Nanolive Experiment Preparation Protocol (Analysis & Translation)
Document type: Nanolive official sample preparation SOP
Original manufacturer: Nanolive SA (Lausanne, Switzerland) — www.nanolive.ch
Structure: 6 chapters / 13 pages / 2 figures (cover + appendix RI table)
1. What does this Protocol solve? 🧪
Nanolive's 3D Cell Explorer + STEVE delivers high-quality RI refractive index data, but only if sample preparation is correct. Any sample prep mistake leads to data failure — optical path attenuation, signal blur, segmentation drift are common consequences.
This Protocol provides customers/operators with step-by-step sample preparation SOP to ensure:
- ✅ Correct instrument operating environment (avoid water mist/vibration)
- ✅ Compatible sample consumables (glass bottom is hard requirement)
- ✅ Reasonable cell confluency (< 80% optimal)
- ✅ Imaging medium refractive index matching (RI 1.33-1.44)
- ✅ Standardized fixed/live cell operations (avoid methanol fixation)
- ✅ Sample cleanliness达标 (no debris/fingerprints/residue)
2. Instrument installation environment 🏠
2.1 Equipment placement (must-read)
| Item | Requirement |
|---|---|
| Space | ≥ 1 m² platform (recommend slightly larger) |
| Surface | Level, sturdy, vibration-resistant |
| Surroundings | Avoid centrifuges and other high-vibration equipment |
| Environment temp | Recommend ≥ 25°C (prevent water mist on workstation/lens) |
2.2 Darkroom (optional)
- Fluorescence imaging: darkroom better (improve fluorescence SNR)
- No darkroom: can still image (but quality affected)
2.3 CO₂ gas (optional)
- With CO₂ tank/line: extend live-cell observation time
- Without CO₂: can still observe (short-term)
3. 5 sample preparation considerations ⚠️
Any single non-compliance significantly affects image quality.
- ⚠️ Cells must be seeded on glass bottom (coverslip or glass-bottom dish) — plastic bottom unusable
- ✅ Suitable for any eukaryotic/prokaryotic cells + thin tissue sections (5-10 μm)
- ⚠️ Cell confluency has limits: monolayer/multilayer both OK, but cannot exceed observable max thickness 30 μm
- ⚠️ Medium RI should be close to living material (≈ 1.35) — PBS optimal; phenol red-containing also works but liquid volume must be controlled
- ⚠️ Optical surfaces must be clean (lens, workstation cover, dish holder)
4. Specifications overview 📋
4.1 Spec table 1 — Slides
| Condition | Nanolive spec | Note |
|---|---|---|
| Coverslip thickness | 170 ± 20 μm | Recommend Hirschmann (hirschmannlab.com) |
| Max observable sample thickness | 30 μm | Recommend 5-10 μm |
| No paraffin embedding | — | Paraffin interferes |
| Aqueous medium RI | 1.33-1.44 | — |
4.2 Spec table 2 — Medium
| Condition | Spec | Note |
|---|---|---|
| Glass-bottom dish diameter | ≥ 35 mm | Complete ibidi compatible list: nanolive.ch/wp-content/uploads/nanolive-ibidi-labware.pdf |
| Max observable sample thickness | 30 μm | Same as Table 1 |
| Max optically clear medium volume | ~1.5 mL (35 mm dish) | Depends on optical properties |
| Max slightly scattering medium volume | ~1 mL (35 mm dish) | Verified: Agarose hydrogel works |
5. Sample preparation — consumables + medium + SOP 🔬
5.1 Compatible consumables
Manufacturer-recommended + domestically verified consumables
5.1.1 Microscope slides and coverslips
- Bottom thickness 130-170 μm
- Recommended slide: Slide SuperFrost (BioSystems)
- Recommended coverslip: Hirschmann (hirschmannlab.com)
- Domestic option: some biosharp confocal dishes verified experimentally workable
5.1.2 ibidi labware (complete list)
| Type | Models |
|---|---|
| Multi-well chambers | µ-Slide 2 Well / µ-Slide 18 Well – Flat |
| Channel Slides | µ-Slide I Luer / y-shaped / III 3in1 / III 3D Perfusion / VI – Flat / VI 0.5 Glass Bottom |
| Microscopy dishes | µ-Dish 35 mm low / high / high Glass Bottom / low Grid-500 / high Grid-500 / high Grid-500 Glass Bottom |
Remove top cover during 3D image acquisition (avoid top cover interfering with optical path)
5.2 Cell confluency
- No explicit limit, but single cells/low confluency typically yield best image quality
- Monolayer/multilayer cells observable, but cannot exceed 30 μm max thickness
- Exceeding thickness limit → low-quality signal alarm or fault message
5.3 Imaging medium (core chapter)
5.3.1 Recommended medium
| Medium | RI value | Use |
|---|---|---|
| PBS solution | 1.334 | ✅ Universal first choice |
| FluoroBrite DMEM (Thermo A1896701) | 1.33+ | Live-cell imaging specific |
| Tris buffer | 1.33+ | OK |
| Phenol red-containing medium | 1.33+ | ⚠️ Experimentally verified only (not recommended) |
⚠️ Key limits:
- Liquid height cannot exceed half of dish height (too high affects image quality)
- Plant/fungal cells typically have RI > 1.44, need glycerol+PBS adjustment to match
- Increased glycerol concentration is toxic to live cells
5.3.2 Coverslip imaging medium (mounting solutions)
| Mounting solution | RI value | Note |
|---|---|---|
| PBS solution | 1.334 | ✅ First choice |
| Fluoromount-G Fluorescent Mounting (Southern Biotech) | 1.389 | Fluorescent mounting |
| ibidi mounting medium | 1.42-1.44 | — |
| 75% glycerol/distilled water | 1.44 | — |
| ProLong® Gold Fresh | 1.39 | — |
| ProLong® Gold Cured 1 day | 1.40 | — |
| ProLong® Gold Cured 4 days | 1.44 | — |
⚠️ OCT/paraffin not fully transparent, interfere with acquisition — must remove before mounting
⚠️ Non-aqueous mounting media generally incompatible
5.4 4 sample preparation & imaging general rules
5.4.1 Adherent live or fixed cells (ibidi 35 mm low-edge dish)
For fixed cells (7 steps):
- Coat with minimum coating material (ibidi AN08 protocol)
- Seed cells per ibidi protocol
- Culture to desired confluency (recommend < 80%)
- Wash with warm (37°C) PBS, within 30 seconds to next step (reduce cell stress)
- Fix with 1 mL 4% PFA, room temp 15 min, wash with PBS 3 times
- ⚠️ Never use methanol!
- Cover with recommended medium (mounting solution)
- Image or store at 4°C
For live cells (5 steps):
1-3 same as fixed cells
4. Replace with pre-warmed live-cell imaging medium
5. Image or store at 37°C incubator
⚠️ After removing cover, can directly observe in dish
5.4.2 Non-adherent cells (bacteria, yeast, RBCs, etc.)
- Add recommended medium volume
- Add a few drops of suspended cells at dish center
- Sediment for 10 minutes (imaging at this stage has poor quality and high failure rate)
- Minimize vibration (let cells stabilize at bottom)
- Image
Fixation aids (prevent drift):
- Protoslo: flagellates/ciliates
- Cell-tak: general purpose
- Poly-D-Lysine: suspended blood cells
5.4.3 Fixed cells on coverslip (11 steps)
- Sterile coverslip at dish bottom
- Coat
- Seed cells
- Culture to desired confluency
- Wash with warm PBS, within 30 seconds to next step
- 2 mL 4% PFA, room temp 15 min
- 2 mL PBS wash 3 times
- Imaging spacer + one drop of mounting solution
- Remove coverslip from PBS, dry with tissue paper at side (warning: do NOT place tissue on top)
- Place coverslip on imaging spacer (cells facing slide)
- Image or store at 4°C
Sealing (2 options):
- ✅ Recommended: imaging spacer tape (SS1X9 SecureSeal, Grace Biolabs #654002)
- ⚠️ Second choice: nail polish (often spills onto coverslip)
5.4.4 Tissue sections
Key conditions:
- ⚠️ OCT/paraffin not fully transparent → remove before mounting
- ✅ 5-10 μm section optimal (max thickness < 30 μm)
- ✅ Aqueous mounting medium, RI 1.33-1.44
- ⚠️ Inverted observation (coverslip facing objective)
6. Sample cleanliness 🧹
Sample cleanliness is key to image quality.
Because of rotating light source system, light beam may pass through debris (floating or out-of-focus):
- Coverslip: use lens tissue (Thorlabs #MC-50E) + few drops ethanol, gently wipe (remove dust/fingerprints, no cotton lint)
- Glass-bottom dish: wash with PBS 3 times to remove dead cells/debris, then add mounting medium + same lens tissue cleaning for external surface (the one contacting objective)
⚠️ Cell dimensions should be smaller than microscope FoV and depth of field (see user manual or nanolive.ch/hardware/ for specifications)
7. Accessories — Live Cell Workstation 🧫
Nanolive's 3D Cell Explorer + Live Cell Workstation together enable days of non-invasive 4D long-term imaging under physiological conditions.
Workstation core functions:
- Temperature control
- Humidity control
- pH control
💡 Detailed partner product list: nanolive.ch/accessories/
8. Appendix — Glycerol/PBS Refractive Index (RI) Reference 📊
Fig 1 — Glycerol/PBS solution RI value reference curve (x-axis: glycerol concentration %; y-axis: RI). This curve lets customers precisely prepare imaging medium with specific RI values.
- Glycerol reference: Sigma Aldrich G5516
- Measurement instrument: AR4 series Abbe refractometer (KRUSS OPTRONIC)
💡 Usage points:
- Find target RI → look up glycerol concentration from curve → prepare solution
- Increased glycerol concentration is toxic to live cells → use only when necessary
- Mammalian cell RI range 1.33-1.44 (most cases PBS suffices)
9. Sales perspective customer Q&A 💼
| Customer question | Answer |
|---|---|
| "My dish is plastic bottom, can I use it?" | No. Must be glass bottom. Some biosharp confocal dishes verified experimentally workable. |
| "My cell confluency is high (> 80%), can I use it?" | Yes, but cannot exceed 30 μm thickness. Recommend < 80% confluency for optimal quality. |
| "Can I fix with methanol?" | Absolutely not! Use 4% PFA (paraformaldehyde). |
| "Will phenol red in medium interfere?" | Slightly, but can image. Recommend FluoroBrite DMEM or PBS. |
| "Do I need to adjust RI with glycerol?" | Generally no (mammalian cell RI ≈ 1.35). Only needed for high-RI samples like plant/fungal. |
| "What section thickness?" | 5-10 μm optimal, max < 30 μm. |
| "How to do days-long live-cell imaging?" | Pair with Nanolive-recommended Live Cell Workstation (temp + humidity + pH three-control). |
| "Recommended consumables?" | ibidi µ-Dish 35 mm series most common (manufacturer-verified). Coverslip: Hirschmann 170 μm. |
| "How to fix suspended cells/blood cells?" | Use Protoslo (flagellates) / Cell-tak (general) / Poly-D-Lysine (blood cells). |
10. Key resources 🔗
- Nanolive official: http://nanolive.ch
- Nanolive accessories: http://nanolive.ch/accessories/
- Nanolive hardware specs: http://nanolive.ch/hardware/
- Nanolive ibidi compatible list: http://nanolive.ch/wp-content/uploads/nanolive-ibidi-labware.pdf
- ibidi dish coating AN08: http://ibidi.com/img/cms/support/AN/AN08_Coating.pdf
- ibidi 35 mm low dish protocol: http://ibidi.com/img/cms/products/labware/open_standard/D_801XX_Dish_35mmlow/IN_8013X_Dish35mm_low.pdf
- ibidi glass-bottom 35 mm dish: http://ibidi.com/labware/185-glass-bottom-dish-35-mm.html
- Hirschmann coverslips: http://www.hirschmannlab.com/
- Customer registration + software download: https://www.nanolive.ch/register
📥 Original PDF
📄 Nanolive Experiment Preparation Protocol PDF (Chinese version, 13 pages, 576 KB)
Contact｜Li Dongwei (李东伟)
📞 18045130660
🌐 Website: https://www.biokingdom.top