Effects of formalin fixation on <i>ex vivo</i> confocal microscopy imaging: towards rapid pathology

Ex vivo confocal microscopy (EVCM) rapidly images excised tissues at near-histopathological resolution, bypassing the time-consuming and labour-intensive tissue processing required for conventional histopathological evaluation. Thus far, the technique has been used to image only fresh or frozen tissues, which are challenging to transport, limiting its use to specialized centres. However, this technique could be extremely beneficial for primary health centres without histopathology laboratories, providing access to rapid pathology. For this, tissues would need to be transported in a preservative to maintain their integrity. Formalin is an excellent preservative that is readily available in clinics. We tested its effect on tissues evaluated by EVCM at 30 min, 1 h, 2 h, 4 h, 6 h, 24 h, 3 days, 5 days and 7 days after fixation and compared the images acquired with images from fresh tissue (baseline). Sixty images were acquired from normal skin tissues and tumours and analysed by a dermatopathologist blinded to the histopathology diagnosis. Normal tissue structures and tumours were correctly identified at all timepoints. Tissue became easier to handle after fixation, and photobleaching was not seen in fixed tissues. Using formalin would also preserve tissue morphology for ancillary studies. Our pilot work sets the stage for future large-scale studies and aids in widespread adaptation of this technique beyond specialized centres.