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 micromachines, retinal imaging, free-space communication, modulating retroreflector, segmented, laser beam, SLM, spatial light modulator, deep tissue microscopy, SPIE, BMC, imaging systems, Photonics West, microscopy, two photon, optical chopper, optical modulator, chopper, Adaptive Optics Scanning Laser Ohphthalmoscope, Joslin Diabetes Center, Mirrors
Just a few weeks ago we arrived back from the Photonics West 2014 exhibition and conference in San Francisco, CA. I wanted to share details and further observations on the show for those present at the show and those not being able to attend this year.
For the first time we made the decision to also attend the BiOS exhibition for the few days prior to PWest. Not being quite sure what to expect for booth traffic, especially since it conflicted with the superbowl, we still generated a good amount of interest for the smaller show. Our main presentations focused on our new adaptive optics-enhanced scanning laser ophthamoscope (AOSLO), the Apaeros Retinal Imaging System, which includes our Multi-DM, and the Superpenetration Multiphoton Microscopy technique, which is enabled by our Kilo-SLM and high speed S-Driver. Although both exhibits generated respectable notice and positive feedback, most people were familiar with the Superpentration Multiphoton work being done. Either wanting to try two-photon microscopy themselves or already in the process of doing so, our Kilo-SLM paired with our high speed S-driver presented data that was intriguing to most.
After wrapping up BiOS, we headed to the opposite side of the South hall at the Moscone Center for a larger booth setup for PWest. Here we had our entire mirror family on display, as well as live demonstrations of the Reflective Optical Chopper and Wavefront Sensorless Adaptive Optics Demonstrator for Beam Shaping (WSAOD-B). For this part of the exhibition, I would say our deformable mirrors produced the most attention, most likely due to our wide assortment of shapes and actuator counts up to 4092. The WSAOD-B live demonstration did generate a great deal of attention, as most people are unaware of how sensorless AO works. Besides our deformable mirror line, I would still say the Multiphoton Microscopy overview was initiating even further interest here as well.
Overall BMC had a great show and it seemed well worth it to expand our exhibit onto BiOS beforehand. Although this was my first time attending the show, I noticed every inch of space at PWest being used for exhibitor tables and booths, even setting up in front of the bathrooms! I hope to see PWest advance even larger, maybe one day expanding to its third space, West Hall. I look forward to next year’s show and hope to reconnect with you all again throughout the year.
If you were not able to attend the show and would like any information on the products mentioned, please visit our website and download our whitepapers.
It has been quite some time since our last blog post due to a great deal going on at BMC! Alongside some new product releases, we recently made a few adjustments and updates to our ophthalmic imaging instrument, the Adaptive Optics Scanning Laser Ophthalmoscope (AOSLO) which we are releasing early next year.
This next-generation instrument allows in vivo retinal imaging on a cellular level and is currently undergoing beta testing at the Beetham Eye Institute at Joslin Diabetes Center, led by Dr. Jennifer Sun and her team. There it is being used to directly quantify features such as cone density, microaneurysm size and measure blood flow through the microvasculature in the retina. By pairing a Scanning Laser Ophthalmoscope (SLO) with advanced Adaptive Optics, it offers the advantage of imaging the retina at a resolution 2-3 times that of a standard SLO.
The AOSLO is also capable of measuring various properties of retinal cone physiology. Due to its enhanced imaging and software, it enables evaluation of the following attributes:
- Cone Density
- Nearest Neighbor Distance
- Voronoi Tessellation Tile Area
- Effective Radius
- Packing Factor
The AOSLO’s ability to measure such features allows early stage detection of visual decline due to diabetes. This can be identified by the decrease in cone regularity, cone mosaic changes, cone reflectance and a decrease regularity of cone spacing. This function of the AOSLO can help determine early treatment plans for patients and generate further investigative studies.
When testing out the AOSLO at Joslin, we found something very interesting out about our CEO, Paul Bierden. The pictures below depict his own retina, discovering that he has a microaneurysm! This was unexpected news, since normally it would be undetectable by any other retinal imaging systems. 30% of the microaneurysms imaged using the AOSLO at Joslin were not visible in fundus photos. The AOSLO is able to accomplish this by evaluating the vascular and neural retinal planes in vivo with cell-scale resolution. The pictures below also point out the microaneurysm attributes that can be measured. They are:
- Presence of lumen clot
- Wall reflectivity
Lastly, the AOSLO is able to measure small-vessel blood flow. This is done with the help of its enhanced imaging qualities, instrument optimization and post-processing software. By stopping a horizontal scan over a blood vessel, it can measure the blood velocity by tracking the moving erythrocytes over a scanning line. With this information, researchers can produce a blood velocity profile for retinal vessels. See the video below to see how it’s done!
If you have any interest in using the AOSLO, let us know! Please give us a call and let us know about your research. We are accepting orders for the new instrument and are open to collaborative grant applications to secure funding. If you are interested in seeing the AOSLO in action, we are setting up appointments now for the next few months. We hope to hear from you soon!
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.
Ultrafast lasers have been extensively used in ground breaking research including two Nobel Prizes. Applications within spectroscopy, photochemistry, laser processing and microscopy are widespread. However, to capitalize on such short laser pulses, a pulse compressor is required to compensate for the dispersion induced by optical elements. Liquid crystal based spatial light modulators are most commonly used in laser pulse compressors. Although a proven technology in display applications, liquid crystals have drawbacks including phase jitter and a limited fill factor. Researchers at the Cui Lab at HHMI’s Janelia Farm Research Campus looked to Boston Micromachines Corporation’s prototype Linear Array Deformable Mirror (DM) to address these challenges.
To evaluate the performance of the pulse compressor, the laser pulses were analyzed with frequency resolved optical gating (FROG) using a commercial instrument (Grenouille, Swamp Optics, Atlanta, GA). In Figure a and b, the temporal and spectral profile of the pulse is shown when a flat wavefront is displayed on the DM. Evidently, the pulse is distorted and the spectral phase is not flat at all (a flat spectral phase is required for a transform limited pulse). Next, the beam returning from the pulse compressor was focused with a concave mirror onto a GaAsP photodiode and the resulting nonlinear signal was used as a feedback for the correction algorithm. After optimization using a technique called Phase resolved interferometric spectral modulation (PRISM), the temporal profile (Figure c) shows a dramatically shorter, Gaussian shaped pulse. The spectral phase is perfectly flat (Figure d) with less than 0.01 radians phase error and is stable in time. These results suggest that the precision and stability of the Linear Array DM allows close to perfect restoration of transform limited laser pulses. For more information on the optimization technique, you can access a scientific publication here.
In our next blog post, we will discuss the results of the use of the Linear Array DM in an interesting two-photon microscopy experiment.
More details can be found in our Linear Array white paper which includes a more detailed description of this application.
This past summer, Boston Micromachines Corporation conducted a survey of nearly 300 members of the business and scientific community to find out what features were valued in a deformable mirror for adaptive optics and other wavefront correction applications. Respondents came from our three major vertical markets: microscopy, astronomy and laser science. In this survey, we asked some fundamental questions and had respondents choose between three DMs with properties varying in categories of actuator count, stroke, response time and price in various combinations. We were able to drill down to what each respondent valued. Here are some of our key findings:
1) Actuator count was the most valued property
Across all verticals, this was true. Overall, respondents preferred an average of 1000 actuators. While microscopists preferred 140 actuators by almost 2 to 1 over other models, those who identified as laser scientists were looking for an average of 1001 actuators and astronomers preferred, on average, 1800 actuators.
This was very interesting to us considering we are the only player in the market to provide deformable mirrors with these actuator counts as standard products or are developing DM systems which meet these specific needs (we have a 2000 element mirror in the works).
2) High speed is important
The most frequently chosen option for response time amongst laser scientists was 50μs and all other disciplines preferred average response better than 300μs. This is great news for the industry considering that most mirror architectures can respond adequately to meet the needs of the users. Our DM architectures are available with response times up to 22μs and we are able to drive these mirrors with our X-Driver (response time down to 4μs), satisfying high speed requirements as well.
3) Low price is desired
As we hear so often, most users were looking for low-priced devices. This was the second preferred property after actuator count. While those of us in the industry talk about lower prices with higher volumes, the volumes just haven’t been there yet to make this prophecy come true. The hope in the future is that the DMs based on scalable technologies, such as MEMS, will take off and lower-priced devices will be available.
We definitely learned a lot from this survey, above and beyond what is mentioned above. If you have any questions about our methods or are interested in discussing more specifics about the responses, I would be glad to chat further. Just contact me at firstname.lastname@example.org.