Fluorescence recovery after photobleaching (FRAP) has been developed to measure molecular diffusion in living cells. However, conventional FRAP using a single stationary beam guided by a pair of galvanometer mirrors is not tailored for raster scanning microscopy. Furthermore, it has been shown that a single point of 2D FRAP only acquires molecular diffusion within a given imaging plane and does not fully capture the full molecular dynamics. Here, we address these limitations with a custom-built 2-photon polygon scanning microscope that features volumetric scanning with a frame rate of 20 fps and 170 nm pixel size. Importantly, our system allows photomanipulation to selectively measure FRAP from the diffusion dynamics of fluorescent molecules in a 3D sample. To demonstrate these capabilities, we performed rapid axial scans of fluorescent beads in suspension, achieving a volumetric scan rate of less than a second. FRAP functionality was verified in vitro on sulforhodamine-labelled giant unilamellar vesicles and diffusion kinetics determined from the rate of fluorescence recovery. The resolution and speed introduced from polygon scanning microscopy coupled with photomanipulation capabilities sets a precedent for 2-photon 3D FRAP imaging.
Cellular imaging in living animal has opened up a wide range of avenues to study cells in its microenvironment. High speed laser scanning microscopy possess the ability to observe fast real-time biological phenomena such as cell movements, cell division, cells death. Due to the anatomical difference of difference organs in small animals, there is a need to engineer a flexible microscope that can readily adapt to different imaging position. For videorate imaging, the design of a flexible microscope depends mainly on scanning devices. Existing multiphoton microscope platforms (i.e. Thorlabs Bergamo® II Series) uses a rotating objective mount to conform of the specimens. This is possible because of the compact resonant mirror scanners. However, for varying imaging speed using a polygon microscope, this approach is not feasible due to high rotating speed. As such, we developed a dual objective microscope system that can achieve both upright and inverted, we termed it as UNI-SCOPE. The integrated platform can achieve flexible scanning speed of up to 120 FPS with an overall footprint of 450mm*600mm*450mm. Using a dual objective approach, users can tailor the platform to the imaging sample.
Multiphoton laser scanning microscopes exhibit highly localized nonlinear optical excitation and are powerful instruments
for in-vivo deep tissue imaging. Customized multiphoton microscopy has a significantly superior performance for in-vivo
imaging because of precise control over the scanning and detection system. To date, there have been several flexible
software platforms catered to custom built microscopy systems i.e. ScanImage, HelioScan, MicroManager, that perform
at imaging speeds of 30-100fps. In this paper, we describe a flexible software framework for high speed imaging systems
capable of operating from 5 fps to 1600 fps. The software is based on the MATLAB image processing toolbox. It has the
capability to communicate directly with a high performing imaging card (Matrox Solios eA/XA), thus retaining high speed
acquisition. The program is also designed to communicate with LabVIEW and Fiji for instrument control and image
processing. Pscan 1.0 can handle high imaging rates and contains sufficient flexibility for users to adapt to their high speed
imaging systems.
Intravital multiphoton microscopy has emerged as a powerful technique to visualize cellular processes in-vivo. Real time processes revealed through live imaging provided many opportunities to capture cellular activities in living animals. The typical parameters that determine the performance of multiphoton microscopy are speed, field of view, 3D imaging and imaging depth; many of these are important to achieving data from in-vivo. Here, we provide a full exposition of the flexible polygon mirror based high speed laser scanning multiphoton imaging system, PCI-6110 card (National Instruments) and high speed analog frame grabber card (Matrox Solios eA/XA), which allows for rapid adjustments between frame rates i.e. 5 Hz to 50 Hz with 512 × 512 pixels. Furthermore, a motion correction algorithm is also used to mitigate motion artifacts. A customized control software called Pscan 1.0 is developed for the system. This is then followed by calibration of the imaging performance of the system and a series of quantitative in-vitro and in-vivo imaging in neuronal tissues and mice.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.