Fluorescent Probes & Labeled Lipids: FAQ Guide

Fluorescent probes are powerful tools for visualizing and quantifying biological processes. At Avanti Research™, we provide a wide range of fluorophore families and fluorescently labeled lipids to support imaging, trafficking, and live-cell studies.

Below are answers to the most frequently asked questions about our fluorescent probes.

What are fluorescent probes and why are they used?

Fluorescent probes (fluorophores) are molecules that absorb light at one wavelength and emit at a longer wavelength. When attached to lipids or biomolecules, they allow sensitive detection of:

• Membrane dynamics

• Lipid trafficking

• Protein localization

• Live-cell imaging

Because fluorescence can be detected at very low concentrations, the right probe enables minimal biological perturbation while maintaining a strong signal-to-noise ratio.

Which fluorophore families do you offer?

NBD (Nitrobenzoxadiazole): Green-emitting, environment-sensitive probe; commonly used in uptake and trafficking assays.

TopFluor™ (BODIPY-like): Bright, photostable, and minimally disruptive; available in green or orange-red (TMR) variants for multi-color imaging.

Cyanines (Cy3/3.5/5/5.5/7/7.5): Cover visible to near-infrared (NIR); ideal for multiplex imaging and FRET due to distinct spectral windows.

SquareFluor™ (Squaraines): Highly photostable, long-wavelength probes; perfect for extended imaging sessions and demanding environments.

Lissamine Rhodamine (LR): Orange-red probe frequently used for membrane and liposome labeling.

DPH (1,6-diphenyl-1,3,5-hexatriene): Hydrophobic probe for membrane order and viscosity measurements.

Pyrene: Excimer-emitting probe used to study membrane fusion and lipid mixing.

Laurdan & Organelle-Targeted Laurdan: Polarity-sensitive probes; available as ER-, Golgi-, lysosome-, mitochondria-, and plasma membrane–specific variants.

Fluorescein (FITC/CF derivatives): Green probes widely used in protein and lipid labeling, compatible with standard GFP filter sets.

How do I choose the best fluorophore for my application?

• Instrument compatibility: Match excitation/emission to your filters and detectors.

• Multiplexing: Select well-separated spectra (e.g., green + far-red) to reduce bleed-through.

• Photostability: For long time-lapse imaging, choose TopFluor™, Cyanines, or SquareFluor™.

• Environmental sensitivity: Use NBD or Laurdan for polarity/packing studies; use DPH for anisotropy.

• Biological fidelity: TopFluor™ labels closely mimic native lipid behavior.

What functional chemistries are available for probe conjugation?

• NHS esters: Label primary amines (proteins, peptides).

• Maleimides: Selective for thiols (cysteines).

• Azides/DBCO: Enable click chemistry (Cu-catalyzed or copper-free).

• Free acids: Can be coupled via amide bond formation.

Do you carry fluorescently labeled lipids?

Yes — Avanti Research™ offers a broad range of fluorescent lipid standards:

• Phospholipids (PE, PC, PS, PI, PA, cardiolipin)

• Sphingolipids (ceramides, sphingomyelin, glycosphingolipids)

• Sterols (cholesterol derivatives such as NBD- and TopFluor™-cholesterol)

• Glycerolipids (mono-, di-, triacylglycerols with fluorescent labels)

• PEG-lipids (DSPE-PEG-N-Cyanine, DSPE-PEG-TopFluor™)

How should fluorescent probes be stored and handled?

• Stock solutions: Prepare in dry solvents (DMSO, ethanol, chloroform).

• Aliquots: Store at −20 °C to avoid repeated freeze–thaw.

• Light protection: Always shield from light.

• Working levels: Use the lowest effective probe concentration.

• Buffer compatibility: Avoid quenching agents or conditions that degrade reactive groups.

Are these probes live-cell compatible?

Yes — many probes such as NBD, TopFluor™, and Cyanines are commonly used in live-cell imaging.

• Validate for your specific cell type.

• Minimize light dose to reduce phototoxicity.

• Use anti-fade agents for extended imaging sessions.

Do you offer organelle-targeted or specialty probes?

Yes, including:

• Organelle-specific Laurdan derivatives (ER, Golgi, lysosome, mitochondria, plasma membrane)

• Trifunctional lipids with alkyne, diazirine, and coumarin groups

• Dual-labeled lipids with fluorophore + quencher

• Fluoro-azasterol for sterol metabolism studies

• Fluorescent PEG-lipids and sterols for delivery and tracking applications

When should I use NBD-cholesterol vs TopFluor™-cholesterol?

• NBD-cholesterol: Suited for fast, high-throughput readouts (plate reader, flow cytometry); less photostable.

• TopFluor™-cholesterol: Best for live-cell confocal and time-lapse studies; brighter, more photostable, and closely mimics native cholesterol.

Why use Cy5/Cy7 in thick samples?

Far-red/NIR probes like Cy5 and Cy7 are recommended for spheroids, organoids, and tissues (>50 µm):

• Deeper penetration

• Reduced autofluorescence

• Cleaner multiplexing with GFP/mCherry

How can I prevent liposome leakage at higher dye loadings?

• Keep probe levels low (0.1–0.5 mol%).

• Use cholesterol (20–40 mol%) to stabilize membranes.

• Remove free dye via size-exclusion chromatography (SEC).

• Match fluorophore acyl chains to host lipids.

• Validate with leakage assays (e.g., carboxyfluorescein release).

Do you provide custom synthesis or support?

Yes. Avanti Research™ offers custom conjugation services and scientific consultation to help you:

• Select the right fluorophore family

• Choose labeling position and chemistry

• Design tailored lipid probes for your experiments

Final Takeaway

From NBD and TopFluor™ to Cyanines, SquareFluor™, and organelle-targeted probes, we provide the fluorescent probes and labeled lipids researchers need for membrane dynamics, lipid trafficking, live-cell imaging, and more!

Explore our full catalog of fluorescent lipids and probes today to find the best fit for your research.