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To examine big-bubble (BB) formation success rates in deep anterior lamellar keratoplasty (DALK) at various corneal depths using real-time guidance from swept-source, microscope-integrated optical coherence tomography (SS-MIOCT).The DALK procedure was performed ex vivo with 34 human donor corneoscleral buttons on pressurized artificial anterior chambers using the BB technique employed by Anwar and Teichmann. We inserted a needle under controlled ex vivo conditions to corneal depths ranging from 40% to ≥90% using real-time guidance from SS-MIOCT and injected air. BB success was then determined for each injection.The average needle depth for successful full BB formation was 79.9% ± 3.0% compared with 66.9% ± 2.6% for partial BB formation and 49.9% ± 3.4% for no BB formation (P < 0.0001). Expressed as stroma below the needle tip, this corresponded to 123.9 ± 20.0 μm for successful full BB formation compared with 233.7 ± 23.8 μm for partial BB formation and 316.7 ± 17.3 μm for no BB formation (P < 0.0001). All other variables tested (sex, race, age, endothelial cell density, air injected, needle angle, and central corneal thickness) did not significantly affect BB formation success rates.BB formation in DALK is more successful if needle insertion and air injection occur at deeper corneal depth. However, ≥90% corneal depth was not necessary in this ex vivo model of DALK. SS-MIOCT can be used to accurately guide the needle in real time.