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Pregnancy Timeline by SemestersDevelopmental TimelineFertilizationFirst TrimesterSecond TrimesterThird TrimesterFirst Thin Layer of Skin AppearsEnd of Embryonic PeriodEnd of Embryonic PeriodFemale Reproductive SystemBeginning Cerebral HemispheresA Four Chambered HeartFirst Detectable Brain WavesThe Appearance of SomitesBasic Brain Structure in PlaceHeartbeat can be detectedHeartbeat can be detectedFinger and toe prints appearFinger and toe prints appearFetal sexual organs visibleBrown fat surrounds lymphatic systemBone marrow starts making blood cellsBone marrow starts making blood cellsInner Ear Bones HardenSensory brain waves begin to activateSensory brain waves begin to activateFetal liver is producing blood cellsBrain convolutions beginBrain convolutions beginImmune system beginningWhite fat begins to be madeHead may position into pelvisWhite fat begins to be madePeriod of rapid brain growthFull TermHead may position into pelvisImmune system beginningLungs begin to produce surfactant
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Fluorescent proteins light up living cells

Tracing proteins in cells is like looking for a needle in a haystack. But, in order to locate such proteins and decipher their function in living cells, researchers can now label them with fluorescent molecules.

A group of researchers from Goethe University (GU), in Frankfurt, Germany — working in close collaboration with US colleagues — has now found a solution to this problem. In the current issue of Nature Communications, researchers use fine-tuned pressure to deliver chemical probes into living cells.

"Although more and more protein labeling methods use synthetic fluorescent dyes, they often suffer from problems such as cell membranes that won't allow liquids to pass through — or low labeling efficiency. Or, they cannot be combined with other protein labeling techniques", explains Ralph Wieneke PhD, from the Institute of Biochemistry at Goethe University.

Recently, researchers led by Wieneke and Robert Tampé PhD, developed a marker that finds selected proteins in cells with nanometre (10-9 meter or one billionth of a meter) precision. This highly specific element consists of a small synthetic molecule called trisNTA and a genetically encoded His-tag. A His-tag is a string of histidines located on one end of recombinant proteins. It is known that His-ags bind tightly to nickel.

In order to place these protein markers into cells, GU researchers along with colleagues from the Massachusetts Institute of Technology (MIT), Cambridge, USA, used a unique procedure. They mixed cells with the protein marker, then forced the combination under pressure through the cell wall.

Called "cell squeezing", cells incorporated the fluorescent probes at an efficiency rate greater than 80 percent, at one million cells per second.

The marker bound specifically to the target protein and its concentration was precisely regulated within the cell. Now researchers are able to record high resolution microscopy images within living cells and trace targeted proteins using only light.

The technique can even be combined with other protein labeling techniques in order to observe in real time several proteins simultaneously. In the future it will be possible at high resolution to follow dynamic processes in living cells in time and space.

"Using cell squeezing, we were able to deliver a number of fluorescent labeled trisNTAs into cells. This tremendously expands the scope of conventional and high resolution microscopy in living cells."

Robert Tampé PhD, Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Germany

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (~1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.

Authors: Alina Kollmannsperger, Armon Sharei, Anika Raulf, Mike Heilemann, Robert Langer, Klavs F. Jensen, Ralph Wieneke & Robert Tampé:

Article: "Live-cell protein labelling with nanometre precision by cell squeezing"; Nature Communications, 7:10372, DOI: 10.1038/ncomms10372

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Feb 15, 2016   Fetal Timeline   Maternal Timeline   News   News Archive   

The nuclear envelope of protein Lamin A was stained with a fluorescent label (green).
Other proteins can be visualized simultaneously within the same cell —
Histon 2B is magenta, Lysosomes in blue, Microtubuli are red.
Image Credit: Goethe University, Frankfurt, Germany




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