Nanolive Digital Staining Training (Analysis & Translation)

Document type: Nanolive official digital staining training manual
Original manufacturer: Nanolive SA (Lausanne, Switzerland) — www.nanolive.ch
Structure: 5 chapters / 20 pages / 21 figures

1. Why need digital staining? 🔬

Live cells are transparent — no sufficient contrast under traditional microscopes. But cells have a valuable natural physical marker: Refractive Index (RI). RI shows how a medium affects light propagation; cell internal RI varies slightly due to composition differences.

Nanolive's 3D Cell Explorer measures RI point-by-point, providing 3D reconstruction of given volumes. Based on this info, organelle/cell differences can be distinguished — thanks to the digital stain process.

This document is a practical training guide containing:

✅ Step-by-step procedure for STEVE software digital staining
✅ Theory explanation (refractive index + RI gradient)
✅ Staining operation examples
✅ Golden advice (improve staining results, avoid typical errors)

💡 All training data available at nanolive.ch/downloads/data/, or via STEVE "Help > Get More Data" menu.

2. STEVE Software Digital Staining 8-Step Procedure 🎨

With Fig 1a-1l practical screenshots, follow step-by-step to complete single-cell/sub-cellular structure digital staining.

2.1 Step overview

Step	Operation	Figure
1	Select information-rich plane in Z stack panel	Fig 1b
2	Click "+" to add new stain	Fig 1c
3	Select name (optional)	Fig 1d
4	Select color	Fig 1e
5	Draw ROI on RI layer	Fig 1f
6	Stain overlays on RI layer	Fig 1g
7	Manipulate stain (opacity/edge softness/move/shape)	Fig 1h
8	Add other stains (repeat 1-7)	Fig 1i

2.2 STEVE software interface overview

Fig 1a — STEVE software interface

Fig 1a — STEVE software interface overview: RI layer Z stack panel on left; D-stain digital stain panel view (RI × RI gradient 2D space) on right; stain picker area + buttons below.

2.3 Detailed operations

Step 1 — Select Z plane

Fig 1b — Z stack selection

Fig 1b — Hold left mouse button and drag up/down on RI layer, or drag the "Slices slider" below the panel, to observe cells at different Z planes. Select the most informative plane as drawing starting point.

Step 2 — Add new stain

Fig 1c — Click + button

Fig 1c — In the D-stain panel's stain picker area, click "+" button to add a new stain.

Step 3 — Select name (optional)

Fig 1d — Naming

Fig 1d — Click the black-gray name input field, type your desired name (e.g., "nucleus", "mitochondria").

Step 4 — Select color

Fig 1e — Color selection

Fig 1e — Click to select your desired color. Optional: drag mouse for more precise saturation/brightness selection.

Step 5 — Draw ROI on RI layer

Fig 1f — Draw ROI

Fig 1f — Click and/or drag mouse on desired ROI. Pixels under cursor light up in panel view — release mouse, software calculates stain range based on selected pixels. Optional: use mouse wheel to zoom RI layer.

Step 6 — Stain overlay

Fig 1g — Stain overlay

Fig 1g — Stain now appears in D-stain panel view, overlaid on RI layer. Optional: move Overlay slider to adjust colored image weight on RI layer.

Step 7 — Stain manipulation

Fig 1h — Stain manipulation

Fig 1h — 4 stain manipulation types:

a. Opacity (Opacity slider OR right-click hold and drag up/down)
b. Edge Softness (Edge Softness slider OR right-click hold on stain edge and drag up/down)
c. Move stain (in D-stain panel, click and drag stain to desired position in RI-Index gradient space)
d. Change shape (select edge/corner points and drag)

Step 8 — Add other stains

Fig 1i — Multi-stain overlay

Fig 1i — Repeat 1-7 to add multiple stains (e.g., simultaneously stain nucleus + cytoplasm + membrane + vesicles).

Extra — Stain management

Fig 1j — Enable/disable
Fig 1k — Delete
Fig 1l — Change color

Fig 1j — Enable/disable stain visualization (eye icon)
Fig 1k — Delete stain (trash icon)
Fig 1l — Change stain color (double-click color button or click palette icon)

3. What are "Refractive Index (RI)" and "RI Gradient"? 📐

Theory chapter — understand the physical basis of digital staining.

3.1 Refractive Index (RI) definition

Refractive Index = speed of light in vacuum / speed of light in the medium:

RI = c / v
  c ≈ 300,000 km/s
  v = speed of light in the medium

Water RI = 300,000 / 225,000 ≈ 1.33

3.2 Common medium RI values (green light 520 nm)

Medium	RI
Waters	1.333
PBS	1.334
DMEM + 10% FBS	1.337
Ethanol	1.364
Urea	1.495
FluoroDish glass	1.525
### 3.3 RI Gradient (	∇RI	) definition

RI gradient describes how RI varies near a pixel:

∇RI = √[(RI(x+1,y,z) - RI(x-1,y,z))² / (2px)² +
         (RI(x,y+1,z) - RI(x,y-1,z))² / (2py)² +
         (RI(x,y,z+1) - RI(x,y,z-1))² / (2pz)²]

Here px,y,z is resolution, RI(x±1,y±1,z±1) is adjacent pixel's RI.

3.4 D-stain panel 4-quadrant theory

Fig 2a — RI gradient formula

Fig 2a — RI gradient geometric meaning: adjacent pixel RI difference computation.

Fig 2b — Digital stain panel 4 quadrants

Fig 2b — D-stain panel's typical 4-quadrant configuration:

Quadrant	RI	RI Gradient	Typical structure
I (top-left)	Low	High	Cell membrane (edge structure)
II (top-right)	High	High	Intracellular vesicles (texture bands)
III (bottom-left)	Low	Low	Background (transparent, uniform)
IV (bottom-right)	High	Low	Cell nucleus (high-density-like structure)

3.5 D-stain applied on RI image

Fig 2c — D-stain applied example

Fig 2c — D-stain applied on cell RI image: original (left) vs applied (right) comparison. Stain box position in RI × RI gradient space determines its coverage in 2D view.

4. Stain fine-tuning operations 🎛️

3 core adjustment dimensions — make stain precisely correspond to your desired biological structure.

4.1 Position adjustment

Fig 3 — Position adjustment

Fig 3 — Move stain box in RI × RI gradient 2D space:

Horizontal move → adjust RI range (different biological components have different RI ranges, e.g., cell membrane vs nuclear membrane)
Vertical move → adjust RI gradient range (edge vs internal)

4.2 Shape adjustment

Fig 4 — Shape adjustment
Fig 4b — Width adjustment

Fig 4 + Fig 4b — Stain box shape adjustment:

Horizontal stretch/compress → control RI range width (how many RI pixels to stain)
Vertical stretch/compress → integrate how many RI gradient changes (edge sharpness)

4.3 Opacity and Edge Softness

Fig 5 — Opacity and Edge Softness
Fig 5b — Panel view

Fig 5 + Fig 5b — Opacity and Edge Softness adjustment effects.

Parameter	Meaning	Range
Opacity	Maximum intensity of colored pixels	0 (transparent) ↔ 100 (opaque)
Edge Softness	Speed of intensity variation within colored pixels	0 (smooth) ↔ 100 (sharp)

Fig 5c — Top view 3D
Fig 5d — Side view 3D

Fig 5c/d — Adjustment effect on 3D view (top view + side view): high opacity + high edge softness = smooth soft color rendering; low values = sharp but grainy rendering.

5. Best staining practices (Golden advice) 🏆

4 core recommendations from the manufacturer — avoid typical errors.

5.1 Avoid imaging artifacts

Fig 6a — Artifact example
Fig 6b — Avoid artifact

Fig 6a — Wrong approach: stain box overlaps background area → stain covers background, spreads above/below cell adhesion plane (green line), imaging artifacts appear in 3D view (circle).

Fig 6b — Correct approach: ensure all stain boxes do NOT overlap background area → digital stain only meaningful sample regions → 3D view clear, no artifacts.

5.2 Avoid single-color staining

Fig 7 — Single-color staining issue

Fig 7 — Avoid same or similar colors — if using same color for different structures, cannot effectively distinguish them. Recommend high-contrast colors (red+green+blue+yellow).

5.3 Overlapping stain boxes

Fig 8a — Sub-stain example
Fig 8b — Overlap stain example

Fig 8 — Generally, simple effective stain panels include well-distinguishable stain boxes (non-overlapping). But 2 exception scenarios:

Fig 8a (Sub-stain): when one structure is inside another larger RI range compartment, use sub-stain to highlight structures inside (e.g., stain organelles and whole cell simultaneously)
Fig 8b (Overlap stain): in specific cases, several overlapping stains can highlight evidence regions otherwise hard to stain

5.4 Stain with specific RI value (e.g., cell membrane)

Fig 9 — Specific RI value stain panel
Fig 9b — Applied result

Fig 9 + Fig 9b — Best method to stain specific RI value (e.g., cell membrane):

Stain box width as narrow as possible (minimum RI range)
Stain box height same as entire panel (no RI gradient-based distinction)
Set Opacity and Edge Softness to maximum 100 — so all matching RI pixels get stained

6. Common errors quick reference 🚨

5 most common pitfalls customers encounter:

Error	Phenomenon	Correct approach
❌ Stain box overlaps background	3D view artifacts	Only draw meaningful regions, don't let box overflow into background
❌ Same color for different structures	Cannot distinguish structures	Use contrast colors (red/green/blue/yellow)
❌ Stain box contains too many pixels	Stain covers unrelated structures	Narrow stain box width
❌ Opacity/Edge Softness values too low	Stain looks faint	Adjust to 80-100 (default insufficient)
❌ Wrong Z plane selection	Low info, poor stain effect	Select plane with clear edge structures as drawing start

7. Sales perspective customer Q&A 💼

7.1 Digital staining value

Customer question	Answer
"What's the essential difference between digital staining and fluorescence staining?"	Digital staining is physical parameter (RI), fluorescence is chemical label. Digital staining is label-free, quantifiable, can distinguish 7 organelles simultaneously; fluorescence only sees a few markers.
"Can digital staining achieve confocal/EM resolution?"	No. Digital staining is based on holographic tomography (lateral 200nm / axial 400nm), cannot compare with EM (nm level). Advantage is live cells + label-free.
"Why doesn't Nanolive just see fluorescence directly?"	Base 3D Cell Explorer only sees RI; fluo version overlays 3-channel fluorescence. But EA segmentation proves fluorescence segmentation is less accurate than RI segmentation — this is the EVE technical note empirical conclusion.
"How to choose digital staining colors?"	Use contrast colors (red/green/blue/yellow), avoid same or similar colors. Colors are for operator viewing, don't change data.
"What affects digital staining precision?"	① Sample preparation quality (clean/flat); ② Z plane selection (most informative); ③ Stain box position (precisely matches RI range).
"How many structures can be stained per cell?"	Theoretically unlimited (STEVE supports up to 7 simultaneous digital stains + 3 fluorescence channels = 10 markers simultaneously).

7.2 Differentiation with 9X Bio portfolio

Dimension	CelVivo ClinoStar	3D Cell Explorer + STEVE digital staining
Staining?	No (see spheroid morphology)	Yes (7 structures digitally)
Need labeling?	No	No
Readout	Spheroid morphology + section staining	RI + 7 digital stains + dry mass
Invasiveness	None	None (extremely low phototoxicity)

STEVE digital staining positioning in 9X Bio portfolio: fills the "label-free + real-time + multi-structure staining" gap. The other two don't excel in multi-structure label-free staining.

8. Key resources 🔗

Nanolive official: http://nanolive.ch
Nanolive training data download: http://nanolive.ch/downloads/data/
STEVE software tutorial videos: via STEVE "Help > Get More Data" menu
Customer registration + software download: https://www.nanolive.ch/register

📥 Original PDF

📄 Nanolive Digital Staining Training PDF (Chinese version, 20 pages, 2.4 MB)

Contact｜Li Dongwei (李东伟)
📞 18045130660
🌐 Website: https://www.biokingdom.top