# Leica STELLARIS 8 Tau-STED FALCON — Super-Resolution Confocal / STED Nanoscopy / FLIM

## System Reference for Imaging & Microscopy Workflows

**Version date:** 2026-09-16 (rev. 2026-09-16T13:47 UTC)
**FILM system ID:** HCF3

This document describes a Leica STELLARIS 8 Tau-STED FALCON confocal/STED/FLIM system built on a DMi8 CS inverted microscope stand. The configuration includes a pulsed White Light Laser (440–790 nm) with Pulse Picker, 405 nm solid-state laser, three STED depletion lasers (592/660/775 nm) for 2D/3D Tau-STED nanoscopy, an 8 kHz tandem (FOV + resonant) scanner, four internal Power HyD spectral detectors (2 × HyD S + 1 × HyD X + 1 × HyD R), the FALCON FLIM/FCS module, Lightning computational super-resolution (including STED Expert), environmental control, and STED-White water-immersion optics. 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

| Component | Code | Description |
|---|---|---|
| DMi8 CS | 155933662 | Inverted research microscope stand; fully motorised; confocal-optimised optics path |
| Transmission Axis | 155933666 | Transmitted-light illumination path |
| Binocular Tube | 155933660 | Eyepiece observation |

⚠️ **Note:** This quotation lists a different DMi8 CS part code (155933662) from the HCF2 system's "DMi8 CS Premium" (155933663). The "Premium" designation is not present in the item description for this system — verify on-site whether this is a functionally distinct stand tier.

### 1.2 Focus System

| Component | Code | Description |
|---|---|---|
| Closed Loop Focus | 158204201 | Closed-loop Z motorisation with AFC (Adaptive Focus Control) hardware autofocus |

The AFC hardware autofocus is confirmed present and functional on this system: the FILM Quick Start Guide for HCF3 shows an active AFC control and status indicator in the LAS X Acquire tab. The part code (158204201) differs from HCF2's explicit "with AFC" code (158204202), but the on-site QSG confirms AFC is operational. AFC provides reflection-based drift correction during time-lapse acquisition — essential for long STED/FLIM sessions.

### 1.3 Transmitted-Light Detection

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

⚠️ **DIC optics added after original quotation.** No Wollaston prisms or DIC sliders appear in the source quotation bill of materials, but DIC hardware was added to this system separately and is confirmed operational (DIC is selectable on the microscope TFT touchscreen per the FILM Quick Start Guide). The 63× W motCORR CS2 objective is manufacturer-rated for TL-DIC contrast.

### 1.4 Widefield Epifluorescence

| Component | Code | Description |
|---|---|---|
| Leica LED3 | 158000677 | Multi-channel LED epifluorescence illumination supply + LED transmitted light for ocular screening |
| Filter Cube DsRed | 15525309 | For DsRed / TRITC / mCherry-range dyes |
| Filter Cube GFP | 15525314 | For GFP / FITC / Alexa 488-range dyes |
| Filter Cube LED 405 | 15525338 | For DAPI / Hoechst-range dyes |

Unlike HCF2 (single triple-band DA/FI/TX cube), this system uses three separate single-band filter cubes for ocular/widefield screening.

### 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 (identical part code to HCF2; specifications shared across the platform):

| 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 |

No dedicated petri-dish insert is itemised in this quotation (unlike HCF2's 36 mm insert) — the universal insert is the only sample-holder line item present.

### 1.7 Vibration Isolation & Mounting

| Component | Code | Description |
|---|---|---|
| Optical Table 900 × 900, actively damped | 158005666 | Active vibration-isolation optical table with integral breadboard |

⚠️ **Note:** This single line item bundles breadboard and active damping (unlike HCF2, which lists a separate breadboard, active isolation frame, and air compressor). The covering summary explicitly states "Compressed Air Required" for this table, but no air compressor appears as a line item in this quotation — compressed air supply is presumably drawn from existing facility infrastructure rather than a dedicated unit.

---

## 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 |
| HC PL APO 20×/0.75 CS2 | 15506517 | 20× | 0.75 | Air (dry) | 0.62 | 0.17 mm (#1.5) | Intermediate resolution, tiling |
| 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×/1.20 W motCORR CS2 | 15506361 | 63× | 1.20 | Water (n = 1.33) | 0.30 | With coverslip (TL-BF, TL-DIC, FLUO rated) | Motorised correction collar (software-controlled via motCORR board); AFC-suitable |
| HC PL APO 86×/1.20 W motCORR STED White | 15506333 | 86× | 1.20 | Water (n = 1.33) | 0.30 | 0.14–0.19 mm | STED-White objective; motorised correction collar; primary STED objective for live samples and aqueous media (including STED-FCS); AFC-suitable |
| HC PL APO 100×/1.40 Oil STED White | 15506378 | 100× | 1.40 | Oil (n = 1.518) | 0.13 | 0.17 mm (#1.5) | STED-White objective; highest NA on system; primary STED objective for fixed/mounted samples; no correction collar; very short WD — glass-bottom #1.5/#1.5H coverslips only |

**Note on 100× Oil STED White sourcing:** This objective does not appear as an itemised line in the source quotation bill of materials. However, both the quotation cover page ("STED White Objectives – 100× 1.40NA Oil + 86× 1.20NA Water"), a separate FILM objectives inventory record, and the FILM HCF3 STED Quick Start Guide ("100x oil immersion for fixed and mounted samples") confirm it is present on the instrument. It was likely supplied through a separate order or included as part of the STED module package.

### 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. The oil-immersion 40× lens has a short WD (0.24 mm) and is designed for standard #1.5 glass coverslips only. The 63× and 86× water-immersion objectives share the same WD (0.30 mm) and both use motorised correction collars, giving more coverslip-thickness tolerance than a fixed lens.

| Vessel / Sample Format | Bottom Thickness | Compatible Objectives (this system) |
|---|---|---|
| Standard glass slide + #1.5 coverslip | 0.17 mm | All six objectives |
| Glass-bottom dish (MatTek, ibidi µ-Dish), #1.5 glass | 0.17 mm | All six objectives |
| Glass-bottom multi-well plate, #1.5 glass | 0.17 mm | All six objectives |
| Thick coverslip (#2, 0.19–0.23 mm) | 0.19–0.23 mm | 10×, 20×, 40× Oil (marginal); 63×/86× W motCORR within collar range; NOT 100× Oil (WD 0.13 mm too short) |
| Plastic-bottom plate (polystyrene, ~1 mm) | ~1.0 mm | 10× only (WD 2.56 mm) |
| Plastic-bottom plate (thin, ~0.17 mm) | ~0.17 mm | All six objectives (dry/water only unless polymer is solvent-resistant; avoid oil objectives on polymer) |

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

- For **STED on fixed samples**: use the 100×/1.40 Oil STED White — highest NA gives highest resolution; requires #1.5 or #1.5H glass coverslip.
- For **STED on live samples** or STED-FCS: use the 86×/1.20 W motCORR STED White — water immersion matches aqueous specimen RI and avoids oil-related drift.
- The 40× and 63× lenses are **not** STED-corrected — do not select them for STED acquisition.
- If the user specifies a plastic-bottom plate, do not select any high-NA objective — only the 10× or 20× have sufficient clearance.
- Both the 63× and 86× water objectives have motorised correction collars controlled through LAS X (software-driven via the motCORR board, unlike a manual ring).
- For STED, use only **#1.5H high-precision coverslips** (0.170 ± 0.005 mm) for optimal performance; standard #1.5 (0.170 ± 0.01 mm) is acceptable; #1 coverslips significantly degrade STED resolution and should be avoided.

### 2.3 STED Objective Field of View

STED nanoscopy with a high-NA, high-magnification objective (86×) has a correspondingly small field of view. Confirm the effective FOV at the working zoom before designing tiled or multi-position STED experiments; large-area STED acquisition typically requires stitching multiple small fields.

### 2.4 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 with PP | 158301101 | Confocal point-scanning scan head with integrated Pulse Picker; 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 |

The "with PP" designation distinguishes this scan head from the HCF2 base unit (158301100): the Pulse Picker allows the White Light Laser's native ~78–80 MHz pulse train to be reduced to 40, 20, 10, or 5 MHz, extending the maximum measurable fluorescence lifetime window and reducing STED-related photobleaching — relevant to both FALCON FLIM and Tau-STED on this system.

### 3.2 Beam Path Overview

The complete excitation–detection beam path through the STELLARIS 8 scan head:

1. **White Light Laser** (WLL, 440–790 nm), **405 nm laser**, and (for STED) the **592/660/775 nm depletion lasers** 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).
3. **AOBS** (Acousto-Optical Beam Splitter) reflects selected excitation and depletion lines toward the sample and transmits fluorescence emission toward the detectors.
4. **FOV / 8 kHz Resonant Tandem Scanner** — software-switchable galvanometric (FOV) or 8 kHz resonant point scanner raster-scans the beam across the specimen.
5. **Scan Optics** (HIVISR) — relay the scanned beam to the objective.
6. **Objective Lens** focuses excitation and depletion beams onto the specimen; for STED, the depletion beam is shaped into a doughnut (2D) and/or bottle beam (3D, via the 3D STED upgrade) superimposed on the excitation focal spot.
7. Fluorescence emission returns through the objective → scan optics → scanner → AOBS (transmitted, not reflected).
8. **Square Confocal Pinhole** — rejects out-of-focus light for optical sectioning.
9. **Fluorifier Disc** with analyser — optional polarisation analysis in the detection path.
10. **Notch Filter Set (VIS full)** — suppresses reflected excitation *and* depletion laser light at all installed lines (405, WLL, 592, 660, 775 nm).
11. **Prism-Based Dispersion** — a prism disperses the emission spectrum across the detector array.
12. **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 + 8 kHz Tandem Scanner

| Component | Code | Description |
|---|---|---|
| 8 kHz Tandem Scanner STELLARIS 8 | 158301131 | Combined FOV (galvanometric) + 8 kHz resonant point scanner, software-switchable |

Unlike HCF2 (FOV scanner only), this system has a dual-scanner head. The 8 kHz resonant scanner trades resolution/flexibility for speed; the FOV (galvo) scanner gives full-resolution, full-field, flexible-dwell-time scanning.

| Parameter | FOV (galvo) mode | 8 kHz resonant mode |
|---|---|---|
| Max line frequency (bidirectional) | 5200 Hz | 8000 Hz (resonant) |
| Max frame rate (512 × 512) | 10 fps | ~28 fps (over a reduced ~13 mm field) |
| Max frame resolution | 8192 × 8192 px | Reduced pixel range (fixed dwell time) |
| Scan field diameter (full FOV mode) | 22 mm | 13 mm (typical for 8 kHz resonant at full frame rate) |
| Scan zoom | 0.75–48× | 0.75–48× |
| Field rotation | 200° optical | 200° optical |
| Dwell time | Adjustable | Fixed (resonant) |
| Use case | High-resolution, flexible acquisition; STED alignment; Lightning | Fast live-cell imaging, high frame-rate time-lapse |

Available scan modes: 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, xzλΛ (lambda-lambda / excitation spectrum scanning).

### 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 and depletion 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; broader emission collection bands than a fixed dichroic.
- Up to 8 simultaneous laser lines in both fluorescence and reflection mode.
- Switching time < 10 µs between line configurations.
- Fully transparent from below 400 nm to beyond 4 µm.
- No mechanical wear; no alignment drift.

### 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 through the scan head |
| Notch Filter Set VIS Full | 158204512 | Full visible-range notch filter set — suppresses reflected excitation *and* STED depletion laser light (405, WLL, 592, 660, 775 nm); broader coverage than the "VIS Base" set on non-STED systems |
| 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 (platform maximum is 5; position 1 is not populated on this system). 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 R | 158301335 | HyD R (Red-optimised) | Extended red/near-IR sensitivity; FALCON-capable |

### 4.2 Detector Specifications

| Property | Power HyD S | Power HyD X | Power HyD R |
|---|---|---|---|
| Sensor type | Multi-pixel silicon photo-multiplier array | GaAsP hybrid detector | Red-optimised hybrid detector |
| Spectral sensitivity range | 410–850 nm | 410–750 nm | ~720/740–850 nm (approximate; manufacturer sources vary on exact cut-on) |
| Typical PDE (@ 500 nm) | 58 % | 46 % | Not verified for this position — request manufacturer datasheet |
| Detection modes | Analog, reflection, counting, fast | Digital, counting | Digital, counting |
| FALCON capability (FLIM/FCS) | No | Yes | Yes |

⚠️ **Note:** With HyD R installed, this system covers the near-red/NIR emission range (beyond ~740 nm) that HCF2 explicitly lacks — relevant for far-red STED dyes and NIR probes (e.g. beyond Cy5/AF647).

### 4.3 Spectral Detection Unit — Summary

| Parameter | Value |
|---|---|
| Spectral detection range | 410–850 nm (HyD S/HyD R); 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) and full FALCON FLIM (§8) during acquisition.

### 4.5 Transmitted-Light Detection

Brightfield images are acquired via the BF detector (158004201) in the stand base, using the LED3 transmitted-light source through the condenser. See §1.3 for the note on DIC hardware absence.

---

## 5. Laser Sources

### 5.1 White Light Laser (WLL) with Pulse Picker

| Parameter | Value |
|---|---|
| Type | Pulsed VIS White Light Laser with Pulse Picker |
| Spectral range | 440–790 nm (continuously tuneable) |
| Tuning step | 1 nm |
| Simultaneous lines | Up to 8 independent lines (selected via AOTF) |
| Native repetition rate | 78 MHz |
| Pulse Picker settings | 80, 40, 20, 10, 5 MHz (software-selectable) |

Power per line (typical, from manufacturer documentation for this laser generation):

| 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 Pulse Picker reduces the effective pulse repetition rate below the native 78 MHz, extending the maximum unambiguous lifetime window (12.5 ns at 80 MHz) for slower-decaying fluorophores, and is used to manage STED excitation dose. The pulsed WLL, combined with the AOBS, enables TauSense lifetime-based contrast, time-gated detection, and full FALCON FLIM without additional excitation hardware.

### 5.2 405 nm Solid-State Laser

| Component | Code | Description |
|---|---|---|
| Laser 405 DMOD | 158202140 | 405 nm diode laser with direct modulation (DMOD); 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 |

⚠️ The 405 nm laser is continuous-wave (not pulsed) and therefore cannot be used for FLIM/FALCON or the semi-quantitative TauSense modes — only the pulsed WLL lines support lifetime-based contrast.

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

### 5.3 STED Depletion Lasers

| Component | Code | Depletion wavelength | Notes |
|---|---|---|---|
| STELLARIS STED 592 | 158305604 | 592 nm | Depletion line for orange/yellow-excited dyes (e.g. ATTO 594, Alexa Fluor 594-range) |
| Laser Upgrade STED 660 A | 158005654 | 660 nm | Depletion line for red dyes (e.g. STAR RED, Alexa Fluor 594/647-range) |
| Laser Upgrade STED 775 A | 158005659 | 775 nm | Depletion line for far-red dyes; matched to the WLL's pulsed output for Tau-STED gating |

All three depletion lines cover the visible-to-far-red spectrum, enabling multicolour STED across most common dye panels. Depletion laser power and pulse timing are managed by LAS X and are not independently specified in the source quotation — request current values from Leica or verify via Device Setup before quantitative work.

⚠️ **Fluorophore caution:** Alexa Fluor 647 is widely reported to photobleach very rapidly under STED depletion on this dye/laser combination — avoid it for STED imaging on this system; STAR RED, CF680R, and ATTO 647N are commonly reported as more STED-stable alternatives in the far-red channel.

### 5.4 Excitation Modulation

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

### 5.5 Available Excitation/Depletion Lines — Summary

| Source | Lines (nm) | Type | Role |
|---|---|---|---|
| White Light Laser (WLL) | 440–790 (any, 1 nm steps, up to 8 simultaneous) | Pulsed supercontinuum, 78 MHz (Pulse Picker: 80/40/20/10/5 MHz) | Excitation; FLIM/TauSense; STED excitation |
| 405 nm DMOD | 405 (fixed) | CW diode, direct modulation | Excitation only (not FLIM-capable) |
| STED 592 | 592 (fixed) | Depletion | STED depletion, orange/yellow dyes |
| STED 660 | 660 (fixed) | Depletion | STED depletion, red dyes |
| STED 775 | 775 (fixed) | Pulsed depletion | STED depletion, far-red dyes; Tau-STED gating |

---

## 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 full data overhead of 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). Lifetime information is independent of fluorophore concentration, making it a method of choice for functional imaging.

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. The zero value of the measurement is calibrated using the reflection signal from the excitation laser reaching each detector. The FPGA scan-head electronics sort detected photons into digitally preset gates depending on their arrival times and 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.

TauSense does not aim to replace FLIM — it provides access to a lifetime-based level of information in a guided way during standard confocal acquisition. For full quantitative FLIM with multi-exponential fitting and phasor analysis, the FALCON module (§8) is used.

### 6.2 TauSense Tools

| Tool | Function | Detail |
|---|---|---|
| **TauContrast** | Maps mean photon arrival time per pixel to a colour scale | Semi-quantitative lifetime contrast relative to a control; real-time; 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; delivers both the gated image and the gated-out image for quality control |
| **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 lifetime-component distribution; produces intensity images in discrete temporal windows |
| **TauSeparation** | Separates spectrally overlapping fluorophores by lifetime | User selects representative mean lifetime components from the online diagram; TauSeparation selects appropriate temporal windows and fits the lifetime-based information to generate separated images; 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 |
| **GateScan** | Differentiates desired and unwanted fluorescence signals | Complements TauGating with scanning-based gating |

### 6.3 TauSense and Tau-STED

On this system, the same pulsed-laser/photon-timing infrastructure that underlies TauSense is also used by **Tau-STED** (§7) to map the STED depletion response of each fluorophore in real time, suppressing residual, incompletely-depleted background and pushing effective resolution beyond intensity-based STED alone — see §7.2.

---

## 7. Tau-STED — Stimulated Emission Depletion Nanoscopy

### 7.1 STED Module & Accessories

| Component | Code | Description |
|---|---|---|
| STELLARIS 8 STED Adapter | 158305600 | Optical/mechanical adapter coupling the STED module to the scan head |
| STED Adapter Kit DMi8 | 158005689 | Mechanical adapter coupling the STED module to the DMi8 stand |
| Notch Filter Set STED 3× | 158004503 | Suppresses reflected depletion laser light in the detection path |
| 3D STED Upgrade for STED 3× | 158005677 | Enables 3D-STED: adds a bottle-beam (axial) depletion pattern alongside the standard doughnut (lateral) pattern for combined lateral + axial resolution improvement |

### 7.2 Tau-STED Principle & Performance

Standard STED confines the fluorescence emission spot below the diffraction limit by overlapping the excitation focus with a doughnut-shaped depletion beam that stimulates emission everywhere except at the very centre. **Tau-STED** additionally exploits the fact that a fluorophore's excited-state lifetime shortens with STED depletion strength: photons detected with a shorter apparent lifetime originate from positions closer to the depletion-beam centre (i.e., higher spatial precision), so time-gating/weighting photons by arrival time recovers extra resolution and suppresses out-of-focus/incompletely-depleted background without simply increasing depletion laser power (which would increase photobleaching).

**TauSTED phasor-based mechanism** (from Alvarez et al., Nature Methods Application Note, June 2021): TauSTED uses phasor analysis in a novel way. The STED process creates a lifetime gradient across the effective PSF — the maximum STED energy sets the shortest lifetime while the minimum (at the doughnut centre) marks the longest. These two values mapped into a phasor plot define a "STED trajectory" containing all photons emanating from the STED process. TauSTED automates the determination of this trajectory using a complex wavelet filter that preserves fine structures even at very low photon budgets. Two parameters control the output:

- **TauBackground Suppression:** Removes photon signals with fluorescence lifetime signatures not correlated to the STED trajectory (noise, background, autofluorescence). Can be toggled on/off to evaluate its effect.
- **TauStrength:** Weights intensity values based on their positions along the TauSTED trajectory — higher values give more weight to longer-lifetime signals (centre of doughnut, higher spatial precision), pushing resolution further. The limit is set by the photon budget and SNR.

**TauSTED Xtend** (available on the STELLARIS platform) combines lifetime-based information with an additional spatial readout, giving a significant resolution gain at lower STED depletion powers and extending multicolour live imaging capabilities at nanoscale.

| Parameter | Value |
|---|---|
| Depletion lines | 592 nm, 660 nm, 775 nm |
| Lateral resolution | Typically < 50 nm (fixed samples); best-case figures under optimal dye/sample conditions reported down to ~20–30 nm (demonstrated on GATTA-Beads R, nominal 23 nm diameter) |
| Axial resolution | Typically < 130 nm (typically ~100 nm with 3D STED); with 3D STED upgrade, improved axial confinement via combined doughnut + bottle-beam depletion |
| Multicolour capability | Multi-channel, multi-colour standard, gated, or Tau-STED; demonstrated with simultaneous 592/660/775 nm depletion (e.g. three-colour TauSTED of mitotic cells) |
| Live-cell STED | Supported; TauSTED reduces excitation and STED light dose — demonstrated at 1 frame/s with 85 % fluorescence retention after 100 consecutive frames |
| STED-capable objectives | 100×/1.40 Oil STED White (fixed samples, highest NA); 86×/1.20 W motCORR STED White (live samples, aqueous media, STED-FCS) |

⚠️ All resolution figures above are approximate and strongly sample-, fluorophore-, and condition-dependent; treat as indicative rather than guaranteed performance.

### 7.3 FALCON Integration for Tau-STED

Because Tau-STED depends on photon arrival-time information, it requires the same FALCON-capable detectors (Power HyD X, Power HyD R) and FPGA electronics used for FLIM (§8). On this system, both are present, so Tau-STED and quantitative FLIM/FALCON can be combined in the same experiment (e.g., FLIM-FRET readout on a STED-resolved structure).

### 7.4 STED Fluorophore Guide

The three depletion lines on this system (592, 660, 775 nm) cover the full visible spectrum for STED-compatible fluorophores. The following recommendations are sourced from Leica's STED sample preparation guide and are rated based on internal and external feedback. Ratings may vary with microenvironment, labelling density, mounting medium, and sample age.

**Top recommended labels for single-colour STED:**

| STED line | Fixed samples (top 3) | Live cells (top 3) |
|---|---|---|
| 592 nm | Oregon Green 488 (excellent), Alexa Fluor 488 (excellent), STAR 488 (excellent) | Citrine / mVenus / mNeonGreen |
| 660 nm | Alexa Fluor 555 (excellent), ATTO 542 (excellent), Cy3 (excellent) | SPY555 probes |
| 775 nm | STAR 635P (excellent), ATTO 647N (excellent*), Alexa Fluor 594 (excellent) | SiR probes / SPY650 |

*ATTO 647N is hydrophobic and may require labelling optimisation to suppress background.

**Top recommended label pairs for dual-colour STED (single STED line):**

| Label #1 | Exc (nm) | Em (nm) | Label #2 | Exc (nm) | Em (nm) | STED (nm) |
|---|---|---|---|---|---|---|
| STAR 440SX | 458/470 | 475–510 | Oregon Green 488 | 514/520 | 523–580 | 592 |
| Alexa Fluor 532 | 532 | 520–565 | TMR / TRITC / Cy3 | 580 | 590–650 | 660 |
| AF 594 / ATTO 594 | 590 | 600–630 | STAR 635P / ATTO 647N | 635/650 | 655–750 | 775 |
| STAR 580 | 575 | 585–640 | STAR 635P / ATTO 647N | 635/650 | 655–750 | 775 |

⚠️ **Critical LLM routing rules for STED fluorophore selection:**

- **Avoid Alexa Fluor 647** — rapid photobleaching under STED depletion. Use STAR RED, STAR 635P, ATTO 647N, CF680R, or SiR instead.
- **Avoid DAPI and Hoechst** for counterstaining — they cause background, especially with the 592 nm STED line. For nuclear staining in STED, use Picogreen (592/660 nm STED) or SiR-DNA (775 nm STED).
- **Avoid mounting media containing DAPI** and avoid Vectashield (alters fluorescence quantum yield of large Stokes-shift dyes).
- For proximity/colocalisation studies, image with a **single STED line** and two labels with different emissions — channels are then intrinsically co-aligned.
- Samples must be **bright and photostable in confocal mode** before switching to STED.
- Use **2–5× higher secondary antibody concentrations** than standard confocal protocols for optimal STED labelling density.
- Consider **nanobody-conjugated dyes** (e.g. from abberior or Jackson ImmunoResearch) for smaller tag size — the ~10 nm IgG antibody size can become the resolution-limiting factor at STED scales.

**Abberior STAR dyes** (from [abberior.rocks](https://abberior.rocks/dyes-labels/abberior-star/)) are specifically optimised for STED and confocal and are an excellent alternative to the Leica-recommended dye panel above. Key dyes compatible with this system's STED lines:

| abberior Dye | Exc (nm) | STED line | Notes |
|---|---|---|---|
| abberior STAR 488 | 488 | 592 | Green; high brightness; available as phalloidin, NHS, nanobody conjugates |
| abberior STAR 580 | 580 | 775 | Orange; excellent dual-colour partner with STAR RED at 775 nm; available as phalloidin, nanobody conjugates |
| abberior STAR ORANGE | ~550 | 775 | Membrane stain and conjugates; good for live-cell STED |
| abberior STAR RED | 635 | 775 | Far-red; top-tier STED performance; available as membrane stain, phalloidin, NHS, nanobody conjugates |
| abberior STAR 635P | 635 | 775 | Far-red; high photostability; excellent for fixed samples |
| abberior STAR 460L | 460 | 775 | Long Stokes-shift; enables 3-colour STED with a single 775 nm depletion line (STAR 460L + STAR ORANGE/580 + STAR RED) |
| abberior STAR GREEN | ~490 | 592 | Green; used for NPC staining |

abberior also offers **LIVE dyes** (cell-permeable organic fluorophores for live-cell STED), **LIVE HaloX** (exchangeable HaloTag ligands for extended live imaging), and **FLUX dyes** (optimised for MINFLUX/STORM blinking). The abberior STAR series is available as NHS esters, maleimides, phalloidin conjugates, nanobody conjugates, and SNAP/Halo-tag ligands from [abberior.shop](https://abberior.shop/).

### 7.5 STED Sample Mounting & Substrate

- **Coverslips:** Use #1.5H high-precision glass (0.170 ± 0.005 mm) for optimal STED performance. Standard #1.5 (0.170 ± 0.01 mm) is acceptable. #1 coverslips significantly decrease image quality and should be avoided.
- **Recommended mounting media:** Prolong Gold, Prolong Diamond (wait ≥ 24 h curing), Mowiol + DABCO (2.5 %), glycerol/PBS mixtures (RI 1.33–1.47). Avoid Vectashield and p-phenylenediamine-based antifade reagents.
- **Match refractive index:** Mounting medium RI should match the immersion medium (n = 1.518 for oil objectives; n = 1.33 for water objectives). RI mismatch degrades the STED doughnut shape.
- **Glass-bottom dishes/chambers:** Lab-Tek Chamber Slide System, ibidi µ-slides/µ-dishes, MatTek glass-bottom dishes with #1.5H glass.
- **Avoid polymer coverslips and embedding resins** — they may absorb at the STED depletion wavelengths (592, 660, 775 nm) and be destroyed during imaging.

### 7.6 STED Beam Alignment

STED depletion laser alignment must be performed at startup after the STED lasers have warmed up for at least 30 minutes. In LAS X, under the Configuration tab → STED, run "Align Beams" (takes approximately 3 minutes). This auto-aligns all active depletion lines to the excitation focal spot — misaligned depletion beams result in asymmetric PSFs and degraded resolution.

### 7.7 Detection Modes for STED

The STED QSG documents three detector operating modes available during STED acquisition on this system, selected per channel in the LAS X detector settings:

| Mode | Operating Mode | Use case |
|---|---|---|
| **Analog** | Analog (intensity-based) | Standard STED — conventional intensity detection; no lifetime information |
| **TauSTED** | Counting | TauSTED — lifetime-weighted STED; automated background suppression via TauStrength and Denoise parameters; adjustable time gate for fluorescence lifetime range selection |
| **TauGating** | Counting | Time-gated STED — manual positioning of lifetime gates to isolate signal; adjustable gate positions on a nanosecond timeline |
| **FLIM STED** | FLIM module | Full quantitative FLIM on STED data — requires the FLIM module and HyD X/R detectors; select "FLIM STED" in the Dye Assistant |

---

## 8. FALCON — Quantitative FLIM / FCS / FCCS / FLCS

### 8.1 FALCON Module

| Component | Code | Description |
|---|---|---|
| STELLARIS 8 FALCON | 158305100 | FAst Lifetime CONtrast — full quantitative FLIM module |
| STELLARIS 8 FCS | 158305101 | Fluorescence Correlation Spectroscopy module |

Unlike HCF2 (TauSense only, no FALCON licence), this system has the full FALCON module installed, enabling quantitative fluorescence lifetime imaging: multi-exponential decay fitting, phasor analysis, high photon-flux FLIM, and FLIM-FRET analysis. FALCON records FLIM in up to 4 spectral channels simultaneously and up to 10 channels sequentially, and is fully integrated into the standard LAS X acquisition workflow (3D stacks, time-lapse, tiling).

The FCS module adds fluorescence correlation spectroscopy for measuring diffusion coefficients, concentrations, and molecular interactions from intensity fluctuations at a fixed point or small region; combined with FALCON's lifetime data this also supports **FCCS** (cross-correlation between two spectral channels, for binding/interaction studies) and **FLCS** (Fluorescence Lifetime Correlation Spectroscopy, using lifetime to filter correlation data), consistent with the covering summary's "FALCON FLIM + FCCS + FLCS" description.

### 8.2 FALCON Technical Architecture

The FALCON FLIM approach is based on the FPGA scan-head electronics (§4.4) and differs fundamentally from traditional TCSPC-based FLIM. Key performance characteristics (from Alvarez et al., Nature Methods Application Note, October 2019):

| Parameter | Value |
|---|---|
| Temporal resolution | 97 ps |
| System dead time | < 1.5 ns |
| Max count rate per detector | 80 Mcps (at 80 MHz pulsed laser) |
| Photon flux for accurate lifetimes | Up to 225 Mcps with 0.2–1.6 % error (high-speed FLIM filter) |
| Photon filter modes | High-speed FLIM filter (default; avoids pile-up), First photon filter (mimics traditional TCSPC), All photons mode |
| Multi-detector approach | Multiple spectral detectors can be combined and treated as one, increasing effective photon flux |
| FLIM speed | Video-rate FLIM acquisition possible; pixel-by-pixel quantification |
| Phasor analysis | Yes (installed per quotation cover summary) |

The high-speed FLIM filter ensures that only single-photon events between laser pulses are used for building the overall decay curve, avoiding the pile-up effect that plagues traditional TCSPC at high count rates. The per-pixel fitting then uses the unfiltered data with the overall mathematical model derived from the filtered decay — combining accurate model fitting with maximum photon statistics.

### 8.3 FALCON Requirements

Quantitative FLIM/FCS requires:
- A pulsed excitation line (WLL only — not the 405 nm CW laser).
- A FALCON-capable detector: Power HyD X (Pos 4) or Power HyD R (Pos 5) on this system.
- The FPGA-based photon-counting electronics (§4.4), standard on this scan head.
- Maximum unambiguous lifetime window: 12.5 ns at 80 MHz; extendable to 200 ns at 5 MHz via the Pulse Picker (§5.1) — select the Pulse Picker setting to match the expected fluorescence lifetime of the sample.
- Keep photon flux below 1 photon per pulse per detector to avoid pile-up; the high-speed FLIM filter tolerates up to ~2.5 photons per pulse.

### 8.3 Software Integration

| Component | Code | Description |
|---|---|---|
| LAS X FLIM Offline Analysis | 158303252 | Offline FLIM data processing and fitting, for a second (non-acquisition) workstation |
| LAS X FCS Offline Analysis | 158303253 | Offline FCS data processing |

---

## 9. Lightning — Confocal Super-Resolution

### 9.1 Lightning Modules

| Component | Code | Description |
|---|---|---|
| LAS X Lightning Expert | 158203204 | Computational super-resolution modality with Expert mode for advanced parameter control |
| LAS X Lightning STED Expert | 158303205 | Extends Lightning deconvolution/adaptive processing to STED datasets |

### 9.2 Principle & Performance

Lightning is a fully automated intelligent information extraction method for confocal data, operating in near real-time using parallel GPU processing (from Reymann, Lightning White Paper, Leica Microsystems, September 2018). The key difference from conventional deconvolution lies in the voxel-precise, on-the-fly evaluation of image properties — an adaptive process that correlates the deconvolution parameter space with local image quality for each volume segment.

**Processing pipeline:**

1. **Pre-Processing:** Local image properties (background and signal-to-noise ratio) are determined with voxel accuracy. A global background estimate is computed and related to the local SNR for each pixel.
2. **Decision Mask:** An n-dimensional adaptive mask (where n = number of acquisition channels) maps the local image quality characteristics (SNR, background) per voxel. This defines voxel-specific deconvolution parameters — higher SNR regions receive more aggressive deconvolution (lower regularisation), lower SNR regions receive stronger regularisation to prevent noise amplification.
3. **Deconvolution:** A Richardson-Lucy algorithm with a physically modelled Point Spread Function (adapted to the imaging method: confocal, STED, etc.) is applied per voxel with the parameters extracted from the Decision Mask. The iteration count is fully automated — terminated when two successive iterations show no significant difference.
4. **Normalisation:** The photon number of the pre-processed image is used to normalise the output, ensuring output images are quantitatively comparable.

**Key properties:**

- Preserves the sum of all intensities and the total photon number between input and output images.
- Original confocal raw data is always retained — Lightning results sit alongside, not instead of, the raw data.
- Non-adaptive mode (global-parameter deconvolution matching conventional methods) is also available.
- Lightning STED Expert applies the same adaptive pipeline to STED data, using a STED-specific PSF model.

| Parameter | Value |
|---|---|
| Confocal Lightning lateral resolution | Down to ~120 nm (at NA 1.40; demonstrated on 120 nm molecular nanorulers) |
| Confocal Lightning axial resolution | Down to ~200 nm (at NA 1.40) |
| Number of spectral channels | All installed channels simultaneously |
| Scanner compatibility | FOV and 8 kHz resonant scanners |
| Detector compatibility | All Power HyD channels |
| Objective compatibility | All objectives (resolution scales with NA) |
| Expert mode | User control over deconvolution parameters; adjustable signal-to-noise vs resolution trade-off |
| Processing | Near real-time via GPU; fully automated or expert-tuneable |
| Quantifiability | Fully quantifiable — photon counts preserved; no intensity-based distortion within PSF correlation width |

---

## 10. Environmental Control

### 10.1 Incubation System — Okolab

| Component | Code | Description |
|---|---|---|
| Okolab Transparent Box Incubator DMi8 | 158206046 | Whole-microscope enclosure; transparent chamber covering the entire DMi8 stand |
| Okolab Sample Chamber Inv SuperZ | 158206037 | Stage-top incubator insert compatible with the SuperZ galvo stage |
| Okolab CO2, Humidity Passive | 158206039 | CO2 gas mixer + passive humidity control |

### 10.2 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 control | Environmental control is selectable in LAS X via the "machine-Climate-Control.xlhw" hardware configuration at startup; the Okolab controller must be powered on before launching LAS X with this option. Manual control via the Okolab controller is also possible independently of the software. |

### 10.3 Sample Holders

The following sample holders are available on this system (documented in the FILM HCF3 Quick Start Guide):

| Holder | Accepts |
|---|---|
| Universal Holder | Standard slides (26 × 76 mm) and 35 mm dishes |
| 35 mm Dish Insert | Adapts the universal holder for 35 mm petri dishes |
| Multiwell Plate Holder | Multi-well plates (6-well to 96-well typical); with environmental lid |
| Multi-Format Holder | Multiple sample formats in one holder; with environmental lid |

Sample holders are secured by small screws (4 turns to loosen; do not completely unscrew). Allen key provided. Do not overtighten.

---

## 11. Software — LAS X

### 11.1 Core Platform

| Module | Code | Description |
|---|---|---|
| LAS X STELLARIS Control Software | 158203200 | Full confocal/STED/FLIM system control: Image Compass user interface; full hardware control |
| LAS X Offline Software | 158203251 | Base offline analysis licence for a second (non-acquisition) workstation |

### 11.2 Application Modules (installed)

| Module | Code | Description |
|---|---|---|
| LAS X Lightning Expert | 158203204 | Adaptive confocal deconvolution (§9) |
| LAS X Lightning STED Expert | 158303205 | Adaptive deconvolution for STED data (§9) |
| LAS X FLIM Offline Analysis | 158303252 | Offline FLIM processing (§8.3) |
| LAS X FCS Offline Analysis | 158303253 | Offline FCS processing (§8.3) |

### 11.3 Core Functionality (included with LAS X STELLARIS Control Software)

The following features are included as part of the core control software and are confirmed functional on this system via the FILM Quick Start Guides:

| Feature | Description |
|---|---|
| TauSense | Integrated lifetime-based contrast (TauContrast, TauGating, TauSeparation, TauScan, TauInteraction) — no additional module |
| Dynamic Signal Enhancement | Maintains optimal SNR at high frame rates |
| Lightning basic detection | Basic adaptive deconvolution (Lightning Expert is the advanced upgrade, installed) |
| Navigator basic | Spiral scan for specimen overview; stitching; mark-and-find; multi-position acquisition; tiling with focus map |
| Lambda scan / Lambda-lambda scan | Emission and combined excitation-emission spectrum acquisition (with 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 |
| Dye Assistant | Database-driven automatic channel setup — select fluorophores and the system auto-assigns excitation wavelength, detector range, and sequential method |
| IPS (Instrument Parameter Settings) | Save/load full acquisition configurations from XML files (stored on E:\ drive); enables reproducible experiment setups across sessions and users |

### 11.4 Navigator — Tiling & Multi-Position Acquisition

The Navigator module (confirmed operational from FILM HCF3 protocols) provides:

- **Spiral overview scan:** Low-magnification (10×/20×) rapid overview of the entire sample using the Spiral or pre-defined region button.
- **Tiling with Focus Map:** Define a tile region, add focus points (for sample tilt/curvature correction), set Z at each focus point, then acquire a stitched mosaic. Raw tiles and merged image are both stored.
- **Tiled Z-stacks:** Combine tiling regions with Z-stack acquisition; "Same Stack Size for all Regions" toggle; Z-galvo or motorfocus selectable; merged 3D volumes may contain interpolated slices from alignment.
- **Multi-position single images:** Mark individual positions on the preview; record Z at each; batch-acquire all positions.
- **Multi-position Z-stacks:** Combine multi-position marking with a fixed Z-stack applied to all positions.
- **Acquisition modes:** xyz, xyt, xyzt, xyλ, xyλt, xyzλ, xyzλt available from the Acquisition Mode selector.

⚠️ **Software modules not present in this configuration** (in contrast to HCF2, where these are itemised as separate paid modules): **LAS X 3D Visualisation**, **LAS X Dye Finder**, **LAS X MicroLab** (dedicated FRAP/FLIP/FRET workflow module), **LAS X FRAP Zoomer**, **LAS X Co-Localisation** (quantitative Pearson's/Manders' analysis), and **LAS X Assay Editor** (automated multi-well/multi-position screening) do not appear as line items in this quotation. Basic 3D viewing, stitching/Navigator, and lambda(-lambda) scanning are expected to be present as part of the core control software (as on HCF2), but the dedicated advanced modules listed above should be assumed absent unless confirmed on-site.

No **Switchable Beam Expander** (FRAP Booster) item appears in this quotation either — sequential ROI-based photobleaching may still be possible through the core acquisition software, but dedicated high-speed/zoomed FRAP support is more limited than on a system with the FRAP Zoomer module.

For workflows needing 3D volume rendering, quantitative colocalisation, dedicated FRAP/FRET protocols, or automated multi-well screening, consider HCF2 (which has these modules) or verify whether they have been added to HCF3 since this quotation.

---

## 12. Operational Procedures & Constraints

This section contains practical operating rules and parameters that affect experiment design. It is derived from the FILM Quick Start Guides for HCF3 (confocal and STED modes).

### 12.1 Power-On Sequence — Confocal Mode

1. Start PC.
2. On the central power unit: switch on Power, then Laser, then turn the Laser Emission Key.
3. **Do not load a sample until the microscope has finished starting up** — the stage will auto-calibrate (move) and fingers can be trapped.
4. Log in to the IC network account.
5. Wait until the TFT screen on the front of the microscope has finished booting.
6. Start LAS X on the desktop. In the startup window, select `machine.xlhw` from the Configuration drop-down. **Ensure the STED option is deselected** for confocal-only sessions.

### 12.2 Power-On Sequence — STED Mode

1. Follow steps 1–5 of the confocal sequence above.
2. On the **STED Laser Power Unit** (separate rack): insert the laser keys and switch on the laser power buttons (do not turn on the keys yet).
3. Place the safety cover over the condenser.
4. Start LAS X. Select `machine.xlhw` and **select the STED option** (ON).
5. In the Configuration tab → Laser Config, turn on lasers starting with the WLL (white light) laser. **The STED 775 laser will not start unless the WLL is already on.**
6. Turn on STED laser keys on the STED Laser Power Unit.
7. Allow STED lasers to warm up for **≥ 30 minutes** before performing the STED beam alignment (§7.6).

### 12.3 Shutdown Sequence

1. In the Configuration tab → Laser Config, **switch off all lasers**.
2. Close LAS X.
3. Remove samples and clean objective lenses with fresh lens tissue.
4. If STED was used: remove safety cover from condenser; on the STED Laser Power Unit, switch off laser keys then power buttons.
5. On the central power unit: switch off Laser Key, then Laser, then Power.
6. Save files onto the server. Shut down PC or sign out for the next user.

### 12.4 Hardware Configurations in LAS X

| Configuration file | Use |
|---|---|
| `machine.xlhw` (STED OFF) | Standard confocal acquisition — no STED depletion lasers |
| `machine.xlhw` (STED ON) | STED nanoscopy — enables STED laser control, beam alignment, and STED detector modes |
| `machine-Climate-Control.xlhw` | Confocal or STED with environmental control — Okolab must be powered on before launching |

### 12.5 Channel Setup Methods

Three methods are available for setting up confocal/STED channels, in order of recommendation:

1. **Dye Assistant (automatic):** Select fluorophores from the database; system auto-assigns excitation wavelength, detector range, and sequential scan method (line sequential or frame sequential). For STED: change the detector mode to "S" (STED) by clicking the detector type selector; use frame sequential for STED (line sequential is not recommended for STED).
2. **Load existing setup:** Load from a previously saved channel configuration file or from a previously acquired image (via the APPLY icon in the Projects tab). Note: objective, pixel number, bit depth, zoom, and averaging are not reloaded and may need re-setting.
3. **IPS (Instrument Parameter Settings):** Load complete acquisition parameters from an All_Users.xml file on the E:\ drive — loads all image settings including scan mode, format, and channel configuration.

### 12.6 STED Acquisition Setup — Practical Rules

From the FILM HCF3 STED Quick Start Guide:

- Find the sample using a high-NA objective: **86× water immersion for live samples** or **100× oil immersion for fixed and mounted samples**.
- Set up the channels using the Dye Assistant (STED mode) or manual setup.
- **Switch off the STED shutter** and use "Fast Live" to find and focus the sample first.
- In the XY window, set acquisition parameters: pixels, zoom, averaging/accumulation/speed. For best results, set pixel size to be **better than Nyquist** (e.g. 1024 × 1024 at zoom 5).
- Try to keep **excitation laser intensity low** (use slower speed and/or accumulation rather than higher laser power).
- Open STED shutter and adjust STED parameters (depletion intensity, 3D STED slider, Smart Pinhole).
- Capture image.

### 12.7 Data Format

Images are stored in the Leica **LIF library format** (.lif files). Each Capture Image or Start press adds the image to the current Library in the "Open Projects" tab, but **images are not auto-saved** — the user must click the Save icon to persist data to disk. Projects can be browsed, renamed, and exported from the Open Projects panel.

---

## 13. Workstation & Display

### 13.1 Workstation

| Component | Code | Description |
|---|---|---|
| Workstation Premium | 158203112 | CUDA-enabled GPU workstation (dedicated HP) for Lightning deconvolution, STED, and FALCON processing |

⚠️ This is a different tier code from HCF2's "Workstation Expert" (158203113); detailed CPU/RAM/GPU specifications are not given in the source quotation and should be requested from Leica or verified on-site if needed for workflow planning (e.g. batch FALCON/Lightning processing throughput).

### 13.2 Monitor

| Component | Code | Description |
|---|---|---|
| High Brilliance Monitor, flat (×2) | 158003151 | Two flat high-brilliance monitors |

Two monitors are itemised (qty 2), a different code and count from HCF2's single curved 37.5″ ultrawide monitor (158003150).

### 13.3 Furniture

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

---

## 14. Capability Summary

This system supports the following imaging modalities and workflows:

| Modality | Key Components |
|---|---|
| **Laser-scanning confocal** | STELLARIS 8 scan head with Pulse Picker, dual FOV + 8 kHz resonant tandem scanner, WLL (440–790 nm) + 405 nm, AOBS (8 lines), 4-ch Power HyD spectral detection (410–850 nm) |
| **Fast/resonant imaging** | 8 kHz tandem resonant scanner (up to ~28 fps @ 512×512 over a reduced field), software-switchable with the FOV scanner |
| **Tau-STED nanoscopy** | 592/660/775 nm depletion lasers, 3D STED upgrade, 100×/1.40 Oil STED White + 86×/1.20 W motCORR STED White objectives, Lightning STED Expert; typical resolution < 50 nm lateral / < 130 nm axial |
| **Quantitative FLIM (FALCON)** | FALCON module, HyD X + HyD R detectors, FPGA photon-counting electronics; up to 4 simultaneous / 10 sequential channels |
| **FCS / FCCS / FLCS** | STELLARIS 8 FCS module + FALCON lifetime data; offline analysis licence |
| **Lifetime-based contrast (TauSense)** | TauContrast, TauGating (16 gates), TauScan, TauSeparation, TauInteraction; no additional hardware beyond the pulsed WLL |
| **Confocal super-resolution (Lightning)** | Lightning Expert + Lightning STED Expert; adaptive online deconvolution |
| **Spectral imaging & unmixing** | Continuous spectral tuning via AOBS + prism detection; lambda scan; lambda-lambda scan (with WLL) |
| **Live-cell imaging** | AFC hardware autofocus (drift correction); Okolab box + stage-top incubator (temp/CO2/humidity); SuperZ galvo stage; motorised correction-collar water objectives |
| **Tiling / large-area** | Motorised XY scanning stage; Navigator module with spiral overview, focus map, multi-position, tiled Z-stacks, and stitching |
| **Widefield fluorescence (ocular)** | LED3 epifluorescence; DsRed / GFP / 405 single-band filter cubes |
| **DIC / Brightfield** | DIC optics (added post-quotation, confirmed operational); BF transmitted-light detector; LED3 transmitted light |

---

## 15. Excitation / Detection Quick-Reference

### 15.1 Available Laser Lines

| Source | Lines (nm) | Role |
|---|---|---|
| White Light Laser (WLL) | 440–790 (any, 1 nm steps, up to 8 simultaneous) | Excitation, FLIM, STED excitation |
| 405 nm DMOD | 405 (fixed) | Excitation only |
| STED depletion lasers | 592, 660, 775 (fixed) | STED depletion |

### 15.2 Widefield Filter Cubes

| Cube | Fluorophore Match |
|---|---|
| Filter Cube LED 405 | DAPI, Hoechst |
| Filter Cube GFP | GFP, FITC, Alexa 488 |
| Filter Cube DsRed | DsRed, TRITC, mCherry |

### 15.3 Detector Spectral Coverage

| Detector | Range | Sensor | FALCON-capable | Channels on this system |
|---|---|---|---|---|
| Power HyD S (×2) | 410–850 nm | Multi-pixel silicon photo-multiplier | No | Internal ch 2, 3 |
| Power HyD X (×1) | 410–750 nm | GaAsP hybrid | Yes | Internal ch 4 |
| Power HyD R (×1) | ~720/740–850 nm (approx.) | Red-optimised hybrid | Yes | Internal ch 5 |
| BF Detector | Transmitted light | — | — | 1 (external) |

---

## 16. Key Specifications at a Glance

| Parameter | Value |
|---|---|
| Microscope type | Inverted (Leica DMi8 CS) |
| Scan head type | Confocal point scanner (STELLARIS 8, with Pulse Picker) |
| Scanner type | Dual: FOV (galvanometric) + 8 kHz resonant (tandem, software-switched) |
| Max frame rate (512 × 512, FOV mode) | 10 fps |
| Max frame rate (512 × 512, resonant mode) | ~28 fps (reduced ~13 mm field) |
| Max frame resolution | 8192 × 8192 px |
| Scan zoom | 0.75–48× |
| Scan field diameter (max) | 22 mm |
| AOBS simultaneous lines | 8 |
| Internal detection channels | 4 (2 × HyD S, 1 × HyD X, 1 × HyD R) |
| Spectral detection range | 410–850 nm (HyD S/HyD R); 410–750 nm (HyD X) |
| Spectral tuning resolution | 1 nm |
| Photon counting time resolution | 97 ps |
| WLL tuning range | 440–790 nm |
| WLL native repetition rate | 78 MHz |
| Pulse Picker settings | 80 / 40 / 20 / 10 / 5 MHz |
| 405 nm laser | CW diode, DMOD |
| STED depletion lines | 592, 660, 775 nm |
| STED lateral resolution (typical) | < 50 nm |
| STED axial resolution (typical) | < 130 nm |
| 3D STED | Yes (upgrade installed) |
| FALCON FLIM | Yes (module installed) |
| FCS / FCCS / FLCS | Yes (module installed) |
| TauSense | Yes |
| Lightning (confocal + STED) | Yes, both Expert modules installed |
| Objective NA (max, STED oil) | 1.40 (100× HC PL APO Oil STED White) |
| Objective NA (max, STED water) | 1.20 (86× W motCORR STED White) |
| Objective NA (max, oil, confocal) | 1.30 (40× HC PL APO Oil CS2) |
| Objective NA (max, water, confocal) | 1.20 (63× W motCORR CS2) |
| DIC optics | Yes — added after original quotation; confirmed operational |
| Hardware autofocus (AFC) | Yes — confirmed present and functional (FILM QSG) |
| FALCON FLIM max count rate | 80 Mcps per detector; accurate to 225 Mcps with < 1.6 % error (high-speed FLIM filter) |
| Environmental control | Temperature + CO2 + passive humidity (Okolab) |
| Vibration isolation | Active optical table, 900 × 900 mm |

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## 17. System Limitations & Notes for Workflow Design

- **DIC optics not in original quotation BOM.** DIC hardware was added separately after the original procurement and is confirmed operational. DIC prism part codes are not documented in this file — if specific prism identification is needed, check on-site.
- **No dedicated FRAP/FRET/3D-visualisation/colocalisation/screening modules itemised.** LAS X MicroLab, FRAP Zoomer, 3D Visualisation, Co-Localisation, Dye Finder, and Assay Editor do not appear in this quotation, unlike HCF2. Route dedicated FRAP, quantitative colocalisation, or automated multi-well screening workflows to HCF2, or confirm these modules have since been added to HCF3.
- **No Switchable Beam Expander (FRAP Booster).** Sequential ROI-based photobleaching may still be possible through the core acquisition software, but dedicated high-speed/zoomed FRAP support is more limited than on a system with the FRAP Zoomer module.
- **Reduced field of view in resonant scan mode.** The 8 kHz resonant scanner's high frame rates are only achieved over a reduced (~13 mm) field, not the full 22 mm FOV scanner range — for full-field high-resolution work, use the FOV (galvo) scanner instead.
- **STED field of view is objective-limited.** The 86× and 100× STED objectives give correspondingly small fields; large-area STED requires tiling/stitching via the Navigator, which should be planned explicitly.
- **Fluorophore photostability under STED.** Alexa Fluor 647 photobleaches very rapidly under STED depletion — use STAR RED, STAR 635P, CF680R, ATTO 647N, or SiR instead. Avoid DAPI/Hoechst as counterstains (especially with 592 nm STED). Avoid Vectashield as mounting medium.
- **Coverslip quality is critical for STED.** Use #1.5H high-precision coverslips (0.170 ± 0.005 mm). #1 coverslips cause significant loss of STED resolution and signal.
- **405 nm laser is not FLIM-capable.** Only pulsed WLL lines support FALCON/TauSense; the 405 nm CW diode is excitation-only.
- **STED lasers require 30-minute warm-up** before beam alignment. Alignment must be performed at the start of every STED session.
- **STED 775 laser dependency.** The STED 775 nm laser will not start unless the WLL laser is already on.
- **Workstation tier and specification not detailed.** "Workstation Premium" (158203112) part number is given without CPU/RAM/GPU specification in the source document — request current specification from Leica if this affects processing-throughput planning.
- **Data is not auto-saved.** Images accumulate in the LIF library in memory but are only written to disk when the user clicks Save. Failure to save before shutdown loses all acquired data.

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## 18. References & Resources

**Source documents used to compile this reference file:**

- Equipment quotation QU-0187961-C (Leica Microsystems, July 2021) — primary bill-of-materials source
- FILM Quick Start Guide: HCF3 Confocal, Leica Stellaris 8 inverted (FILM, April 2022)
- FILM Quick Start Guide: HCF3 STED, Leica Stellaris 8 inverted (FILM, April 2022)
- FILM Navigator protocols: Tiling, Tiled Z-stacks, Multi-position imaging (FILM)
- FILM Leica objectives inventory (51�Թ��� FILM)
- Leica STELLARIS Technical Documentation (December 2024)
- Alvarez et al., "SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM," Nature Methods Application Note, October 2019
- Roberti et al., "TauSense: a fluorescence lifetime-based tool set for everyday imaging," Nature Methods Application Note, September 2020
- Alvarez et al., "Pushing STED beyond its limits with TauSTED," Nature Methods Application Note, June 2021
- Reymann, "LIGHTNING: Image Information Extraction by Adaptive Deconvolution," Leica Microsystems White Paper, September 2018
- Leica Microsystems, "The Guide to STED Sample Preparation," Application Report, March 2021
- Abberior, "abberior STAR dyes" product information ([abberior.rocks/dyes-labels/abberior-star/](https://abberior.rocks/dyes-labels/abberior-star/))

**Video tutorials (Leica Microsystems workshop series, January 2021):**

- Workshop 5: Tau-STED — application of lifetime measurement in super-resolution microscopy ([youtube.com/watch?v=PmoA5oLkC0E](https://www.youtube.com/watch?v=PmoA5oLkC0E))
- Workshop 8: Data analysis in depth II — Phasor analysis with LAS X ([youtube.com/watch?v=WWEVuhMnhwg](https://www.youtube.com/watch?v=WWEVuhMnhwg))
- Lecture 8: Enrico Gratton — The phasor plots for FLIM and FRET ([youtube.com/watch?v=mELWi2I-jkE](https://www.youtube.com/watch?v=mELWi2I-jkE))

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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), FALCON application note (Nature Methods, October 2019), TauSense application note (Nature Methods, September 2020), TauSTED application note (Nature Methods, June 2021), Lightning white paper (Leica, September 2018), STED sample preparation guide (Leica, March 2021), FILM HCF3 Quick Start Guides, and abberior product documentation.*