DAPI & Hoechst: avoiding photoconversion artefacts

DAPI & Hoechst most commonly used dyes to visualize nuclei

In fluorescence microscopy,?DAPI (4′,6-diamidino-2-phenylindole) and Hoechst 33342 are the two most popular counterstaining dyes to visualize nuclei and chromosomes. The reason for that is both DAPI and HOE can bind to DNA AT-rich regions in the minor groove and?their fluorescence increases upon binding.

DAPI & HOE have a slight difference, though:?

DAPI has difficulties penetrating living specimens and requires cell fixation and or permeabilization for better staining; Hoechst 33342 (HOE) is lipophilic and, therefore cell-permeant molecule used for living samples.

You can use DAPI properties to your advantage if trying to detect dead cells vs living cells or studying apoptosis-related membrane depolarization. As for staining protocols, keep in mind that when using higher DAPI staining concentrations (e.g., with living samples), DAPI might change its binding properties and become toxic for cells (you can recognize it when the nucleus shows abnormal granulated morphology and "patchy" signal).

What is cross-talk (bleed-through) in microscopy fluorescence imaging

We often use nuclear counterstaining simultaneously with other fluorescent probes and/or proteins in multi-color experiments. A very common situation would be when DAPI or HOE are combined with any green probe (e.g., Alexa Fluor 488, EGFP) and/or red (mCherry). This scenario can cause a?dye crosstalk (or bleed-through) [Fig. 3].

Crosstalk happens for two reasons:

• dyes excitation/emission spectra overlap

• dye photoconversion occurs

In the first scenario, we can correct it when carefully selecting and combining multiple fluorescent probes, which spectra DON'T overlap, matching the properties of the in-house microscope (available lasers, filters, etc.); and applying sequential scanning (image them individually one after another, longer wavelength to shorter wavelength) in digital microscopy.

What is a dye photoconversion in fluorescence microscopy imaging

When exposed to a specific light, as a result, the spectral properties of a fluorophore can change. For example, dyes DAPI and HOE can undergo UV-induced?photoconversion and become green- and red-emitting fluorophores. A very intuitive figure illustrates DAPI photoconversion below [Fig. 4].

In practice, when you use UV-excitation to check the nuclei in the eyepiece first, and shortly after you check the remaining eGFP/mCherry using green/red excitation light, you might notice a dimmer signal resembling the DAPI nuclear pattern in the green or red channels, respectively. Voila! You caused DAPI/Hoechst photoconversion and also collected artifacts interfering with eGFP/mCherry-specific signals.

General recommendations to avoid DAPI & HOE photoconversion

Luckily, the second scenario can also be prevented, simply by following these general recommendations:

• before switching to DAPI/HOE, image green fluorescence first (or red in case of mCherry)
• if the sample was exposed to UV light first, then move your sample to an unexposed region before imaging the green (or red) channel
• avoid using glycerol-based mounting medium supplemented with DAPI or HOR as a counterstain
• when using epifluorescence to focus on your sample in the eyepiece, try to use other than DAPI/HOE signals

Lastly, ALWAYS prepare single-dye/probe and no-dye/probe controls, especially when checking your experimental conditions during a test run. Simply, separate the samples?

• stained with each probe by itself (e.g., with fluorescent stains or fluorescent-labeled antibodies)

or

• expressing single-color fluorescent protein (especially if your goal is to check co-expression or co-localization)

and use designed imaging settings to check if an non-specific signal bleeds through into the other, not designated, channel.

Useful resources

I like this tool to check the fluorescence spectra or design and plot multi-color experiments:?https://app.fluorofinder.com/dyes

As for fluorescent tags/proteins, this is a great tool available online: https://www.fpbase.org/

Cross-talk also can be corrected after data was collected. I prefer using SVI crosstalk corrector tool is available from Scientific Volume Imaging Huygens commercial software.?

Disclaimer: I would like to clarify that I have not been involved in any paid partnerships nor am I affiliated with the companies mentioned in this article.

References:

?urek-Biesiada D. et al. (2013). UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry, 83A: 441–451. doi:10.1002/cyto.a.22260

Karg T.J. and Golic K.G. (2018). Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation. ?Chromosoma 127 (2): 235–245.

Nuclear stains: Biotinum

Tech tip: Biotinum

What is crosstalk: SVI Huygens

DAPI profile: FluoroFinder

Cell cycle analysis: DNA binding regions

Hoechst 33342 profile: FluoroFinder

Labeling nuclear DNA with Hoechst 33342

How can I access the quality of the nucleus?