Our ImagePatch approach is not really something new but still a pretty powerful tool to study subcellular calcium signaling. We have utilized this techniques the first time to investigate the importance of the proximity of intracellular organelles to plasma membrane ion channel activity in endothelial cells (Frieden et al). Using a high-resolution microscope (in our experience the most suitable is an adapted array confocal laser scanning microscope) that allows positioning of the pipette in the proximity of e.g. the mitochondria. If you want to see a movie on such procedure please click here.
Our most investigated endothelial cells do not contain voltage-gated calcium channels but so called store operated (capacitative) Ca2+ channels. These channels seem to open upon depletion of the endoplasmic reticulum and allow Ca2+ to enter the cell according the driving gradient, which is mainly established by the concentration gradients between outside and inside and the membrane potential. Consequently, membrane depolarization significantly diminishes this driving force for Ca2+ to enter the cell. However, most cell-attached patch clamp approaches work by depolarizing the cell membrane using high K+ buffers (left panel). Our approach is to use conventional extracellular buffer in the bath but high K+ buffers only in the pipette (right panel). This approach allows patch clamp experiments without depolarizing our cells. This technique was firstly described in Malli et al.
Moreover to avoid cell depolarization, we intended to utilize Ca2+-activated K+ channels (BKca) to measure subplasmalemmal Ca2+ concentration right at the mouth of the channel. To achieve the "physiological patch clamp" approach to measure spatial Ca2+ concentration just beneath the plasma membrane, we calibrated in inside-out configuration the channel's open probability (Po) vs the actual membrane potential applied (Vwc) and the Ca2+ concentration that is at the mouth of the channel (panel). This calibration allows us to calculate the channels Po at any existing membrane potential and Ca2+ concentration. In other words, measuring the channel's Po and knowing the actual membrane potential at the patch, one is able to calculate the Ca2+ concentration at the mouth of the channel.
That's how it finally works (panel): We do know the applied membrane potential provided by the patch amplifier but not that which is at the mouth of the channel as the cell's membrane potential is not 0 as it is normally in the conventional patch clamp technique. However, by measuring the conductance of the channel and utilizing its I/V curve (1), we calculate the actual membrane potential at the patch (Vpm) (2). Considering the applied membrane potential provided by the patch amplifier, we are now able to calculate the cell's membrane potential (Vwc) during the whole experiments (3). Moreover, considering the Po of the channel at given time (4) and membrane potential of the patch (Vpm) (2), and by utilizing our equation extracted from the calibration procedure described above, the actual Ca2+ concentration at the mouth of the channel can be calculated (5).
For more detail please see Malli et al.
Utilizing FRET techniques and multi dye labeling of living cells, we are often facing the problem of overlapping emission spectra of the fluorescent proteins/dyes used. This harms the quality of the images and represents an error if one is performing quantitative measurements. That's why we have come up with our multi-emission separation (MES) algorithm that allows quantitative channel separation in any conventional fluorescence microscope as well as (array) confocal laser scanning microscope (ACLSM). It can also be utilized to calculate online FRET efficiency and dynamic FRET changes. Currently we are using MES in our ACLSM as well as our Zeiss LSM410.