Background: Visualisation of neurons labeled with fluorescent proteins or\ncompounds generally require exposure to intense light for a relatively long\nperiod of time, often leading to bleaching of the fluorescent probe and\nphotodamage of the tissue. Here we created a technique to drastically shorten\nlight exposure and improve the targeting of fluorescent labeled cells that is specially\nuseful for patch-clamp recordings. We applied image tracking and mask overlay to\nreduce the time of fluorescence exposure and minimise mistakes when identifying\nneurons.\nMethods: Neurons are first identified according to visual criteria (e.g. fluorescence\nprotein expression, shape, viability etc.) and a transmission microscopy image\nDifferential Interference Contrast (DIC) or Dodt contrast containing the cell used as a\nreference for the tracking algorithm. A fluorescence image can also be acquired later to\nbe used as a mask (that can be overlaid on the target during live transmission video). As\npatch-clamp experiments require translating the microscope stage, we used pattern\nmatching to track reference neurons in order to move the fluorescence mask to match\nthe new position of the objective in relation to the sample. For the image processing\nwe used the Open Source Computer Vision (OpenCV) library, including the\nSpeeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720)\nwas analyzed under normal conditions of acquisition and with influence of noise\n(defocusing and brightness).\nResults: We validated the method in dissociated neuronal cultures and fresh brain\nslices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer\nTomato (tdTomato) proteins, which considerably decreased the exposure to\nfluorescence excitation, thereby minimising photodamage. We also show that the\nneuron tracking can be used in differential interference contrast or Dodt contrast\nmicroscopy.\nConclusion: The techniques of digital image processing used in this work are an\nimportant addition to the set of microscopy tools used in modern electrophysiology,\nspecially in experiments with neuron cultures and brain slices.
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