# Leica STELLARIS 8 — Super-Resolution Confocal

## System Reference for Imaging & Microscopy Workflows

**Version date:** 2026-08-28  
**FILM system ID:** HCF2

This document describes a Leica STELLARIS 8 confocal point-scanning system built on a DMi8 CS Premium inverted microscope stand. The configuration includes a pulsed White Light Laser (440–790 nm), 405 nm solid-state laser, Acousto-Optical Beam Splitter (AOBS), FOV laser scanner, four internal Power HyD spectral detectors (3 × HyD S + 1 × HyD X), Lightning computational super-resolution, Tau Sense lifetime-based contrast, environmental control, and a full DIC outfit. It is intended as a machine-readable reference for designing acquisition workflows, analysis pipelines, and experiment planning with LLM-based tools.

---

## 1. Microscope Stand

### 1.1 Main Body — DMi8 CS Premium

| Component | Code | Description |
|---|---|---|
| DMi8 CS Premium | 155933663 | Inverted research microscope stand; fully motorised; CS-designated optics path optimised for confocal scan heads; 19 mm FOV standard at all camera ports |
| Transmission Axis | 155933666 | Transmitted-light illumination path |
| Binocular Tube | 155933660 | Eyepiece observation |

### 1.2 Focus System

| Component | Code | Description |
|---|---|---|
| Closed Loop Focus with AFC | 158204202 | Closed-loop Z motorisation with Adaptive Focus Control (hardware autofocus) |

Focus system specifications (from manufacturer technical documentation):

| Parameter | Value |
|---|---|
| AFC type | Reflection-based hardware autofocus |
| AFC sampling rate | 15 Hz |
| Motorfocus minimum step | 50 nm |
| Motorfocus step increments | < 4 nm (z-wide) |
| Closed-loop repeatability | < 20 nm, without hysteresis |
| Image-based autofocus | Available for transmitted light and fluorescence; combinable with AFC |

### 1.3 Transmitted-Light Detection

| Component | Code | Description |
|---|---|---|
| BF Detector for DMi | 158004201 | Brightfield and DIC transmitted-light detector |

### 1.4 Widefield Epifluorescence

| Component | Code | Description |
|---|---|---|
| Leica LED3 | 158000677 | Multi-channel LED epifluorescence illumination supply + LED transmitted light for ocular screening |
| Filter Cube DA/FI/TX | 15525321 | Triple-band filter cube: DAPI / FITC / Texas Red |

### 1.5 Stage & Z-Drive

| Component | Code | Description |
|---|---|---|
| Scanning Stage Inv. Universal | 158004141 | Motorised XY scanning stage (inverted, universal) |
| Cable Scanning Stage | 15500332 | 3 m, 90° connector |
| XY Advanced Board | 15525226 | Advanced control board for scanning stage |
| SuperZ Galvo Stage | 158004421 | Galvanometer-driven Z stage |

SuperZ galvo stage specifications:

| Parameter | Value |
|---|---|
| Travel range | 1500 µm |
| Minimum step size | 20 nm |
| Step increments | < 1.5 nm |
| Z modes | Galvo Flow and discrete steps |
| XZY scan mode | Real-time XZ slices for correction-collar setup and axial resolution assessment |
| Scan modes enabled | xzy, xzt, xzyt, xzλ, xzλt |

### 1.6 Sample Holders

| Component | Code | Description |
|---|---|---|
| Insert Universal Inverse SuperZ | 158004424 | Universal sample insert for SuperZ stage: accepts slides, dishes, and plates |
| Petri Dish Insert 36 mm for SuperZ | 158004136 | 36 mm petri dish adapter for SuperZ stage |

### 1.7 Vibration Isolation & Mounting

| Component | Code | Description |
|---|---|---|
| Base Plate DMi8 | 158000640 | Mounting base plate |
| Sealed-Holes Metric Breadboard | 8108754 | 750 × 750 × 60 mm optical breadboard |
| Active Vibration Isolation Frame | 8108762 | 700 series, 750 × 750 mm active anti-vibration table |
| Air Compressor | 8109984 | 220/240 V, 50 Hz, UK power; supplies active table |

---

## 2. Objective Lenses

### 2.1 Installed Objectives

| Objective | Code | Mag | NA | Immersion | Free WD (mm) | Coverslip | Notes |
|---|---|---|---|---|---|---|---|
| HC PL APO 10×/0.40 CS2 | 15506424 | 10× | 0.40 | Air (dry) | 2.56 | 0.17 mm (#1.5) | General overview, low-mag tiling; generous WD accommodates thick vessels |
| HC PL APO 20×/0.75 CS2 | 15506517 | 20× | 0.75 | Air (dry) | 0.62 | 0.17 mm (#1.5) | Intermediate resolution, tiling; good for tissue sections |
| HC PL APO 40×/1.30 Oil CS2 | 15506358 | 40× | 1.30 | Oil (n = 1.518) | 0.24 | 0.17 mm (#1.5) | High-resolution, fixed samples; short WD — incompatible with plastic-bottom plates |
| HC PL APO 63×/1.40 OIL CS2 | 15506350 | 63× | 1.40 | Oil (n = 1.518) | 0.14 | 0.17 mm (#1.5) | Highest NA; ideal for Lightning super-resolution; very short WD — glass-bottom dishes with #1.5 coverslip only |
| HC PL APO 63×/1.20 W CORR CS2 | 15506346 | 63× | 1.20 | Water (n = 1.33) | 0.30 | 0.14–0.18 mm (CORR) | Correction collar; optimised for live-cell and deep-tissue imaging; see §2.2 for collar calibration |

### 2.2 Working Distance & Coverslip Guide

**Why this matters for workflow design:** The free working distance (WD) is the gap between the front element of the objective and the top surface of the coverslip. Choosing an objective whose WD is shorter than the total thickness of the vessel bottom will physically prevent focusing. Oil-immersion lenses on this system have very short WDs (0.14–0.24 mm) and are designed exclusively for standard #1.5 glass coverslips (0.17 mm ± 0.005 mm).

| Vessel / Sample Format | Bottom Thickness | Compatible Objectives (this system) |
|---|---|---|
| Standard glass slide + #1.5 coverslip | 0.17 mm | All five objectives |
| Glass-bottom dish (e.g. MatTek, ibidi µ-Dish), #1.5 glass | 0.17 mm | All five objectives |
| Glass-bottom multi-well plate, #1.5 glass | 0.17 mm | All five objectives |
| Thick coverslip (#2, 0.19–0.23 mm) | 0.19–0.23 mm | 10×, 20×, 40× Oil (marginal), 63× W CORR (within collar range); NOT 63× Oil (WD too short) |
| Plastic-bottom plate (polystyrene, ~1 mm) | ~1.0 mm | 10× only (WD 2.56 mm); none of the high-NA objectives |
| Plastic-bottom plate (thin, ~0.17 mm, e.g. ibidi polymer) | ~0.17 mm | All five objectives (use dry or water objectives to avoid solvent damage to polymer); oil objectives only if polymer is solvent-resistant |
| Chamber slide (glass, #1.5) | 0.17 mm | All five objectives |

**Rules of thumb for an LLM selecting objectives:**

- If the user specifies a plastic-bottom plate, do not select the 40× Oil, 63× Oil, or 63× Water objectives — only the 10× (WD 2.56 mm) or 20× (WD 0.62 mm) have sufficient clearance, and neither reaches high-NA resolution. Suggest switching to a glass-bottom plate.
- If the user specifies a #0 or #1 coverslip (0.08–0.13 mm), only the 63× W CORR can compensate (collar range starts at 0.14 mm — still marginal). The oil objectives are corrected for exactly 0.17 mm and will show spherical aberration.
- If the user needs to image deep into tissue (> 50 µm below the coverslip), prefer the 63× W CORR over the 63× Oil to avoid refractive-index mismatch between oil (n = 1.518) and aqueous tissue (n ≈ 1.33–1.38).

### 2.3 Correction Collar Guide — HC PL APO 63×/1.20 W CORR CS2

The correction collar on this objective compensates for spherical aberration introduced by variations in coverslip thickness, temperature, and immersion medium. Correct collar setting is essential for optimal resolution and signal — an incorrectly set collar can degrade axial resolution by 2–5× and reduce peak intensity by > 50 %.

**Collar range:** 0.14–0.18 mm (marked on the objective barrel).

**What the collar compensates for:**

| Variable | Effect on optimal collar position | Typical shift |
|---|---|---|
| Coverslip thickness | Thicker glass requires higher collar value | Dominant factor; set collar to match measured coverslip thickness |
| Temperature | Heating from RT (23 °C) to 37 °C expands the coverslip and changes the refractive index of water | Shift collar ~0.01 mm higher at 37 °C compared to RT for a #1.5 coverslip |
| Imaging depth | Deeper focus into aqueous medium introduces additional mismatch | Minor for water immersion (n_immersion ≈ n_specimen); significant only at > 100 µm depth in high-RI tissue |
| Immersion medium | Water (n = 1.33) vs saline or culture medium (n ≈ 1.335–1.34) | Negligible; collar position unchanged |

**Calibration procedure (recommended before each session):**

1. **Mount sample and apply water immersion** to the objective front lens. Ensure no air bubbles between the objective and the coverslip.
2. **Set the collar to nominal coverslip thickness.** For a standard #1.5 coverslip, start at 0.17. If coverslip thickness is known (e.g. from batch data or measurement with a micrometer), start there.
3. **Switch to a bright, point-like feature** (e.g. a sub-resolution fluorescent bead, a bright punctate structure, or a reflection from the coverslip surface).
4. **Use the SuperZ xzy scan mode** (LAS X: select xzy scan). This produces a real-time XZ cross-section that directly shows axial PSF shape. A well-corrected PSF appears symmetric above and below the focal plane; an aberrated PSF shows an asymmetric flare (comet-tail) in Z.
5. **Rotate the collar in small increments** (~0.005 mm) while observing the XZ image. Adjust until the PSF is symmetric in Z and the peak intensity is maximised.
6. **At 37 °C** (live-cell imaging with the Okolab incubator), allow the system to equilibrate thermally for ≥ 30 minutes before calibrating. Recalibrate the collar after temperature changes.
7. **Record the collar setting** in the LAS X experiment notes for reproducibility.

**Quick-reference collar starting positions:**

| Condition | Coverslip | Temperature | Starting collar position |
|---|---|---|---|
| Fixed sample, standard | #1.5 (0.17 mm) | RT (23 °C) | 0.17 |
| Fixed sample, thin glass | #1 (0.13–0.15 mm) | RT (23 °C) | 0.15 (at lower edge of collar range) |
| Live cell, glass-bottom dish | #1.5 (0.17 mm) | 37 °C | 0.18 |
| Live cell, thin-bottom dish | Custom (0.15 mm) | 37 °C | 0.16 |

These are starting points; always verify with the XZ PSF check (step 4–5 above).

### 2.4 DIC Wollaston Prisms

| Component | Code | Position |
|---|---|---|
| IC Condenser Prism K3 | 15555017 | Condenser-side |
| IC Condenser Prism K6 | 15521521 | Condenser-side |
| IC Condenser Prism K10 | 15521524 | Condenser-side |
| IC Prism C | 15555009 | Objective-side |
| IC Prism D | 15555010 | Objective-side |
| IC Prism E | 15555046 | Objective-side |

A complete set of condenser and objective-side prisms is installed, enabling DIC across the full range of objectives. The optical outfit includes automated, encoded, and motorised DIC path switching.

### 2.5 Immersion Media

| Item | Code | Description |
|---|---|---|
| Type F Immersion Liquid | 15513859 | ISO 8036; for oil-immersion objectives |

---

## 3. STELLARIS 8 Scan Head

### 3.1 Main Unit

| Component | Code | Description |
|---|---|---|
| STELLARIS 8 | 158301100 | Confocal point-scanning scan head; prism-based spectral detection; fully integrated with AOBS and Power HyD detector family |
| STELLARIS 8 DMi8 Adapter | 158301140 | Mechanical adapter coupling scan head to DMi8 stand |
| Scan Optics HIVISR DMi8/DM8 | 158301121 | High-visibility super-resolution scan optics |

### 3.2 Beam Path Overview

The complete excitation–detection beam path through the STELLARIS 8 scan head (component numbers reference the manufacturer scan-head poster):

1. **White Light Laser** (WLL, 440–790 nm) and/or 405 nm laser enter the scan head.
2. **AOTF** (Acousto-Optical Tunable Filter) selects excitation wavelengths and controls intensity for WLL lines. The 405 nm laser uses direct modulation (DMOD) or its own AOTF.
3. **AOBS** (Acousto-Optical Beam Splitter) reflects selected excitation lines toward the sample and transmits fluorescence emission toward the detectors.
4. **Switchable Beam Expander** (FRAP Booster) — increases laser power density at the sample for photobleaching.
5. **FOV Scanner** — galvanometric X-Y mirror pair raster-scans the beam across the specimen.
6. **Scan Optics** (HIVISR) — relay the scanned beam to the objective.
7. **Objective Lens** focuses excitation onto the specimen.
8. Fluorescence emission returns through the objective → scan optics → scanner → AOBS (transmitted, not reflected).
9. **Square Confocal Pinhole** — rejects out-of-focus light for optical sectioning.
10. **Fluorifier Disc** with analyser — optional polarisation/DIC analysis in the detection path.
11. **Notch Filters** — suppress reflected laser light near excitation wavelengths.
12. **Prism-Based Dispersion** — a prism disperses the emission spectrum across the detector array.
13. **SP Detection** — up to 5 Power HyD detectors (this system: 4 detectors) collect spectrally separated emission with software-tuneable bandwidth per channel.

### 3.3 FOV Laser Scanner

| Component | Code | Description |
|---|---|---|
| FOV Scanner STELLARIS 8 | 158301130 | Field-of-View galvanometric point scanner |

Scanner specifications (from manufacturer technical documentation, 12.2024):

| Parameter | Value |
|---|---|
| Scanner design | X2Y-scanner with optically correct scanning at low inertia |
| Max line frequency (bidirectional) | 5200 Hz |
| Min line frequency | 1 Hz |
| Line frequency selection | Freely selectable in steps of 1 Hz (unidirectional), 2 Hz (bidirectional) |
| Max frame rate (512 × 512) | 10 fps |
| Max frame rate (512 × 16) | 216 fps |
| Max frame resolution | 8192 × 8192 px |
| Scan zoom | 0.75–48× |
| Panning | Yes |
| Field rotation | 200° optical |
| Scan field diameter | 22 mm |
| Beam park | Yes |

Available scan modes on this system: xyz, xt, xyt, xyzt, xyλ, xyλt, xyzλ, xyzλt. With the SuperZ galvo stage: xzy, xzt, xzyt, xzλ, xzλt (real-time Z sectioning). With the WLL: xyΛ, xzΛ, xyzΛt, xyλΛ, xz λΛ (lambda-lambda / excitation spectrum scanning).

This configuration has only the FOV scanner. It does not include a tandem (resonant + FOV) scanner. Systems with the optional 8 kHz resonant scanner can reach 28 fps at 512 × 512 (16,000 lines/s bidirectional) over a 13 mm field; systems with the 12 kHz resonant reach 40 fps.

### 3.4 AOBS — Acousto-Optical Beam Splitter

The AOBS replaces all conventional dichroic and multichroic mirrors with a single programmable crystal-based acousto-optical element.

**Operating principle:** Beam splitting is based on acousto-optical diffraction in a TeO₂ (tellurium dioxide) crystal. An applied radio-frequency acoustic wave creates a tuneable refractive-index grating within the crystal. Excitation light at an angle satisfying the Bragg condition (νλ = 2δ sin α) is diffracted into first order and co-aligned with the optical axis toward the sample. Fluorescence emission is Stokes-shifted and passes through the crystal unaffected.

Key properties:

- Freely programmable for any combination of visible laser lines — no filter changes, no alignment.
- Reflection band width ~2 nm per line (vs ~20 nm for a typical dichroic); this leaves broader emission collection bands and yields ~30 % higher detection efficiency compared to multichroic mirrors.
- Up to 8 simultaneous laser lines in both fluorescence and reflection mode.
- Switching time < 10 µs between line configurations — fast enough for line-sequential scanning of living specimens without spatial correlation loss.
- Can operate as a tuneable 50/50 beam splitter for reflected-light imaging at any chosen wavelength, while simultaneously using up to 7 additional lines for fluorescence excitation.
- Fully transparent from below 400 nm to beyond 4 µm.
- No mechanical wear; no alignment drift; future-proof for any new laser line.

The AOBS is the enabling technology for the White Light Laser: because the WLL can select any wavelength in 1 nm steps, only a freely tuneable beam splitter can match all permutations.

### 3.5 Pinhole

| Parameter | Value |
|---|---|
| Type | Stable single square confocal pinhole (maintenance-free) |
| Diameter control | Motorised by software |
| Automatic mode | Wavelength-dependent automatic pinhole sizing |

### 3.6 Additional Beam-Path Components

| Component | Code | Description |
|---|---|---|
| Fluorifier Disc Basis incl. Analysator | 158204510 | Base disc with analyser for polarisation / DIC through the scan head; 2 fluorifier disc positions with numerous filter options |
| Notch Filter Set VIS Base | 158204511 | Visible-range notch filters to suppress reflected laser light; improves signal-to-noise for emission detection near excitation wavelengths |
| Switchable Beam Expander | 158202220 | Scan head beam expander; switchable for FRAP boost — increases laser power density at the sample for photobleaching and photoactivation experiments |
| SP Light Trap | 158201310 | Spectral light trap for unused wavelengths |

---

## 4. Detectors

### 4.1 Internal Detection Channels — Power HyD Family

This system has **4 internal spectral detection channels** using Leica's Power HyD detector technology. All channels use prism-based dispersion (filterless) with software-tuneable emission bandwidth.

| Position | Detector | Code | Type | Key Characteristics |
|---|---|---|---|---|
| 2 | Power HyD S | 158301312 | HyD S (Spectral) | Highest dynamic range; photon counting; suitable for high-light-level and standard confocal |
| 3 | Power HyD S SP (Core Unit) | 158301313 | HyD S (Spectral) | Same as Pos 2; core-unit position |
| 4 | Power HyD X | 158301324 | HyD X (eXtreme sensitivity) | Optimised for FLIM (FALCON-capable), FCS, and extremely dim samples; single-photon counting |
| 5 | Power HyD S | 158301315 | HyD S (Spectral) | Same as Pos 2 |

### 4.2 Detector Specifications (from manufacturer technical documentation, 12.2024)

| Property | Power HyD S | Power HyD X |
|---|---|---|
| Sensor type | Multi-pixel silicon photo-multiplier array | GaAsP hybrid detector |
| Spectral sensitivity range | 410–850 nm | 410–750 nm |
| Typical PDE (@ 500 nm) | 58 % | 46 % |
| Detection modes | Analog, reflection, counting, fast | Digital, counting |
| TauSense capability | Yes | Yes |
| FALCON capability (FLIM) | No | Yes |
| FALCON capability (FCS) | No | Yes |
| Max detectors per type (platform) | 5 (1 mandatory) | 4 |

Note: The HyD X spectral range extends only to 750 nm (not 850 nm). For near-infrared detection (720–850 nm), the optional Power HyD R detector would be required; it is not installed on this system.

### 4.3 Spectral Detection Unit — Summary

| Parameter | Value |
|---|---|
| Spectral detection range | 410–850 nm (HyD S); 410–750 nm (HyD X) |
| Emission separation | Prism-based spectral detection |
| Maximum simultaneous tuneable channels | 4 (on this system; platform maximum 5) |
| Spectral tuning resolution | 1 nm across full spectrum |
| Minimal detection bandwidth | 5 nm |
| Tunability of emission bands | Yes, all channels independently |

### 4.4 Photon Detection Electronics

| Parameter | Value |
|---|---|
| Photon counting time resolution | 97 ps |
| Photon counting scheme | Power counting: 0, 1, or 2 (double) photons identified per clock cycle |
| Digitisation resolution | 8, 12, or 16 bit |
| Sampling frequency (digital/counting modes) | 10.3 GHz |
| Sampling frequency (analog mode) | 80 MHz |
| Scanner control | Digital (FPGA, field-programmable gate arrays) |

The FPGA-based electronics sort detected photons into digitally preset gates by arrival time, enabling all TauSense functions (§6) during standard acquisition without additional hardware.

### 4.5 Transmitted-Light Detection

Brightfield and DIC transmitted-light images are acquired via the BF detector (158004201) in the stand base, using the LED3 transmitted-light source through the condenser.

---

## 5. Laser Sources

### 5.1 White Light Laser (WLL)

The STELLARIS 8 White Light Laser is a pulsed supercontinuum source and the primary excitation laser for this system.

| Parameter | Value |
|---|---|
| Type | VIS White Light Laser |
| Spectral range | 440–790 nm (continuously tuneable) |
| Tuning step | 1 nm |
| Simultaneous lines | Up to 8 independent lines (selected via AOTF) |
| Repetition rate | 78 MHz |

Power per line (from manufacturer technical documentation, 12.2024):

| Wavelength | Minimum power per line |
|---|---|
| 440 nm | > 1.1 mW |
| 488 nm | > 1.6 mW |
| 560 nm | > 2.0 mW |
| 630 nm | > 2.6 mW |
| 790 nm | > 3.5 mW |

The pulsed nature of the WLL (78 MHz) enables TauSense lifetime-based contrast and time-gated detection without additional hardware. Combined with the AOBS, any excitation wavelength can be matched exactly to the peak absorption of each fluorophore, minimising photo-damage and maximising signal. The WLL supports lambda-lambda scanning (excitation spectrum acquisition) in combination with the AOBS and spectral detectors.

### 5.2 405 nm Solid-State Laser

| Component | Code | Description |
|---|---|---|
| Laser 405 DMOD | 158202140 | 405 nm diode laser with direct modulation (DMOD); 50 mW; covers the UV-excited spectral range not addressed by the WLL |
| Laser Port 405/UV | 158301200 | Scan-head input port for the 405 nm laser |

Typical applications: DAPI, Hoechst, BFP, and other UV-excitable dyes; photoactivation; FRAP at 405 nm.

### 5.3 Excitation Modulation

| Modulation type | Specification |
|---|---|
| AOTF VIS (for WLL) | Up to 8 channels |
| Direct modulation (DMOD) | For 405 nm laser |

### 5.4 Available Excitation Lines — Summary

| Source | Lines (nm) | Type |
|---|---|---|
| White Light Laser (WLL) | 440–790 (any wavelength, 1 nm steps, up to 8 simultaneous) | Pulsed supercontinuum, 78 MHz |
| 405 nm DMOD | 405 (fixed) | CW diode, 50 mW, direct modulation |

---

## 6. TauSense — Lifetime-Based Contrast

TauSense is Leica's integrated lifetime-sensing technology, built into the STELLARIS platform. It uses the pulsed WLL and the FPGA-based photon-detection electronics to measure photon arrival times and extract fluorescence-lifetime information during standard confocal acquisition — without the data size or computational overhead of full FLIM.

### 6.1 Principle

The fluorescence lifetime is the characteristic time a molecule stays in the excited state (S₁) before returning to the ground state (S₀) and emitting a photon. It is typically sub-nanosecond to nanosecond and is sensitive to changes in the fluorophore's local environment within ~10 nm (pH, ion concentration, FRET, binding).

TauSense measures the **average arrival time (AAT)** of photons for each pixel — the difference between the time a photon is detected and the time of the corresponding WLL excitation pulse (calibrated from the reflection signal). The FPGA electronics in the scan head compute the AAT on the fly; the resulting images contain both intensity and arrival-time information per pixel without carrying the full single-photon time-tagged data, yielding smaller files and lower computational load than FLIM.

### 6.2 TauSense Tools

| Tool | Function | Detail |
|---|---|---|
| **TauContrast** | Maps mean photon arrival time per pixel to a colour scale | Provides lifetime contrast in real time; semi-quantitative relative to a control; independent of fluorescence intensity; useful for pH sensing, membrane dynamics, endosome maturation tracking |
| **TauGating** | Time-gates detection to include/exclude photons by arrival time | Up to 16 tuneable digital time gates, simultaneously; isolates signal of interest from autofluorescence, pigments, or scattered light with short arrival times |
| **TauScan** | Scans the lifetime-component distribution across the photon arrival time range | Uses digitally preset gates followed by multi-exponential component fitting; generates an online view of the distribution; produces intensity images in discrete temporal windows |
| **TauSeparation** | Separates spectrally overlapping fluorophores by lifetime | Uses the lifetime-component distribution to assign photons to distinct fluorophore species; solves multiplexing problems where spectral windows overlap (e.g. GFP + green mitochondrial stain) |
| **TauInteraction** | Detects FRET or binding events via lifetime shifts | Lifetime-based FRET measurement (requires LAS X MicroLab) |
| **GateScan** | Differentiates desired and unwanted fluorescence signals | Complements TauGating with scanning-based gating |

### 6.3 TauSense vs FALCON (quantitative FLIM)

TauSense provides qualitative to semi-quantitative lifetime-based information during any confocal acquisition on this system. Full quantitative FLIM — including multi-exponential decay fitting, phasor analysis, high photon-flux FLIM, and FLIM-FRET analysis — requires the **FALCON** module and at least one **Power HyD X** detector. This system has the HyD X detector (Position 4) but does **not** have the FALCON software licence installed. Upgrading to FALCON would enable full quantitative FLIM without hardware changes.

---

## 7. Lightning — Confocal Super-Resolution

### 7.1 Lightning Module

| Component | Code | Description |
|---|---|---|
| LAS X Lightning Expert | 158203204 | Computational super-resolution modality with Expert mode for advanced parameter control |

### 7.2 Principle & Performance

Lightning is a fully automated, adaptive super-resolution process that combines optimised confocal acquisition (reduced pinhole, typically 0.5 AU) with information-content-aware deconvolution. The high photon-detection efficiency of the Power HyD detectors allows the pinhole to be closed to 0.5 AU while maintaining sufficient signal-to-noise for deconvolution, effectively halving the widefield diffraction limit.

| Parameter | Value |
|---|---|
| Lateral resolution | Down to 120 nm (at 1.40 NA) |
| Axial resolution | Down to 200 nm (at 1.40 NA) |
| Resolution improvement | ~2× vs conventional widefield diffraction limit |
| Number of spectral channels | All 4 channels simultaneously |
| Scanner compatibility | FOV (galvanometric) scanner |
| Detector compatibility | All Power HyD channels |
| Objective compatibility | All objectives (resolution scales with NA) |
| Processing | Fully automated online during acquisition; adaptive image quality determination and reconstruction |
| Expert mode | User control over deconvolution parameters; adjustable signal-to-noise vs resolution trade-off |

Lightning works in all spectral channels simultaneously and is compatible with Z-stacks, time-lapse, tiling, and multi-position experiments. Dynamic Signal Enhancement maintains optimal signal-to-noise ratio at up to 420 fps (with resonant scanner; FOV-scanner limited on this system).

---

## 8. Environmental Control

### 8.1 Incubation System — Okolab

| Component | Code | Description |
|---|---|---|
| Transparent Box Incubator DMi8 | 158206046 | Whole-microscope enclosure; transparent chamber covering the entire DMi8 stand; maintains temperature uniformity; suitable for very light-sensitive applications |
| Sample Chamber Inv SuperZ | 158206037 | Stage-top incubator insert compatible with SuperZ galvo stage |
| Insert Multi Well Plate SuperZ | 158206031 | Multi-well plate adapter for stage-top incubator on SuperZ |
| CO2, Humidity Passive | 158206039 | CO2 gas mixer + passive humidity control |
| LAS X Environmental Control | 158203214 | Software module for integrated incubator control from LAS X |

### 8.2 Environmental Parameters

| Parameter | Capability |
|---|---|
| Temperature | Regulated via box incubator; typically 37 °C for mammalian cells |
| CO2 | Premixed gas via Okolab mixer; typically 5 % |
| Humidity | Passive humidification to reduce evaporation |
| Software integration | Full LAS X control of temperature and gas parameters; logging and monitoring |

The dual-layer environmental control (whole-microscope box + stage-top insert) provides maximum thermal stability for long time-lapse experiments. The transparent enclosure allows transmitted-light imaging without removing the chamber.

---

## 9. Software — LAS X

### 9.1 Core Platform

| Module | Code | Description |
|---|---|---|
| LAS X STELLARIS Control Software | 158203200 | Full confocal system control: Image Compass user interface; one-click-per-fluorophore multicolour setup; automatic optimal acquisition settings; full hardware control |

### 9.2 Core Functionality (included)

| Feature | Description |
|---|---|
| Image Compass | Simplified user interface; automatic optimal acquisition settings per fluorophore |
| TauSense | Integrated lifetime-based contrast (TauContrast, TauGating, TauSeparation, TauScan, TauInteraction) |
| Dynamic Signal Enhancement | Maintains optimal SNR at high frame rates |
| Lightning (basic) | Automated super-resolution to 120 nm (basic detection package) |
| Navigator (basic) | Spiral scan for specimen overview; stitching; mark-and-find |
| Lambda scan | Emission spectrum acquisition using spectral detectors |
| Lambda-lambda scan | Combined excitation and emission spectrum acquisition (requires WLL) |
| Z intensity compensation | Laser power and/or detector gain adjustment within Z-stacks |
| Sequential scanning | Frame-by-frame or line-by-line sequential acquisition to eliminate cross-talk |
| 6D imaging | X, Y, Z, time, lambda, position |

### 9.3 Application Modules (installed)

| Module | Code | Description |
|---|---|---|
| LAS X 3D Visualisation | 158203202 | GPU-based 3D rendering of large stacks; clipping tool; MIP; surface rendering; movie export |
| LAS X Lightning Expert | 158203204 | Customisable adaptive deconvolution parameters (see §7) |
| LAS X Dye Finder | 158203206 | Database-driven dye selection; automatically suggests optimal excitation/emission settings |
| LAS X MicroLab | 158203207 | FRAP, FLIP, photoconversion, FRET workflows |
| LAS X FRAP Zoomer | 158203208 | High-speed FRAP with zoomed bleaching ROI; simultaneous bleaching and imaging at different zoom levels |
| LAS X Co-Localisation | 158203209 | Quantitative co-localisation: Pearson's, Manders', scatter plots, co-localisation maps |
| LAS X Assay Editor | 158203211 | Stage Navigator & Assay Editor for automated multi-position, multi-well plate experiments; pre-configured specimen carriers; spiral scan overview; tiling and stitching |

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## 10. Workstation & Display

### 10.1 CUDA Expert Workstation

| Component | Code | Description |
|---|---|---|
| Workstation Expert | 158203113 | CUDA-enabled GPU workstation (dedicated HP) for parallel GPU processing; Lightning deconvolution and 3D rendering |

### 10.2 Monitor

| Component | Code | Description |
|---|---|---|
| High Brilliance Monitor | 158003150 | 4K-wide LED monitor, 37.5″, 21:9 aspect ratio; high colour accuracy for fluorescence image assessment |

### 10.3 Furniture

| Component | Code | Description |
|---|---|---|
| Computer Table incl. Rack | 158204700 | Dedicated desk with equipment rack |

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## 11. Capability Summary

This system supports the following imaging modalities and workflows:

| Modality | Key Components |
|---|---|
| **Laser-scanning confocal** | STELLARIS 8 scan head, FOV scanner (up to 8192 × 8192, 10 fps @ 512²), WLL (440–790 nm) + 405 nm, AOBS (8 lines), 4-ch Power HyD spectral detection (410–850 nm) |
| **Confocal super-resolution (Lightning)** | Lightning Expert module; down to 120 nm lateral / 200 nm axial at 1.40 NA; online adaptive deconvolution during acquisition |
| **Lifetime-based contrast (TauSense)** | TauContrast (AAT mapping), TauGating (16 gates), TauScan, TauSeparation (species unmixing by lifetime), TauInteraction (FRET); pulsed WLL (78 MHz) + 97 ps photon-counting electronics; no additional FLIM hardware required |
| **FRAP / FLIP** | LAS X MicroLab + FRAP Zoomer; switchable beam expander (FRAP Booster); 405 nm laser for photobleaching |
| **FRET** | LAS X MicroLab FRET workflow; spectral detection for sensitised-emission FRET; TauInteraction for lifetime-based FRET |
| **Spectral imaging & unmixing** | Continuous spectral tuning via AOBS + prism detection; lambda scan; lambda-lambda scan (excitation spectrum with WLL); linear unmixing for spectrally overlapping fluorophores |
| **DIC / Brightfield** | Full automated DIC outfit (all objectives); BF transmitted-light detector; LED3 illumination |
| **Widefield fluorescence (ocular)** | LED3 epifluorescence; DA/FI/TX triple-band filter cube |
| **Live-cell imaging** | AFC hardware autofocus (15 Hz); Okolab box + stage-top incubator (temp / CO2 / humidity); SuperZ galvo stage; 63× water-immersion objective with correction collar |
| **Tiling / large-area** | Motorised XY scanning stage; LAS X Assay Editor with spiral scan overview and automatic stitching |
| **Co-localisation** | LAS X Co-Localisation module: Pearson's, Manders', scatter plots |
| **3D visualisation** | LAS X 3D Visualisation: GPU rendering, MIP, clipping, surface rendering, movie export |
| **Multi-well / screening** | Assay Editor; multi-well plate insert for SuperZ; automated multi-position acquisition |
| **Reflected-light imaging** | AOBS in 50/50 mode for any chosen wavelength; simultaneous with up to 7 fluorescence lines; useful for collagen fibres, lipid conglomerations, cytoskeleton |

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## 12. Excitation / Detection Quick-Reference

### 12.1 Available Laser Lines

| Source | Lines (nm) | Type | Power |
|---|---|---|---|
| White Light Laser (WLL) | 440–790 (any, 1 nm steps, up to 8 simultaneous) | Pulsed supercontinuum, 78 MHz | 1.1–3.5 mW per line (wavelength-dependent) |
| 405 nm DMOD | 405 (fixed) | CW diode, direct modulation | 50 mW |

### 12.2 Widefield LED Channels

| Channel | Fluorophore Match |
|---|---|
| DA | DAPI |
| FI | FITC / GFP |
| TX | Texas Red / mCherry |

(LED3 also provides LED transmitted light for brightfield screening.)

### 12.3 Detector Spectral Coverage

| Detector | Range | Sensor | PDE | Channels on this system |
|---|---|---|---|---|
| Power HyD S (×3) | 410–850 nm | Multi-pixel silicon photo-multiplier | 58 % @ 500 nm | Internal ch 2, 3, 5 |
| Power HyD X (×1) | 410–750 nm | GaAsP hybrid | 46 % @ 500 nm | Internal ch 4 |
| BF Detector | Transmitted light | — | — | 1 (external) |

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## 13. Key Specifications at a Glance

| Parameter | Value |
|---|---|
| Microscope type | Inverted (Leica DMi8 CS Premium) |
| Focus: closed-loop repeatability | < 20 nm, no hysteresis |
| Focus: AFC sampling rate | 15 Hz |
| SuperZ galvo range | 1500 µm |
| SuperZ galvo minimum step | 20 nm (increments < 1.5 nm) |
| Scan head type | Confocal point scanner (STELLARIS 8) |
| Scanner type | FOV (galvanometric) only |
| Max line frequency (bidirectional) | 5200 Hz |
| Max frame rate (512 × 512) | 10 fps |
| Max frame rate (512 × 16) | 216 fps |
| Max frame resolution | 8192 × 8192 px |
| Scan zoom | 0.75–48× |
| Scan field diameter | 22 mm |
| Field rotation | 200° optical |
| AOBS simultaneous lines | 8 |
| AOBS switching time | < 10 µs |
| AOBS reflection band width | ~2 nm |
| Internal detection channels | 4 (3 × HyD S + 1 × HyD X) |
| Spectral detection range | 410–850 nm (HyD S); 410–750 nm (HyD X) |
| Spectral tuning resolution | 1 nm |
| Minimum detection bandwidth | 5 nm |
| Photon counting time resolution | 97 ps |
| Sampling frequency (counting) | 10.3 GHz |
| Digitisation | 8, 12, or 16 bit |
| WLL tuning range | 440–790 nm |
| WLL tuning step | 1 nm |
| WLL repetition rate | 78 MHz |
| WLL power (488 nm) | > 1.6 mW per line |
| WLL power (790 nm) | > 3.5 mW per line |
| 405 nm laser | CW diode, DMOD, 50 mW |
| Lightning lateral resolution | Down to 120 nm (at NA 1.40) |
| Lightning axial resolution | Down to 200 nm (at NA 1.40) |
| Objective NA (max, oil) | 1.40 (63× HC PL APO OIL CS2) |
| Objective NA (max, water) | 1.20 (63× HC PL APO W CORR CS2) |
| Objective NA (max, air) | 0.75 (20× HC PL APO CS2) |
| Vibration isolation | Active optical table, 750 × 750 mm |
| Environmental control | Temperature + CO2 + passive humidity (Okolab) |

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## 14. System Limitations & Notes for Workflow Design

- **No resonant scanner.** This configuration has only the FOV (galvanometric) scanner. Maximum frame rate is 10 fps at 512 × 512. Systems with the optional 8 kHz tandem scanner reach 28 fps at 512 × 512 over a 13 mm field; 12 kHz resonant reaches 40 fps. If high temporal resolution is critical, consider whether 10 fps is sufficient or whether an alternative FILM system with a resonant scanner is needed.
- **No FALCON / quantitative FLIM.** TauSense provides qualitative to semi-quantitative lifetime contrast (TauContrast, TauGating, TauSeparation, TauScan) but full quantitative FLIM with multi-exponential decay fitting, phasor analysis, and FLIM-FRET requires the FALCON software licence (not installed). The HyD X detector at Position 4 is FALCON-capable hardware — a software upgrade would enable FLIM without hardware changes. For FLIM now, use HCF3 (Leica Stellaris STED FALCON) or HCF4 (Nikon AX R MP with PicoQuant).
- **No STED.** This system does not have STED depletion lasers. Super-resolution is limited to Lightning (down to 120 nm lateral). For STED nanoscopy (< 50 nm lateral, < 130 nm axial), use HCF3 (Leica Stellaris STED FALCON).
- **No multiphoton.** There is no IR pulsed laser or non-descanned detection. Two-photon and three-photon excitation are not available. For multiphoton imaging, use HCF4 (Nikon AX R MP).
- **4 detection channels (not 5).** Some STELLARIS 8 configurations have 5 detectors (including Power HyD R for extended red/near-IR). This system has 4 channels: 3 × HyD S + 1 × HyD X. The HyD X range ends at 750 nm (not 850 nm). For dyes emitting beyond 750 nm (e.g. Cy7, IRDye 800CW), a HyD R detector would be needed.
- **Oil objectives require #1.5 coverslip.** The 40× and 63× oil objectives (NA 1.30 and 1.40) are designed for #1.5 coverslips (0.17 mm). The 63× water objective has a correction collar for variable cover-glass thickness (0.11–0.23 mm).
- **AOBS operates in the visible range.** The AOBS crystal is transparent from below 400 nm to beyond 4 µm, but beam splitting is optimised for visible wavelengths. UV (355 nm) and IR laser lines require separate input ports and are not installed on this system.

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*Document generated for LLM-assisted workflow design. All pricing, personal identifiers, and contact information have been removed. Specifications sourced from the original equipment quotation, Leica STELLARIS Technical Documentation (12.2024), STELLARIS 8 Scan Head poster (MC-0001385), TauSense Application Note (Nature Methods, September 2020), and AOBS brochure.*