentially, which is done with current technology. This saves a lot of time. FlexiStain is also more flexible, as the customer chooses which antibody is stained with which color, and even very small quantities can be labeled.

The technology to label cells with antibodies is already used in research, in drug development and in clinical practice, all major and growing areas in Life science.

“New technological advances in tissue staining in combination with strong scientific and clinical advances in immunotherapy in drug development have led to a positive spiral where both areas are pushing each other forward“, says Louise Warme, Investment Manager at Almi Invest. “Kromnigon’s new technology and broad product portfolio solve a known need and are well positioned in a rapidly expanding market.“

Kromnigon has four different products in cell staining on the market, and more are in the pipeline.

“Thanks to this investment, we can take the next step in our expansion and start developing additional products in our portfolio“, says Per Fogelstrand, CEO of Kromnigon.

Kromnigon joins AstraZeneca’s BioVentureHub

In March 2021, Kromnigon joined the AstraZeneca’s BioVentureHub. Immunofluorescence microscopy is an imaging technique that can help visualise many proteins or cells in a single tissue section. The rapid expansion of immunofluorescence imaging within drug and diagnostics development is driven by the need to more accurately map immune cell influx into diseased tissues, such as tumours. This makes it easier to screen patients to determine personalized treatment therapies and to evaluate clinical responses.

Henric Olsson, Head of Target Science, Research and Early Development, Respiratory and Immunology, BioPharmaceuticals R&D at AstraZeneca, says, “It’s great to have Kromnigon co-locating with us at our R&D Gothenburg site through the BioVentureHub. We are keen to learn more about their tools and reagents for staining, visualizing and localising multiple proteins in the same tissue section, which may enable simultaneous extraction of more data from fewer samples.

Per Fogelstrand, CEO at Kromnigon, says, ”We are very excited to be part of the creative and collaborative life science community that is being catalysed in the BioVentureHub. We are eager to share our expertise in immunofluorescence microscopy with fellow scientists at AstraZeneca and in the other BioVentureHub companies, and to being inspired to expand the development of our company and or products.

Magnus Björsne, CEO for AstraZeneca’s BioVentureHub, says, “Kromnigon will – literally – add even more colour to our vibrant BioVentureHub innovation environment. We’re looking forward to working with Per and the Kromnigon team to help them accelerate the growth of their imaging innovations and to facilitate mutually beneficial scientific interactions between Kromnigon and AstraZeneca/BioVentureHub company scientists.

GU Ventures invest in Kromnigon AB

Simultaneous detection of multiple structures in biological samples using fluorescently labeled antibodies – multistaining – is a rapidly growing market in biological research and clinical diagnostics. GU Ventures has therefore made an investment in Kromnigon AB, that provide whole solutions in multistaining.

The company comprises two patented innovations that complement each other:

SpecraSplit; a combination of light filter sets – that can be added to regular microscopes – which enables simultaneous and clearer detection of up to eight labeled structures.
FlexiStain; a toolbox that enables fast and flexible antibody-based multistaining with no cross-binding.

SpectraSplit is a fully developed product at early stage market launch, FlexiStain is half way through VINNOVA’s commercial support program called “VINN-verifiering”. The commercial focus will be on acquiring customer validation for the system as well as securing strategic partnerships with microscopy providers in order to increase sales.

– We are looking forward to working with our market launch and expanding sales for SpectraSplit throughout 2016 and onward, said inventors and co-founders Per Fogelstrand and Ulf Yrlid.

– Potential customers have responded very positively to FlexiStain as well, and we’ve already got a listing of researchers who are willing to be initial customers, said Alexander Lagerman, who recently became Operations Manager.

– We have supported the project in early stage and believe in the commercial potential that we have verified in the project. The business idea fits our incubation process and investment criteria well and we looking forward to develop the business in our incubator, aid Lorna Fletcher at GU Ventures.

Kromnigon AB is a Swedish biotechnology company based in Gothenburg with key researchers from University of Gothenburg and the Sahlgrenska Academy. The team consists of Per Fogelstrand, Assistant Professor at the Wallenberg Laboratory, Associate Professor Ulf Yrlid at the Department of Microbiology & Immunology and IP Strategists & Business Developers Dr. Lorna Fletcher (GU Ventures) and Alexander Lagerman (Lagerman Subsidium).

Contact information:

Alexander Lagerman
Operations Manager,
+46 (0)70 525 32 39

GU Ventures:
Lorna Fletcher
Business Developer & IP strategist
+46 (0)73 416 70 84

GU Ventures finansierar och utvecklar nya företag baserade på forskningsresultat och annan akademisk spetskompetens från Göteborgs universitet.

Link to the webpage:

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