**Background:**
The diagnosis of cutaneous manifestations of deep mycoses, including invasive aspergillosis, relies on histopathological and direct examinations, but current criteria miss many cases. Dynamic full-field optical coherence tomography (D-FF-OCT) is a label-free interference microscopy technique that provides high-resolution structural and metabolic contrast imaging of biological tissues without sample preparation. This study aims to evaluate D-FF-OCT for live-cell imaging and monitoring of growth stages in Aspergillus fumigatus.
**Methods:**
The A. fumigatus ATCC 204305 quality-control strain was cultured on Sabouraud dextrose agar at 30°C for 24–72 h in a humidified chamber. Fungal growth was monitored with D-FF-OCT for up to 5 h at room temperature, using a commercial Light-CT Scanner (LLTech – Aquyre Biosciences) with 10× water immersion objectives (NA 0.3), achieving axial and transverse resolution of approximately 1–1.5 µm. Images were acquired at 1-µm axial steps up to 50 µm depth. Dynamic contrast imaging (DCI) was performed by analyzing pixel time series via fast Fourier transform, splitting the power spectrum into three equal-energy bands: high frequencies (fast movements, red), medium frequencies (intermediate movements, green), and low frequencies (slow movements, blue). Antifungal susceptibility testing was performed using Etest strips for voriconazole, amphotericin B, and caspofungin on RPMI agar, with MICs determined by EUCAST methodology. Image analysis was done with ImageJ 1.50e.
**Key Results:**
- D-FF-OCT allowed visualization of conidiophores (diameter 2–4 µm on FF-OCT, 4–6 µm on D-FF-OCT), septate hyphae, phialides, conidia, and vesicles. In-depth imaging up to 45 µm depth showed conidial heads of approximately 30–50 µm.
- Dynamic contrast within conidial head vesicles changed from green (intermediate movements) to red (fast movements) over 240–300 min, indicating faster intracellular movements. Semiquantitative analysis (Figure 3B) showed a progressive decline in blue channel (slow movements) and increase in red channel (fast movements) over 360 min, with stable green channel.
- Live monitoring over 100 min revealed serpentining DCI signal along conidiophores, vacuole trafficking and coalescence, and conidial head formation with vesicle DCI shifting from green to yellow (faster movements). Hyphal growth speed was approximately 50 µm/h.
- Longer incubation (48–72 h) led to thicker, bendier hyphae on FF-OCT and loss of DCI segmentation, with complete D-FF-OCT signal extinction after 72 h, associated with agar drying.
- Antifungal treatment: MICs were 0.094 mg/L (voriconazole), 0.38 mg/L (amphotericin B), and 0.016 mg/L (caspofungin). Voriconazole and amphotericin B decreased hyphal concentration and shifted DCI from warm green–yellow to pale blue–green (slower movements). Caspofungin produced dense, packed microcolonies with predominant blue DCI signal (slow movements), consistent with paradoxical growth effect.
**Clinical Implications:**
D-FF-OCT provides label-free, real-time structural and metabolic imaging of A. fumigatus, enabling monitoring of growth phases, vacuole dynamics, and antifungal effects without staining. This could aid experimental mycology and potentially improve diagnosis of superficial fungal infections and invasive aspergillosis via direct examination of biopsies or surgical specimens. The technique's ability to assess fungal viability and metabolic activity may complement conventional methods, though further validation with organelle-specific staining and higher-resolution setups is needed.