No / The purpose of this research was to correlate in vivo spatial and spectral morphologic changes of short- to long-pulse 532 nm Nd:YAG retinal laser lesions using Fourier-domain optical coherence tomography (FD OCT), autofluorescence (AF), fluorescein angiography (FA), and multispectral imaging. Ten eyes with treatment-naive preproliferative or proliferative diabetic retinopathy were studied. A titration grid of laser burns at 20, 100, and 200 milliseconds was applied to the nasal retina and laser fluence titrated to produce four grades of laser lesion visibility: subvisible (SV), barely visible (BV, light-gray), threshold (TH, gray-white), and suprathreshold (ST, white). The AF, FA, FD-OCT, and multispectral imaging were performed 1 week before laser, and 1 hour, 4 weeks, and 3 and 6 months post-laser. Multispectral imaging measured relative tissue oxygen concentration. Laser burn visibility and lesion size increased in a linear relationship according to fixed fluence levels. At fixed pulse durations, there was a semilogarithmic increase in lesion size over 6 months. At 20 milliseconds, all grades of laser lesion were reduced significantly in size after 6 months: SV, 51%; BV, 54%; TH, 49%; and ST, 50% (P < 0.001), with retinal pigment epithelial proliferation and photoreceptor infilling. At 20 milliseconds, there was healing of photoreceptor inner segment/outer segment junction layers compared with 100- and 200-millisecond lesions. Significant increases in mean tissue oxygenation (range, four to six units) within the laser titration area and in oxygen concentration across the laser lesions (P < 0.01) were detected at 6 months. For patients undergoing therapeutic laser, there may be improved tissue oxygenation, higher predictability of burn morphology, and more spatial localization of healing responses of burns at 20 milliseconds compared with longer pulse durations over time / Optimedica Corp., the Manchester Academic Health Sciences Centre, and the NIHR Manchester Biomedical Research Centre. JD was funded by a College of Optometrists PhD Studentship, United Kingdom.