Abstract
We have demonstrated a combined imaging system, where the physiology of biological specimens can be imaged and profiled at 10–20 frames per second whilst undergoing femtosecond laser irradiation. Individual GH3 cells labeled with the calcium fluorophore Fluo-3 were stimulated using a counter-propagating focused femtosecond beam with respect to the imaging system. As a result of the stimulation, calcium waves can be generated in COS cells, and laser-induced calcium oscillations are initiated in the GH3 cells. Single-photon fluorescence images and intensity profiles of the targeted specimens are sampled in real-time using a modified PerkinElmer UltraView LCI microscope.
Highlights
Continued development of optical systems for simultaneous observation and manipulation of live biological specimens has produced advances in understanding cell physiology
Fluorescence signals of cells can be linked to the overall health and integrity of those cells [1], with fluctuations in the signals indicating effects such as changes in dye loading, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), cell activation, and cell destruction
The fluorescence light is collected with the same objective and passed back through the rotating pinhole array where the dichroic mirror (DM) reflects the fluorescence into the charge coupled detector (CCD)
Summary
Continued development of optical systems for simultaneous observation and manipulation of live biological specimens has produced advances in understanding cell physiology. Imaging samples in real time (typically 25–30 frames per second) have a couple of advantages over standard beam scanning based modalities. The Nipkow disk [20] reduces the high levels of photobleaching associated with beam scanning configurations whilst allowing millisecond (ms) monitoring of fluorescence intensity and/or spectrum across the desired samples. We demonstrate the use of real-time singlephoton fluorescence monitoring of cells that are activated using a femtosecond laser beam. Compared with standard commercial fluorescence microscopes that use a beam scanning method coupled with photomultiplier tubes (PMTs), International Journal of Biomedical Imaging. Traditional beam scanning microscopes can take up to one second to produce an image, which could lead to distorted images of relatively high-speed cellular activity. The fast imaging times used allow this system to monitor the speed by which calcium waves traverse a cell following a short femtosecond laser pulse. We provide examples of calcium ion (Ca2+) oscillations being instigated as a result of femtosecond laser pulses
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