Beyond being the core control center for the cell, the nucleus is one of the most important targets in bioimaging research.1 For example, looking at the nucleus can tell researchers about cell viability, DNA damage, cell cycle status, and localization of nuclear molecules. Fluorescent imaging allows researchers to peer into a cell’s inner workings, shedding light on nuclear processes and beyond.2 Although scientists have developed many different fluorescence-based tools for visualizing the nucleus, deciding which reagents will work best for a given experiment can pose challenges. Increasingly, researchers turn to innovative stains and multiplexed assays to fine-tune nuclear fluorescent imaging.

Blue fluorescent dyes such as DAPI and Hoechst have long been the reagents of choice for nuclear imaging, but these dyes face imaging artifact challenges.
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Feeling Blue: DAPI and Hoechst Dyes
Scientists often use blue fluorescent dyes such as diamidino-2-phenylindole (DAPI) and Hoechst for nuclear labeling.2 These DNA-specific dyes bind the minor-groove of double-stranded DNA and offer simplicity and cost-effectiveness when staining live or fixed cells.1 DAPI and Hoechst dyes have minimal fluorescence in solution but become brightly fluorescent upon binding to DNA, can be used to stain cells without a wash step, and provide stable, low toxicity staining in most cell types.3 Since their inception in the 1970s, DAPI and Hoechst have been the nuclear dyes of choice for researchers performing cellular imaging assays, but these blue counterstains face limitations that scientists must address when designing fluorescence-based experiments in the 21st century.4
Artifact Challenges: Nuclear Dye Limitations
Nonspecific staining
DNA-staining dyes such as propidium iodide (PI) and DAPI commonly exhibit nonspecific binding with RNA due to similar base-pairing behaviors in DNA and RNA, and both molecules exhibit minor grooves within their secondary structures. Moreover, a small quantity of mitochondrial DNA exists outside of the nucleus, which can yield non-nuclear staining and nonspecific readouts.2
Channel restrictions
DAPI and Hoechst nuclear counterstains make use of the blue fluorescence channel, which is frequently avoided for detecting other targets because of high intrinsic background fluorescence in cells and tissues.4 Additionally, depending on the cell type and assay, relying on blue fluorescence for nuclear detection can be problematic because this channel necessitates high-energy UV laser excitation. This can potentially cause artifacts such as DNA damage and cell death that may confound experimental interpretation.2 Staining thick tissue sections with blue dyes such as Hoechst also has limitations related to light scattering and loss of signal intensity due to blue light’s short emission wavelength.5
Cross-talk
The fluorescent signal of DAPI and Hoechst dyes can bleed through into the green channel because of their broad fluorescence emission, leading to imaging artifacts.4 This cross-talk between fluorescence channels is an important concern for multicolor imaging experiments, potentially resulting in artifactual co-localization of signals.6
UV photoconversion has been more recently recognized as an additional source of fluorescence cross-talk, emphasizing the need for careful nuclear labeling reagent selection.4 Blue dyes can undergo spectral shifts when exposed to UV light, which is commonplace when researchers begin epifluorescence microscopy analysis with UV excitation to locate blue-stained nuclei.4,6 During this process, DAPI and Hoechst can emit fluorescence not only in their primary blue channel but also in the green and red channels.7-9 Although this signal is weaker than the expected blue fluorescence, it may interfere with the detection of low-abundance fluorescent probes or labeled antibodies. Furthermore, UV-induced photoconversion may be enhanced by glycerol-based mounting media, which often include DAPI as a counterstain.6
Beyond Blue Dyes: Multiplexing with Nuclear Stains
There is growing demand for new nuclear labeling options, including long-wavelength fluorescent dyes in the red and near-infrared region (NIR).2 Biotium’s NucSpot® Nuclear Stains are convenient alternatives for researchers facing photoconversion issues or those whose experimental design requires options beyond DAPI and Hoechst.
These bright and sensitive nuclear counterstains with narrow emission ranges were developed to address channel bleed-through from DAPI and Hoechst that is caused by a broad emission spectrum and photoconversion. NucSpot® Nuclear Stains offer scientists more flexibility with a wider selection of nuclear imaging colors, from green to NIR. The stains are cell-membrane impermeant and designed for nuclear-specific staining in fixed cells or selectively staining dead cells in live culture. They are available in 7 different colors for flexible multiplexing: FITC, Cy®3, PE, Texas Red®, Cy®5, APC, Cy®5.5, and Cy®7 channels. NIR-emitting dyes like NucSpot® NIR dyes are more advantageous for tissue staining because they are less susceptible to light scattering and the intrinsic background fluorescence of tissues than short-wavelength blue emitting dyes.
Scientists used NucSpot® Nuclear Stains to stain PFA-fixed, Triton® X-100 permeabilized HeLa cells in PBS and imaged the nuclei by confocal microscopy without a wash step. All stains were used at 1X concentration except for NucSpot® 750/780, which was used at 5X concentration in order to image using 640 nm excitation.
Image provided by Biotium
In addition, unlike other green or far-red nucleic acid dyes that stain both the nucleus and cytoplasm, NucSpot® Nuclear Stains selectively stain the nucleus in fixed and permeabilized cells without the need for washing or RNase treatment. NucSpot® Nuclear Stains can also be used for selective staining of dead cells in unfixed cell cultures for analysis by flow cytometry or fluorescence imaging. Several stains can be continuously incubated with cells for multi-day imaging, further expanding researchers’ toolkits for nuclear fluorescence imaging. Additionally, Biotium’s NucSpot® Live Cell Nuclear Stains are cell membrane-permeant DNA dyes with similar strengths that specifically stain nuclei in live or fixed cells. They have excellent DNA specificity without the need for a wash step, allowing researchers to perform low toxicity live cell nuclear imaging in green or far-red fluorescence channels.
- Hu C, et al. Recent advance in nucleus-targeted fluorescent probes for bioimaging, detection and therapy. Chemosensors. 2023;11(2):125.
- Li Z, et al. Next-generation red ultra-bright fluorescent dyes for nuclear imaging and peripheral blood leukocytes sorting. Chem Sci. 2024;15(42):17524-17532.
- Hoechst & DAPI Staining Protocols – Cell Staining with Hoechst or DAPI Nuclear Stains. Biotium. Accessed December 13, 2024.
- Roberts LM. Tips for avoiding artifacts from UV photoconversion of DAPI. Micros Today. 2019;27(5):18-24.
- Han X, et al. Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex. Nat Commun. 2024;15(1):6648.
- Tech Tip: Avoiding Artifacts from UV Photoconversion of DAPI and Hoechst. Biotium. Accessed December 13, 2024.
- Piterburg M, et al. Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation. J Microsc. 2012;246(1):89-95.
- Jež M, et al. The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem. Histochem Cell Biol. 2013;139(1):195-204.
- Zurek-Biesiada D, et al. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A. 2013;83(5):441-51.
