Dr. Meng Cui at the Howard Hughes Medical Center has recently pioneered Super Penetration Multi-Photon Microscopy (S-MPM) at the Cui Lab. He has successfully reported on focusing light through static and dynamic strongly scattering media using our segmented 492-DM (See more on the application here). By using the iterative multi-photon adaptive compensation technique (IMPACT), he since reported new results on in vivo fluorescence microscopy, providing a unique solution to noninvasive brain imaging. I HIGHLY encourage everyone to read his paper for in depth details of his technique here.
As of today, IMPACT has been the only technique used for in vivo microscopy. Due to the complicated wavefront distortion encountered in highly scattering biological tissue, IMPACT has the highest success rate in enabling neuron imaging through intact skulls of adult mice. Through Dr. Cui's testing, he has proven that even with the unpredictable motion of awake mice, IMPACT using the segmented 492-DM were able to perform wavefront measurements and improve the image quality.
Dr. Cui used the BMC segmented 492-DM as both the wavefront modulation and correction device. The IMPACT measurement works by splitting the DM’s pixels and running parallel phase modulation with each actuator at a unique frequency. Modulating only a portion of the pixels while keeping the rest stationary, a linear phase shift is then used as a function of time over the entire 2π phase range. The unique modulation frequency then becomes the unique phase slope value. At the end of the modulation, a Fourier transform is used in IMPACT to determine the correction phase values. Dr. Cui then goes on to explain in detail how to determine what fraction of the pixels should be modulated, how to split the pixels into two evenly distributed groups and how the Nyquist-Shannon sampling theorem is integrated.
The imaging starts by setting the laser beam at the point of interest. The parallel phase modulation begins at one half at a time. As the measurement progresses, the laser focus becomes stronger, and laser power is gradually reduced to preserve the fluorescence signal from the sample. At the conclusion of the measurement, the compensated wavefront is displayed on the DM, and laser scanning in a conventional scope is begun. Figure 1 below shows the test setup for the experiment.
Figure 1. Setup of the multiphoton microscope integrated with IMPACT.
Dr. Cui used IMPACT for imaging the dendrites and spines of layer 5 pyramidal cells, in vivo at 650-670um under the dura in the mouse S1 cortex. In Media 1 below, you can see they are hardly resolvable with system correction only. In Media 2, you can see the dendrites and spines are clearly determined when full compensation has been applied.
Media 1. Hardly resovable dendrites and spines Media 2. Resolved dendrites and spines
For the first time, IMPACT enabled in vivo two-photon fluorescence imaging through the intact skull of adult mice. The technique also improved the fluorescence signal by a factor of ~20, along with overall resolution and contrast, this has proven to be a much greater adaptive optics imaging method than any other before. Dr. Cui also concluded that through these experiments, he found it worked well for awake, head-restrained animal imaging, providing a new and innovative solution for noninvasive studies of the mouse brain.
For more information on research going on at the Cui Lab, click here.
If you are interested in finding out more information on how the segmented 492-DM can help you achieve fluorescent imaging, please contact us here!