Most infrared sources are dim, however, so the information fromtypical IR set-ups is limited in resolution and has a lowsignal-to-noise ratio. Infrared from the ALS’s synchrotron lightsource is a hundred to a thousand times brighter. Previously Holman and her colleagues have used IR beamline 1.4.3,managed by Berkeley Lab’s Michael Martin and Hans Bechtel, toobtain spectra from living organisms in rock, soil, and water. Theyhave monitored ongoing biochemistry within living bacteria adaptingto stress , and more recently within individual skin connective tissue cells(fibroblasts) from patients with mitochondrial disorders.(Mitochondria are the cellular organelles commonly known as the”power-plants” of the cell.) The present study was done with a line of cultured cells calledPC12.
When nerve growth factor, a small protein, is introduced intoa PC12 cell, the cell begins to send out neurites resembling theprojections from nerve cell bodies. Although originally derivedfrom a tumor of the rat’s adrenal gland, PC12 has become, rathercounterintuitively, a valuable model of how nerve cellsdifferentiate from their unspecialized progenitors. Berkeley Lab postdoctoral fellow Liang Chen began the currentexperiments by introducing nerve growth factor to groups of PC12cells to induce them to differentiate; one group of cells was leftuntreated as a control. The cells were cultured on gold-coatedslides in chambers maintained at body temperature in a humidifiedenvironment and supplied with nutrients.
Individual cells of agroup were positioned under the infrared beam at the beamline 1.4.3endstation. FTIR spectra were collected before and after the nerve growthfactor was introduced. After stimulation, the spectra were takenfirst at short intervals, from two to sixty minutes apart.Additional spectra were collected of cells in other groups on thethird, fifth, and seventh day of continued stimulation. The first day’s spectra revealed spikes in phosphorylation activitywithin minutes after the addition of the nerve growth factor, inconcert with changes in the ratios of such important chemicalcontents of the cell as proteins, carbohydrates , and lipids. Vandal Proof Dome Camera
Phosphorylation subsequently waned, then picked upagain in another burst of activity on Day 3, just as the cellsbegan to extend neurites. By comparing results with quantum chemistry simulations by BerkeleyLab’s Zhao Hao – predicting what should be observed from firstprinciples – as well as with results from partial studies usingother methods, the researchers confirmed the monitoring ofphosphorylation phases, their timing, and their target proteins,along with associated changes in other substances in the cell. A new technique takes off “This experiment was a proof of the concept,” says Liang Chen. “Wedemonstrated the dynamics of protein phosphorylation in controllingdifferentiation in this biological system using synchrotroninfrared spectromicroscopy, and we pointed the way to answering themany questions a biologist has to ask about measuring thecoordination of specific processes in real time.” Although in this first experiment the team was not able to followindividual cells continuously, they were able to monitordifferentiation in groups of cultured PC12 cells in real time,without labeling or any other invasive procedure. It was the firststep in an ambitious inquiry into the real-time biochemistry ofliving mammalian cells over the long term. Dome Infrared Camera Manufacturer
At beamline 1.4.3., with the help of new team members KevinLoutherback and Rafael Gomez-Sjoberg, the team is designingequipment to maintain mammalian cells in a thin layer of culturemedia that will keep them healthy yet not interfere with theinfrared beam, while automatically monitoring and adjustingtemperature, humidity, and nutrient ratios, and removing wasteproducts. This will allow data on individual cells to be gatheredcontinuously throughout the entire phosphorylation process. Meanwhile the Berkeley Synchrotron Infrared Structural Biologyprogram at ALS beamline 5.4 is building multimodal facilities thatwill monitor cell development in human cells, bacteria, and plants,within soils, minerals, and other environments, via”hyperspectromicroscopy” – from the ultraviolet through visiblelight and deep into the infrared. Researchers will be able tochoose the frequency window (or combination of windows) best suitedto the sample and the conditions – in Holman’s words, “to watchalmost everything at once.” Says Holman, “Many researchers from the medical communities areinterested in using the technology, and we are particularlyinterested in collaborating with university centers and privatefirms that are seeking a broad view of how promising drugs actwithin specific cells.” Some of the projects will target Alzheimer’s disease , macular degeneration of the retina in diabetes , and mitochondrial diseases in children. In addition, specificprocesses like protein glycation can also be identified. China DVR Kits
Sincedifferent cells and different organisms respond differently, theeventual goal is to develop specific ways to screen the mechanismsof individual medicines. Additional References Citations.