Abstract
BackgroundVisualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons.MethodsNeurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness).ResultsWe validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy.ConclusionThe techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.
Highlights
Patch-clamp experiments in cultures or brain slices require a careful assessment of targeted cells before placing the patch electrode [1]
The microscope stage is constantly moved in both xy axis as in z and targeted cells can often be missed during the patch electrode lowering
Some back-illuminated cooled Electron multiplying charge coupled device (EM-CCD) (Electron Multiplying Charge Coupled Device) cameras can have quantum efficiencies very close to 100%
Summary
Patch-clamp experiments in cultures or brain slices require a careful assessment of targeted cells before placing the patch electrode [1]. To minimise bleaching and photodamage, modern microscope objectives have a very high numerical aperture and more sensitive detection systems that virtually approach the ideal photon detection limit by converting every photon reaching the sensor into an electron. Detection of fluorescence emanating from synthetic compounds or proteins still requires high intensity excitation, which damages the tissue (when applied for long periods) especially in epifluorescence systems, often used in patch-clamp experiments [6,7]. Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. We created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons
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