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Using the PatchStar micromanipulator for a variety of electrophysiology research
The PatchStar is an ultra-stable motorised manipulator. It's versatility and ease of use make it ideal for a variety of electrophysiology recordings, as well as microinjection and other techniques that require precise positioning.
Here are examples of different research techniques and samples that have utilised the PatchStar.
1. Plumbagin-induced oxidative stress leads to inhibition of Na+/K+ -ATPase (NKA) in canine cancer cells.
Sample: Canine cancer cells
Technique: Whole-cell patch clamp electrophysiology
Researchers at the University of Wisconsin-Madison used Scientifica’s PatchStar micromanipulator to investigate the effect of drug-induced oxidative stress on the Na+ /K+ -ATPase protein complex. The team found that plumbagin, a natural product, increases oxygen radicals through the inhibition of oxidative phosphorylation. Treatment with plumbagin resulted in decreased production of ATP and a rapid increase in intracellular oxygen radicals, leading to apoptosis of canine cancer cells due to oxidative stress.
The PatchStar was used to carry out whole-cell patch clamp electrophysiology to investigate the effects of plumbagin exposure on canine cancer cells. The team found that 4 minutes of plumagin exposure resulted in a 48% decrease in outward current at +50 mV. Even when exogenous ATP was supplied to the cells, plumbagin treatment resulted in 46% inhibition of outward current through NKA at +50 mV. In contrast, when the canine cancer cells were pre-treated with the oxygen radical scavenger, N-acetylcysteine, the NKA inhibitory activity of plumbagin was abrogated.
These results suggest that plumbagin, an anti-cancer agent that causes cell death in prostate, breast, ovarian and colon cancer cells, could also be used as an anti-cancer agent for dogs.
2. Using the PatchStar to study the mechanical properties of brain tissue
Sample: Mouse brain tissue
Technique: Ferrule-top indentation
Nelda Antonovaite used the PatchStar micromanipulator during her PhD at Vrije University Amsterdam to perform ferrule-top indentation. This technique enabled the mechanical properties of brain tissue to be studied, as well as structural brain changes that occur in neurodegenerative diseases.
3. Angular approach scanning ion conductance microscopy
Sample: Human umbilical vein endothelial cells and Human mesenchymal stem cells seeded on nanoneedle arrays, islets of Langerhans, cultured organs of Corti, primary hippocampal neurons and intercalated disks of isolated cardiac myocytes
Technique: Scanning ion conductance microscopy
Researchers at Imperial College London, King’s College London, the University of Oxford and University of Cambridge mounted a scanning ion conductance microscope (SICM) head onto a PatchStar micromanipulator to enable the super-resolution imaging technique to be used with an adjustable approach angle. The team used this angular approach SICM to obtain topographical images of live cells and tissues with a resolution of ~10nm.
The type of SICM used was hopping probe ion conductance microscopy (HPICM). This is a version of SICM that is used to visualise specialised, differentiated cells and tissues that often have convoluted structures that are inaccessible for standard SICM.
The design of SICM setups require the piezo assembly to be mounted on an inverted microscope, above the objective, which means upright microscopes, high numerical aperture condensers, phase contrast and differential interference contrast (DIC) cannot be used.
The researchers therefore built an HPICM setup that has an adjustable nanopipette approach angle that can be integrated into any patch clamp setup, including those with an upright microscope. The PatchStar enabled the HPICM nanopipette to approach and position over a 20mm range in X, Y and Z directions as well as the complete withdrawal of the nanopipette. This HPCIM with an angular approach also enables cells grown on non-transparent substrates to be imaged and DIC and phase contrast to be used.
The true potential of angle scan HPICM has been demonstrated by imaging of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscopes. Angle scan approach also enabled mapping of functional sodium channels distinct clusters in intercalated disc of cardiac myocyte cells that may influence intercellular adhesion strength - a study published in Nature Communications journal (DOI: 10.1038/ncomms10342)
4. Dual patch voltage clamp study of low membrane resistance astrocytes in situ
Sample: Mouse brain slices
Technique: Dual whole-cell voltage clamp electrophysiology recordings
Two PatchStars were used in a dual patch configuration by scientists at Ohio State University to perform dual patch single astrocyte recordings.
The dual patch configuration was used to simultaneously record the membrane current and membrane potential from single astrocytes, in order to study their membrane conductance. Due to the low membrane resistance of these cells, it is not possible to achieve adequate voltage-clamp quality using conventional whole-cell patch clamp recordings.
The PatchStar micromanipulators enabled this previously time-consuming technique to be carried out routinely, with 3-8 cells being studied in 6 hours.
5. Using the PatchStar to investigate the intrinsic excitability of neurons
Sample: Pyramidal cells and interneurons
Technique: Dual patch clamp recordings
Dmitri used PatchStar micromanipulators as part of the SliceScope Pro 6000 electrophysiology rig to investigate how the intrinsic exciability of neurons can change and how this affects whole networks of neurons.
6. Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behaviour
Sample: Human cell culture
Technique: Colloidal probe microscopy
Colloidal probe microscopy was used by scientists at Aalto University to quantify the interactions of two human culture cell lines with biomaterials. The cell lines consisted of human pluripotent stem cell line WA07 and human hepatocellular carcinoma cell line HepG2. The researchers were probing the interactions of these cells with natural, xeno-free biomaterials of different chemistry, morphology, and origin. This is important in order to understand how the interaction of cells with surrounding biomaterials affects cell behaviour in vitro (e.g. in cell cultures and scaffolds for tissue engineering applications) and in vivo (e.g. in cancer metastasis and tumour progression).
The interactions between the different biomaterials and cell lines were measured with an atomic force microscope using the colloidal probe technique (colloidal probe microscopy). The PatchStar micromanipulator was used to prepare the colloidal probes, that is, to attach spherical microparticles at the end of tipless AFM cantilevers. The colloidal probes were then coated with different biomaterials. The two cell lines showed distinctly different interactions with the different biomaterials and the adhesion energy increased as contact time between the cells and biomaterials increased.
7. Cation dependent electroosmotic flow in glass nanopores
Technique: Using nanopore sensors to measure electro-osmotic flow
Nanopore sensors contain a nano-scale hole which a current is run-through. The nanopore measures changes in current and is able to sense single molecules, proteins, sequence DNA and detect single nucleotide polymorphisms. Scientists at the University of Cambridge and Gettysburg College mounted nanopore sensors onto the PatchStar to enable their movement to be programmed.
The researchers used this setup to measure the effects of electroosmosis on the functioning of nanopore sensors. Nanopores made from Si3N4 and glass have a negative surface charge in solution at biological pH. This makes positive ions build up close to the surface. Applying an electric field to drive an analyte through the nanopore also causes the charges at the surface to move. This movement couples to the fluid medium resulting in electroosmotic flow (EOF). The EOF contributes to the magnitude and direction of the total force that a target molecule experiences in a nanopore.
The team developed a highly sensitive method for quantifying nanopore EOF and used this to determine the effect of different cation species on the hydrodynamic environment of nanopores.
The results showed that electrokinetic phenomena have a considerable impact on the fluid environment surrounding a nanopore.
The most versatile motorised manipulator for electrophysiological studies. Designed with leading physiologists, the PatchStar is also perfect for microinjection and other demanding positioning requirements.
Banner image credit: Dr. Hovy Wong from the lab of Dr. Jesper Sjöström.
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