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| Thanks to the following for materials & support |
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Imaging
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| Rationale: The BioCurrents Research Center (BRC) designs and develops integrated experimental platforms for the study of cellular dynamics. One application driven development is the real time monitoring of cellular metabolism at the single cell level. Micro electrochemical electrodes, operating in the modulation format, termed self-referencing, already allow us to examine real time glucose transport and oxygen utilization. Confocal microscopy, in conjunction with voltage dyes, permit observation of mitochondrial dynamics. However, to measure the efficiency of energy production through the glycolytic and TCA cycle a direct measure of ATP activity is needed. This is possible by utilizing the light generating interaction of Luciferase with Luciferin and ATP. This is a luminescent reaction that releases photons at comparatively low density. This study is a collaborative effort between the laboratories of Liz Jonas (Yale Medical School), with an interest in how the anti-apoptotic protein Bcl-xl modulates ATP generation (click here for Jonas study), and George Holz (SUNY, Syracuse), with an interest in the metabolic and permissive control of insulin secretion (click here for Holz study).
Problems: One problem when collecting photons, relating to ATP activity, is the variable expression of Luciferase. This enzyme has to be introduced to the cell through viral transfection, with an inevitable variability in the expression level. Even when expressed, the amount of light released is low, requiring exceptionally sensitive light detectors, with related engineering that will ensure total darkness during collection. The associated programming also needs to be sophisticated enough to retain information with spatial and temporal fidelity. Only then can the biology be studied.
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| Methods: Prior to attempting single cell detection, we examine the signal strength from populations of transfected cells. To optimize the probability of signal detection, a photomultiplier tube (PMT: Hamamatsu R464) is used, with the culture dish placed directly on the surface of the photo cathode optical window. The PMT accepts a wide distribution of vector angles, losing spatial information but increasing sensitivity. We are currently developing an LED system, clamped to a defined output through a constant current source, to calibrate the device. |
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To image the ATP activity at the single cell level, photon release must be captured with both spatial and temporal information conserved. This requires single photon detection with XY coordinates and a time stamp. Single photon events are imaged using a Hamamatsu Image Intensifier CCD camera (ICCD). The ICCD is mounted underneath the microscope via the drop port. Apart from the objective, there are no optical components between the preparation and the detector, optimizing signal strength. The objective confines the vector angles collected, although only a photon entering the lens perpendicular to its surface will be correctly placed on the CCD video camera. |
| The microscope is fitted with retractable fluorescence filters and a remote short wavelength light source coupled via a liquid light pipe. This allows prescreening of the cells for fluorescent reporters, such as GFP, indicating successful transfection. |
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Like a photomultiplier tube, an ICCD uses a photo-cathode to detect photons. When a detected photon strikes the charged cathode material, an electron is freed and enters the micro-channel plate where electron multiplication takes place. By this mechanism, a single photon gives rise to multiple electrons. When these strike the phosphor coating on the optical window, the phosphor glows. This luminescent signal is imaged with a CCD camera. Spatial information about incoming photons, relative to the signal position on the window, is recorded in the form of pixel coordinates in a video signal. The information is then transferred to the processing software and electronics.
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| In order to convert the photon data from a video signal to a list of XY coordinates with temporal information, we use a comparator which detects the appropriate voltage level in the video signal. The camera’s pixel clock and sync signals are used to increment and reset counters corresponding to the current XY location in the video signal. When the comparator detects an event, the values in the counters are sent to memory. Custom software then stores the event to disk and updates the image on the monitor. All logic is implemented in a Field Programmable Gate Array. The software and stored data are used to reconstruct images with variable integration times from the photon events. |
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| Early Results: Using Chinese Hamster Ovary cell (CHO), Luciferase was successfully transfected in an adenovirus construct along with green fluorescent protein (GFP). Cells transfected could be identified on the basis of the GFP expression (See picture at top of page). Levels of transfection were sufficient to image single cells after the addition of the cell permeant cofactor Luciferin. CHO cells exhibited oscillations in the cytosolic levels of ATP. Each red spot in the figure above is a single cell. Neonatal cardiac muscle cells have also been successfully transfected, as have hippocampal neurons. We are now focusing on generating a mitochondrial targeted Luciferase (in collaboration with Chris Rhodes, PNRI). As with all our techniques, low light imaging will be integrated with other methodologies for tracking cell metabolism. |
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| • Luminescent imaging: Targeting ATP >> |
| • Development of extracellular sensors >> |
| • Low light and luminescent detection >> |
| • Mitochondrial ATP Production >> |
| • Fiber optic / PMT interface >> |
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| Heart, E., Cline, G.W., Collis, L.P., Pongratz, R.L. Gray J. and Smith, P.J. 2009. Role for malic enzyme, pyruvate carboxylation and mitochondrial malate import in the glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab. (In Press) |
| Danuser, G. and Oldenbourg, R. 2000. Probing f-actin flow by tracking shape fluctuations of radial bundles in Lamellipoda of motile cells. Biophysics Journal, 79: 191-201. |
| Danuser, G., Tran, P.T. and Salmon, E.D. 2000. Tracking differential interference contrast diffraction line images with nanometer sensitivity. Journal of Microscopy, 198(1): 34-53. |
| Oldenbourg, R., Katoh, K. and Danuser, G. 2000. Mechanism of lateral movement of filopodia and radial actin bundles across neuronal growth cones. Biophysical Journal, 78: 1176- 1182. |
| Danuser, G. 1999. Photogrammetric calibration of a stereo light microscope. Journal of Microscopy, 193(1): 62-83 |
| Katoh, K., Hammar, K., Smith, P.J.S. and Oldenbourg, R. 1999. Birefringence imaging directly reveals architectural dynamics of filamentous actin in living growth cones. Molecular Biology of the Cell, 10: 197-210. |
| Katoh, K., Hammar, K., Smith, P.J.S. and Oldenbourg, R. 1999. Arrangement of radial actin bundles in the growth cone of Aplysia bag cell neurons shows a short history of filopodial behavior. Proceedings of the National Academy of Sciences, USA 96: 7928-7931. |
| Danuser G. and Stricker M. 1998. Parametric Model Fitting: From Inlier Characterization to Outlier Detection. IEEE Transactions on Pattern Analysis and Machine Intelligence, 20(3): 263-280. |
| Katoh, K., Langford, G., Hammar, K., Smith, P.J.S. and Oldenbourg, R. 1997. Observation of actin bundles in neural growth cone with the Oldenbourg Pol-Scope. Biological Bulletin, 193: 219-220. |
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Equipment
