**Background:** Cigarette smoking is a major preventable cause of morbidity and mortality, with a global prevalence of 22.3% in 2020. Smoking negatively affects cellular function and metabolism throughout the body, including ocular tissues, and increases the risk of age-related macular degeneration, glaucoma, uveitis, and Grave's ophthalmopathy. However, the influence of smoking on retinal metabolism in young healthy eyes is not fully understood. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a non-invasive diagnostic tool that measures the fluorescence lifetime (FLT) of intrinsic retinal fluorophores, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which change their FLT depending on protein-binding states and cellular energy metabolism. FLT can be altered by structural changes or metabolic alterations. Previous studies have shown FLT changes in patients with type 2 diabetes without diabetic retinopathy and Alzheimer's disease without structural changes, suggesting FLIO's potential to detect early metabolic disease mechanisms. This study aimed to compare FLIO measurements in healthy eyes of smokers and non-smokers to detect potential metabolic alterations induced by smoking.
**Methods:** This cross-sectional study was conducted at the Department of Ophthalmology, University Medical Center Schleswig–Holstein, Campus Lübeck, Germany, between April and September 2021. Fifty-four participants (26 non-smokers, 28 smokers) aged 20-40 years with healthy retinal findings and no systemic diseases (e.g., diabetes, hypertension, thyroid dysfunction) were enrolled. Smokers were defined as those who smoked ≥5 cigarettes daily for at least two years. All subjects underwent slit lamp microscopy, intraocular pressure (IOP) measurement, refractometry, best corrected visual acuity (BCVA) assessment, and optical coherence tomography (OCT) of the macula. FLIO was performed using a 473 nm picosecond-pulsed excitation laser, detecting fluorescence in a short spectral channel (SSC: 498-560 nm) and a long spectral channel (LSC: 560-720 nm). FLT parameters (τ1, τ2, τm) were analyzed using a biexponential fitting model. An ETDRS grid (central area, inner ring, outer ring with subareas) was used for regional analysis. Statistical analysis included t-tests, ANOVA, post hoc tests, and Pearson correlation. The smoker group was further divided into two subgroups based on the median pack-year (7.11): Group 1 (pack-year <7.11, n=14) and Group 2 (pack-year ≥7.11, n=14).
**Key Results:** The mean age of non-smokers was 26.7±4.1 years and smokers 28.5±4.7 years (p=0.130). BMI was significantly higher in smokers (26.6±5.4 kg/m²) than non-smokers (22.8±2.6 kg/m², p=0.002). IOP was significantly higher in smokers (right eye: 16.3±2.8 mmHg; left eye: 16.1±2.5 mmHg) compared to non-smokers (14.6±3.3 and 14.5±3.2 mmHg, p<0.05). No significant differences in retinal thickness were found between groups. In the SSC, smokers showed significantly longer τm in the inner ring and its subareas (N1, S1, T1, I1) compared to non-smokers (p<0.05). Post hoc analysis revealed significant τm prolongation in Group 2 (heavy smokers) compared to Group 0 (non-smokers) at the fovea (C: 143±22.4 vs. 129±17.7 ps, p=0.022), inner ring (226±15.8 vs. 210±17.7 ps, p=0.004), and all inner ring subareas. Significant positive correlations were found between pack-year and τm in the fovea (r=0.424, p<0.01) and inner ring areas (r=0.403, p<0.01). In the LSC, no significant differences were found between smokers and non-smokers overall, but Group 2 showed significantly shorter τm in the outer ring (233±12.1 vs. 242±11.2 ps, p=0.048) and subareas T2 and S2 compared to non-smokers. For τ1 in SSC, smokers had significantly longer values in all inner ring areas, with dose-dependent increases in Group 2. Moderate positive correlations were found between pack-year and τ1 in most regions. For τ2 in SSC, no significant differences were found between smokers and non-smokers, but Group 1 (light smokers) showed significantly shorter τ2 in the fovea and inner ring compared to both Group 0 and Group 2. No significant correlations were found between retinal thickness and smoking-related FLT changes.
**Clinical Implications:** This study demonstrates that cigarette smoking induces detectable changes in retinal fluorescence lifetime in healthy young adults, even in the absence of structural retinal changes on OCT. The dose-dependent prolongation of τm and τ1 in the SSC, particularly in the inner macula, suggests that smoking may alter retinal metabolism, possibly through effects on flavin coenzymes (FAD, FMN), macular pigment density, or vascular density. The paradoxical shortening of τm in the LSC outer ring in heavy smokers may reflect increased A2E (a bisretinoid) due to nicotine-induced isomerization of all-trans-retinal. These findings highlight FLIO's potential as a non-invasive biomarker for early, preclinical retinal damage caused by smoking and other lifestyle factors. FLIO could facilitate early detection of retinal diseases and monitoring of treatment efficacy. Limitations include the small sample size, potential confounding by BMI and age differences between subgroups, and the inability to differentiate between cumulative dose and acute nicotine effects. Future studies with larger, BMI-matched cohorts and detailed smoking history are warranted to confirm these findings and elucidate underlying mechanisms.