5-HMSiR-Hoechst: Live-Cell STED Super-Resolution Imaging Guide

5-HMSiR-Hochest is a fluorogenic, cell-permeable silicon-rhodamine–Hoechst conjugate designed for live-cell STED super-resolution imaging of chromatin, combining the high DNA-binding selectivity of the Hoechst motif with the far-red, photostable fluorescence of HMSiR.

Product SKU

H1450374

Also known as

HY-D2222 (MCE reference)

Fluorophore class

Silicon-rhodamine (SiR) derivative — far-red fluorogenic dye

Targeting moiety

Hoechst (bisbenzimidazole) — selective minor-groove DNA binder

Recommended microscopy modality

STED (stimulated emission depletion) super-resolution microscopy

Cell compatibility

Live-cell compatible; cell-permeable without fixation

Principle and Mechanism

5-HMSiR-Hoechst is a two-component molecular probe consisting of a hydroxymethyl silicon-rhodamine (HMSiR) fluorophore conjugated to a Hoechst bisbenzimidazole DNA-binding unit. In aqueous solution the dye exists predominantly in a non-fluorescent, ring-closed (spirolactone) form, making it fluorogenic: fluorescence is activated upon binding to double-stranded DNA in the AT-rich minor groove, where the equilibrium shifts toward the ring-open, highly emissive form. This fluorogenic behaviour minimises background fluorescence from unbound probe and eliminates the need for wash steps, greatly simplifying live-cell workflows.

The HMSiR fluorophore emits in the far-red region (excitation ~650 nm, emission ~670 nm), well separated from common cellular autofluorescence and compatible with the depletion lasers (typically 775 nm) used in commercial STED systems. The Hoechst targeting unit provides selective, high-affinity binding to chromatin, enabling nanoscale visualisation of nuclear DNA architecture in living cells.

Reagents and Equipment

• 5-HMSiR-Hoechst (H1450374) — prepare a stock solution (e.g., 1–10 mM) in anhydrous DMSO; aliquot and store at −20 °C protected from light.
• Appropriate live-cell imaging medium (phenol-red-free, e.g., FluoroBrite DMEM or equivalent).
• Cell culture consumables: 35 mm glass-bottom dishes (No. 1.5 coverslip, 0.17 mm) or equivalent high-NA compatible chambers.
• STED microscope equipped with a ~650 nm excitation laser and ~775 nm CW or pulsed depletion laser (e.g., Leica SP8/SP8 LIGHTNING STED, Abberior STEDYCON, or equivalent).
• High-NA oil-immersion objective (≥1.4 NA recommended).
• Temperature and CO₂ control stage insert for live imaging.
• DMSO (anhydrous, ≥99.9%) for stock preparation.

Note: Avoid freeze–thaw cycles. Protect all solutions from light at all times.

Step-by-Step Staining and Imaging Protocol

1. Cell Preparation

Seed cells on glass-bottom dishes 24–48 h before imaging to achieve 60–80% confluency. Aspirate culture medium and replace with pre-warmed (37 °C), phenol-red-free imaging medium.

2. Probe Dilution and Staining

Dilute the 5-HMSiR-Hoechst stock to a working concentration in imaging medium. A starting concentration in the range of 100–500 nM is typical for live-cell STED; optimise empirically for your cell type and STED system. Add the working solution directly to cells and incubate for 30–60 min at 37 °C, 5% CO₂. Because the probe is fluorogenic, a wash step is generally not required, but one gentle rinse with imaging medium may be performed to further reduce residual background if desired.

3. STED Imaging Parameters

Transfer the dish to the pre-warmed microscope stage. Recommended starting parameters: excitation 650 nm (5–10% laser power), STED depletion 775 nm (50–100% depletion power, adjust to balance resolution and phototoxicity), detection window 660–720 nm, line or frame accumulation 4–8×. Use time-gated detection (0.5–6 ns gate) if available to further improve signal-to-background.

4. Acquisition and Live-Cell Considerations

Keep total acquisition time per field minimal to reduce phototoxicity. Monitor cell morphology (nuclear envelope integrity, mitotic figures) as viability indicators. For time-lapse experiments, reduce depletion power and increase frame intervals to extend cell viability over the experiment.

Troubleshooting

Observation	Likely Cause	Recommended Action
High cytoplasmic background	Excess unbound probe or incomplete ring-closure equilibration	Allow longer incubation; add one gentle wash with imaging medium
Weak or no nuclear signal	Working concentration too low; DMSO stock degraded	Increase probe concentration; prepare fresh stock from a new aliquot
Rapid photobleaching under STED	Depletion power too high; too many accumulations	Reduce STED power; use time-gating; limit accumulations
Cell rounding or detachment	Phototoxicity or probe cytotoxicity at high concentration	Lower probe concentration; reduce laser exposure; verify cell viability before imaging
Poor STED resolution improvement	Objective or coverslip mismatch; refractive index error	Confirm No. 1.5 coverslip; correct collar adjustment; use immersion oil matching system specs

Storage, Handling, and Safety

Storage: Store lyophilised or solid 5-HMSiR-Hoechst at −20 °C, desiccated and protected from light. DMSO stock solutions should be stored in tightly sealed vials at −20 °C; avoid repeated freeze–thaw cycles (use single-use aliquots).

Handling: Weigh and dissolve under subdued light. Use appropriate PPE (nitrile gloves, lab coat, safety glasses). DMSO enhances skin penetration of dissolved compounds — minimise skin contact with working solutions.

Stability: Lyophilised material is stable for ≥12 months under recommended storage conditions. DMSO stock solutions are stable for ≥6 months at −20 °C when stored as described. Aqueous working solutions should be freshly prepared and used within the same day.

Waste disposal: Dispose of all dye-containing waste in accordance with local regulations for chemical waste. Do not pour fluorescent dye solutions down the drain without verification of local permitting.

Frequently asked questions

Can 5-HMSiR-Hoechst be used with fixed cells in addition to live cells?

While the probe is optimised for live-cell imaging, it can be applied to paraformaldehyde-fixed, permeabilised cells. However, fixation may alter the equilibrium between the spirolactone and open forms and could affect signal intensity. Empirical optimisation of concentration and incubation conditions is recommended for fixed-cell applications.

Is a wash step required after staining with 5-HMSiR-Hoechst?

Due to the fluorogenic (wash-free) design of the probe, a wash step is generally not required. Fluorescence is substantially activated only upon DNA binding, keeping background low. A single gentle rinse with imaging medium can be added if residual cytoplasmic signal is observed.

What STED depletion laser wavelength is compatible with 5-HMSiR-Hoechst?

The HMSiR fluorophore is compatible with 775 nm continuous-wave (CW) or pulsed depletion lasers, which are standard on most commercial STED systems such as the Leica SP8 STED and Abberior platforms. A 650 nm excitation source is typically used for this far-red dye.

What is the recommended working concentration for live-cell STED imaging?

A starting working concentration of 100–500 nM is generally recommended; however, the optimal concentration depends on cell type, incubation time, and the sensitivity of the specific STED system. Always titrate the probe empirically to balance signal intensity, background, and cell viability.

Can this probe be multiplexed with other fluorescent labels?

Yes. The far-red emission (~670 nm) of 5-HMSiR-Hoechst is well-separated from green (e.g., GFP, ~510 nm) and orange-red (e.g., mCherry, ~610 nm) fluorophores, enabling multi-colour imaging. Ensure appropriate filter sets and laser lines are available on your STED or confocal system, and verify minimal spectral cross-talk empirically.

References

1. Uno SN, Kamiya M, Yoshihara T, Sugawara K, Okabe K, Tarhan MC, Fujita H, Funatsu T, Okada Y, Tobita S, Urano Y. A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Nature Chemistry. 2014;6(8):681–689. doi:10.1038/nchem.2002
2. Lukinavicius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T, Mueller V, Reymond L, Corrêa IR Jr, Luo ZG, Bharat T, Bharat TA, Rees EJ, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Hell SW, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T, Bharat TA, Bharat T. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chemistry. 2013;5(2):132–139. doi:10.1038/nchem.1546
3. Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z, Revyakin A, Patel R, Macklin JJ, Bhalla D, York AG, Bhatt RA, Singer RH, Lavis LD. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature Methods. 2015;12(3):244–250. doi:10.1038/nmeth.3256

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