Blue Nuclear Label Red-shift

Sometimes the bleed-through of the blue nuclear label into the green channel seems higher than usual. There is a real phenomenon behind this increase; it's called photoconversion.

It is known for the blue nuclear labels DAPI, Hoechst and Vybrant DyeCyle Violet, but can happen to other labels too. Red-shifts of up to 80 nm have been observed in real samples, e.g. cy5-Beta-catenin on polyvinylidene (PVDF) membrane.

Note that the in-solution values stated in reference may differ from spectra in other conditions.

DAPI, Hoechst 33258 and Vybrant DyeCyle Violet Exhibit Photoconversion

• Photoconversion by exposure to UV light
  • The 405 nm laser is far enough from the UV to not cause this
• Photoconverted derivatives have low photostability
  • Bleached easily
  • Photobleaching of DAPI & Hoechst 33258 recovers 50% in 60 min
  • Photoconverted derivatives bleach at the same rate
• The photoconversion is reversible
  
  • After the photoconversion reversal an equilibrium state lasts for at least 2 more hours
• The photoconversion is dose dependent
  • The dose response of DAPI is stronger
  • 4x increase in 100 s
  • (Hoechst 2x in the same time)
• Hydroxen peroxide induces similar photo-oxidation
  • Red-shift
  • Takes 4 h for Hoechst
  • 1.5 h for DAPI
  • Reversible

Spectral Characteristics of the Red-shifted Label

• Excitation at 450-490 nm
• Emission at 480-600 nm
  • Maxima at 540 nm

Too High Label Concentrations Can Diminish Signal

• High concentrations of DAPI & Hoechst 33258 diminish blue signal
• Self-quenching
• Signal in RNA increased

DAPI Spectral Shift

• DAPI binding with DNA is solution specific
• It can change from groove-binding to intercalation
• It can also induce polymer-dye adducts
• Emission maximum blue-shifts when complexed with DNA

DAPI Spectral Characteristics

• Excitation maxima at 364 nm
• Emission maximum at 454 nm

DAPI Red-shift Conditions

• DAPI also binds to RNA
• Emission maximum red-shifts when bound to RNA
  
  • Binding to RNA minor groove
  • Complexation with polyA-polyU
• Dissociation 100 times faster than from DNA
• High dye/phosphate ratio (1:10) reveals a new binding form
• New emission maximum 540 nm
• Binding of DAPI to sites in polynucleotides in proximity to previously bound DAPI
  
  • Disappears if background electrolyte is 0.4 M KCl
  • Distrupts DAPI-DAPI electrostatic interactions
  • DAPI-polyadenylic acid (polyA)
  • DAPI-Polyphosphate (polyP)
    • Excitation 415 nm
    • Emission 550 nm
  • Inositol phosphates (IP5 & IP6)
    • Emission 550 nm
  • Heparin
    • Emission 550 nm
  • Amorphous calcium phosphate (ACP)
    • Emission 550 nm
    • Not Hydroxyapatite (HAp)
• “Red-shifted DAPI fluorescence is not due to specific substrate chemistry, but indicates the presence of a high density of negatively-charged surfaces or molecules that locally concentrate DAPI. This local, increased DAPI concentration enables DAPI–DAPI interactions and its resultant red-shifted fluorescence.”
• High concentrations of glycerol (as mounting media) are proportional to the level of DAPI photoconversion
• DAPI photoconversion rate has two components
  • Fast half-life < 10 s
  • Slow half-life > 60 s
  • Both are UV illumination intensity dependent (non-linear)

Hoechst 33258 Red-shift

• Exposure to UV causes both photobleaching and photoconversion
  • New emission maxima 540 nm
• Protonation of the dye
  • Also by exposure to hydrogen peroxide
• Similar properties in acidic environments (pH 0.5-3.0)
  • Quantum yield drops 80-fold
• QY increased 20-fold in pH 4.5
• Acid treatment reversible
• Equilibrium at 60 min past UV exposure
• Red-shifted protonated form shows up in nucleoli
  • RNA-binding
    • Excitation 369 nm
    • Emission 437 nm

Hoechst Spectral Characteristics

• Excitation 355 nm
• Emission 465 nm

Vybrant DyeCyle Violet

 Vybrant DyeCyle Violet also exhibits the photoconversion red-shift of it's spectra.

Mitigating the Red-shift

• Minimize UV intensity when imaging the blue nuclear labels.
  • Total dose or exposure time
• Use low label concentration
• Use low glycerol concentration in mounting media.
• Acquire blue nuclear channels last to minimize green channel false positives
• Use alternate nuclear dyes like DRAQ5 or RedDot which fluoresce in the far red region

Sources

• “A cautionary (spectral) tail: red-shifted fluorescence by DAPI–DAPI interactions”; Sidney Omelon, John Georgiou, Wouter Habraken; Biochemical Society Transactions Feb 09, 2016, 44 (1) 46-49; DOI: 10.1042/BST20150231
• “UV-induced Spectral Shift and Protonation of DNA Fluorescent Dye Hoechst 33258”; Dominika Żurek-Biesiada & Piotr Waligórski & Jurek W. Dobrucki; J Fluoresc (2014) 24:1791–1801; DOI 10.1007/s10895-014-1468-y
• “UV-Activated Conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle Fluorescent Dyes into Blue-Excited, Green-Emitting Protonated Forms”; Dominika Zurek-Biesiada, Sylwia Ke˛dracka-Krok, Jurek W. Dobrucki; Cytometry Part A (2013) 83A: 441-451; DOI: 10.1002/cyto.a.22260
• “High UV Excitation Intensity Induces Photoconversion of DAPI During Wide-Field Microscopy”; Stefan Rodic, Claire Brown, Erika (Tse-Luen) Wee; McGill Science Undergraduate Research Journal 9.1 (2014)
• Tanious, Farial A., et al. "DAPI (4', 6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites." Biochemistry 31.12 (1992): 3103-3112.
• “Inositol phosphates induce DAPI fluorescence shift.”; Kolozsvari, Parisi, Saiardi; Biochem J. 2014 Jun 15;460(3):377-85.; doi: 10.1042/BJ20140237.
• “Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342”; Timothy W. Petersen, Sherrif F. Ibrahim, Alan H. Diercks, and Ger van den Engh; Cytometry Part A (2004): 173-181; DOI: 10.1002/cyto.a.20058