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