Tags: deformable mirror, adaptive optics, boston micromachines, resolution, segmented, SLM, spatial light modulator, biological imaging, deep tissue microscopy, Howard Hughes Medical Institute, Janelia Farm Research Campus, BMC, imaging systems, microscopy, two photon, fluorescence
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 resovalbe 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!
Tags: deformable mirror, adaptive optics, Boston University, SLM, spatial light modulator, deep tissue microscopy, Howard Hughes Medical Institute, BMC, imaging systems, two photon, fluorescence
Scattering media can be a real headache if you are looking to achieve high-resolution, deep tissue in vivo images. Without adaptive optics, do not anticipate having the optical control you need to correct for scattering media effectively. But no need to worry, we have a solution.
Since standard Multiphoton Microscopy just wasn’t cutting it, the Cui Lab at Howard Hughes Medical Center pioneered a new technique that Boston University also recently developed, called Superpentation Multi-Photon Microscopy (S-MPM). Each group uses a different optimization scheme but the outcome is the same: The enhanced technique permits active compensation of wavefront aberrations in a scanning beam path through the use of a BMC MEMS Spatial Light Modulator (SLM), allowing for increased depth imaging.
Developed at Boston University and commercialized by Boston Micromachines, the enabling components are the Kilo-SLM and the high speed S-driver. With these components incorporated into the test bed shown in Fig. 1, images of 1 µm diameter fluorescent beads through 280 µm thick mouse skull can be achieved at depths of about 500 µm. The SLM corrected low order spherical aberrations as well as higher order scattering effects. Signal enhancement with higher resolution and contrast were improved by 10x-100x. The optimized SLM phase improves imaging over a field of view of 10-20 µm for samples tested to date with techniques currently in the works to improve upon this.
With 600 nm of stroke and 60 kHz of maximum frame rate, the Kilo-S System comes in a variety of options to fit your needs at a much reduced cost over our standard 1000 channel system. Contact us today for more information on our Kilo-S or any of our other systems!
In our last blog post (Fast and Precise Laser Pulse Compression with the Linear Array DM) we discussed research being done in the Cui Lab at HHMI’s Janelia Farm Research Campus that used our Linear Array DM for laser pulse compression. In part two we examine a two photon fluorescence microscopy project led by associate Reto Fiolka at Janelia Farm that illustrates the use of the Linear Array’s potential as a pulse compressor for imaging applications using the phase resolved interferometric spectral modulation (PRISM) optimization technique.
The Linear Array pulse compressor setup was used to restore the laser pulse to its transform limited state, thus improving the ability to excite fluorescence by two photon absorption. A sample consisting of 10 micron diameter fluorescence beads (emission: 465 nm) was prepared and spread on a cover-slip. The laser beam first propagated through the pulse compressor and was subsequently focused on the sample using a 20X NA 0.5 Nikon objective. A 2D image was obtained by translating a motorized sample stage. Without spectral pulse shaping, only a weak fluorescence signal could be obtained (See figures a and c). Since the objective adds significant additional dispersion to the laser pulse, the spectral phase correction that had been determined previously using the photodiode could not be used. Therefore PRISM optimization was repeated using the fluorescence signal coming from the beads itself as a feedback signal.
Janelia Farm’s results show a dramatic increase in fluorescence signal for the optimized spectral phase (see figures b and d). The signal strength was increased by a factor of ~6.5
According to Fiolka, “The tested device represents a promising alternative to liquid crystal displays, since the MEMS technology enables high filling factor, high efficiency and operation speed, exceptional phase stability and accuracy and can be used over a very broad wavelength spectrum.”
We're very excited about these results and we are currently working with other groups interested in reproducing these results on tissue samples. Thanks again to Dr. Fiolka and the Janelia Farm group for their efforts in improving two photon imaging techniques!!
More details can be found in our Linear Array white paper which includes an application overview of this exciting project. You can also link to the research directly using the links to the Cui Lab and the scientific publication above.