We developed a novel addressable multiregional multiphoton microscope that employs a fast one-dimensional discrete-line scanning approach based on a spatial light modulator (SLM). The phase-only SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple specific discrete-lines, which are designed according to the sizes and locations of the target samples. Only the target-sample areas of are scanned
one-dimensionally, resulting in an efficient use of the laser’s power. Compared with conventional multiphoton microscopies, this technique shortens scanning time and minimizes photodamage by concentrating scanning energy and dwell time on the areas of interest. Additionally, our discrete-line-focus design eliminates the cross-talk that occurs in conventional one-dimensional line-scanning multiphoton microscopes, thus enhancing the lateral and axial resolutions of the line-scanning imaging system.
Using hybrid TPEF-SHG imaging and immunocytological techniques, we studied dedifferentiation of adult
cardiomyocytes. First, the myofibrils shrank to shorten the sarcomere length. At the cell ends, the striated pattern of
myosin filaments began to dissociate; at the center of the cell, the striated pattern of alpha-actinin first faded away and
reappeared near the cell membrane during dedifferentiation. The results suggest that when freshly isolated adult
cardiomyocytes are used to model cardiac muscle, the end-to-end connection may be important to maintain their striated
myofibrillar structure and rod-shape morphology.
Through a combination of a deflective phase-only diffractive spatial light modulator (SLM) and galvo scanners, an addressable multiregional and multifocal multiphoton microscope (AM-MMM) is developed. The SLM shapes an incoming mode-locked, near-infrared Ti:sapphire laser beam into multiple beamlet arrays with addressable shapes and sizes that match the regions of interest on the sample. Compared with conventional multifocal multiphoton microscope (MMM), AM-MMM achieves the effective use of the laser power with an increase of imaging rate and a decrease of photodamage without sacrifice of resolution.
Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second
harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes
during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of
existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed
mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled
onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an
important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length
We developed a novel addressable scanless multifocal multiphoton microscope. This microscope works in a fast scanless
mode. Subjectively selected sample (or multiple samples located in separated areas) in a large field of view can be
imaged by illuminating only the area (or areas) where the target sample (or samples) locate(s). In this way, by precisely
designing the multiple foci according to the size and position of the area of interest, we can concentrate all the laser
energy and dwell time on that area of the sample, making full use of the available laser power and avoiding photodamage
in other areas. Since no scanning is involved, the acquisition time of a multiphoton image is decided only by the
sensitivity and readout time of the CCD camera. Moreover, the interfocal distance of the multiple foci matches the lateral
resolution of the imaging system, so that the two-photon image was recorded with high lateral resolution. However,
crosstalk (spatial interference) on out-of-focus planes occurs between adjacent points when they are too close, degrading
the resolution, especially the axial resolution of the imaging system.
We developed a hybrid two-photon excitation fluorescence-second harmonic generation (TPEF-SHG) imaging system with an on-stage incubator for long-term live-cell imaging. Using the imaging system, we observed the addition of new sarcomeres during myofibrillogenesis while a cardiomyocyte was spreading on the substrate. The results suggest that the TPEF-SHG imaging system with an on-stage incubator is an effective tool for investigation of dynamic myofibrillogenesis.
We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire
laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement
high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The
SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this
imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF
in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG
and TPEF in the research of myofibrillogensis.