Automated Microscopes and Microbalances

Automated microscopes, such as the very recently launched Cell-IQ® (Chip-man Technologies, Tampere, Finland) or IncuCyte™ instruments (Essen Instruments, Ann Arbor, MI), offer unattended, label-free, and real-time kinetic analysis of cell proliferation, cell migration, or neurite outgrowth in a quantitative way. Both instruments are the result of continued development of classical phase contrast microscopy combined with sophisticated software for image analysis. Both instruments offer a technical solution to automatically find or display the required focal plane. Both are also compatible with classical 96-well and various other assay plate formats, and they are thus able to acquire data in low to mid-throughput. Besides their obvious advantage of measuring microscopic changes in cell behavior, automated microscopes find increasing use in cell quality control to improve the reliability of downstream cellular assays especially when using primary cells.

The IncuCyte instrument collects phase contrast images of live cells in microplates or other cell culture vessels, and is placed in a conventional cell culture incubator. Image acquisition is accomplished by taking autofocused images at user-defined times and locations within the sample drawer. Once the digital images are collected, custom image processing software calculates image metrics, such as monolayer confluence, providing a quantitative and consistent measure of cellular proliferation. A novelty of the IncuCyte instrument is the possibility to perform so-called scratch wound assays of cell mono-layers using a special plate design and a Woundmaker™ (Essen Instruments, Ann Arbor, MI), which is essentially a mechanical device that produces well-defined scratch wounds using sterile tips. Scratch wound assays are commonly used to assess the involvement of GPCRs in cellular proliferation and/or migration associated with wound closing [19] . This instrument offers a very convenient way to quantify these effects in a label-free format.

In contrast to the IncuCyte, which is housed in a conventional cell culture chamber, the Cell-IQ device is a fully integrated, self-contained solution offering continuous live cell imaging and analysis. The instrument contains a cell growth environmental chamber, where microplates and other cell culture flasks can be maintained under optimal growth conditions, and features a built-in microscope, light source, and camera. The Cell-IQ system scans through the cells (z-stack), taking images at regular intervals, for example, 1 |M distance. These images are then combined to produce an all-in-focus single resultant image.

Another recently introduced label-free technology (Q -Sense E series) is based on the principle of thin quartz discs that are placed between two gold layers (Q-Sense, Vastra Frolunda, Sweden). The device is primarily designed to measure cell adhesion or cell proliferation. Application of an alternating current (AC) leads to an excitation and oscillation of the quartz crystal, which can be influenced by associating cells. Measuring the resonance, decaying, and dissipative frequencies can accurately quantify this influence. So far, only limited information is available to demonstrate wide applicability in cell biological experiments, and there are currently no published records demonstrating GPCR - related applications.

Despite the obvious potential of quartz sensors or automated microscopes as a means to measure GPCR activation or inhibition, and the consequences at the microscopic level, they carry the disadvantage that changes in proliferation or cell migration are not universal readouts in GPCR biology, and furthermore, in absence of pharmacological intervention (e.g., pertussis toxin), such readouts have so far not been indicative of the activation of specific G proteins, effectors, or pathways.

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