Flexible polygon-mirror based laser scanning microscope platform for multiphoton in-vivo imaging.
Commercial microscopy systems make use of tandem scanning i.e. either slow or fast scanning. We constructed, for the first time, an advanced control system capable of delivering a dynamic line scanning speed ranging from 2.7 kHz to 27 kHz and achieve variable frame rates from 5 Hz to 50 Hz (512 × 512). The dynamic scanning ability is digitally controlled by a new customized open-source software named PScan1.0. This permits manipulation of scanning rates either to gain higher fluorescence signal at slow frame rate without increasing laser power or increase frame rates to capture high speed events. By adjusting imaging speed from 40 Hz to 160 Hz, we capture a range of calcium waves and transient peaks from soma and dendrite of single fluorescence neuron (CAL-520AM). Motion artifacts arising from respiratory and cardiac motion in small animal imaging reduce quality of real-time images of single cells in-vivo. An image registration algorithm, integrated with PScan1.0, was shown to perform both real time and post-processed motion correction. The improvement is verified by quantification of blood flow rates. This work describes all the steps necessary to develop a high performance and flexible polygon-mirror based multiphoton microscope system for in-vivo biological imaging.
Research School of Engineering, College of Engineering and Computer Science, Australia National University, North Road, Canberra ACT, 2601, Australia.
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Li
John Curtin School of Medical Research, Australian National University, Garran Road, Canberra ACT, 2601, Australia.
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Gautam
John Curtin School of Medical Research, Australian National University, Garran Road, Canberra ACT, 2601, Australia.
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Brüstle
John Curtin School of Medical Research, Australian National University, Garran Road, Canberra ACT, 2601, Australia.
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Cockburn
John Curtin School of Medical Research, Australian National University, Garran Road, Canberra ACT, 2601, Australia.
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Daria
John Curtin School of Medical Research, Australian National University, Garran Road, Canberra ACT, 2601, Australia.
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Gillespie
Australia- EMBL Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney NSW, 2052, Australia.|Australia Research Council Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Australia.
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Gaus
Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, 02114, USA.
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Alt
Research School of Engineering, College of Engineering and Computer Science, Australia National University, North Road, Canberra ACT, 2601, Australia.|Australia Research Council Centre of Excellence in Advanced Molecular Imaging, Australian National University, Australia.
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Lee
D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D017785:Photons; D011058:Pollen
Intravital microscopy; high-speed scanning; neuron signaling; real-time motion correction