Multiple optic devices are designed for the various experimental requirements, such as stimulating neuron of C. elegans, training C. elegans and inducing C. elegans' to move in a certain direction. They can regulate and output the optical signal varying with not only time but also space.

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Modulate Projector Light Source

Biology background in our project: We need 395nm light to activate Calcium indicator protein GEM-GECO for neuron imaging. For the independent excitation of neurons (AWA and AWB) in C. elegans, channelrhodopsins CoChR and Chrimson are activated by blue light and red light respectively ^[1]. Due to these proteins' limited sensitivity to the wavelength of light, we need to purify the light from the projector.

Fluorescence microscope plays an important role in our experiment. The Zeiss Company creates a perfect visual animation to show how the fluorescence microscope works, and you can find it on their [http://zeiss-campus.magnet.fsu.edu/tutorials/basics/axioobserver/indexflash.html web]. However, typical fluorescence microscopes can only illuminate the specimen ( C. elegans) with the same light in whole visual field. Thus, we modify a consumed projector to a new light source, which can illuminate specimens locally, in multi-color, in multi-points and dynamically. The Projector Light Source also has short response time and high light intensity, which will be an effective and promising tool for synthetic biology.

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The design of optics

GEM-GECO is activated by 395nm light from LumencorSpectra LED Illuminator.

We use projector [http://www.epson.com.cn/products/projectors/239/CB-X03/ EPSON CB-X03] as red light& blue light source for CoChR & Chrimson. Projector's 3LCDs(red, green and blue channel) functions as 1024*768 electronic shutters in each channel. Projector's super high pressure mercury lamp functions as a high-intensity light source[Fig. 2].

Firstly, we need to purify the light of projector. Although the original filter can separate white light into red light and blue light, the bandwidth of red light or blue light is not narrow as what our optical sensory proteins(CoChR & Chrimson) require . We install additional red filter Chroma ET630/20X and blue filter Chroma ET480/20X outside 3LCDs. (Fig. 3B) According to the principle of fluorescence microscope, corresponding Di-Mirror 89402bs and emission filter 89402m are installed inside the microscope filter wheel(Fig. 3C). You can see all filters we use in our project (Fig. 3).

Secondly, the imaging scale of projector should be shrinked. Becausing of using 395nm light, red light and blue light at the same time, they are combined by a device, called double LH Adapter(it contains a semi-transparent mirror). The double LH Adapter is connected to microscope. (Fig. 5) However, based on the optical principle of microscope, the light should focus on the plane of Double LH adapter. To project the whole image (from projector) to microscope, it should be about 5cm*5cm on plane. The focus distance of [http://www.ebay.ph/itm/141444870051 Dukane 3 inch/2.5] is(a cheaper lens than original one) 3 inches(75mm). Every pixel in the LCD is considered as a single point light source. According to the formula below: \frac{1}{f_{len}}=\frac{1}{D_{len-LCDs}}+\frac{1}{d_{len-adapter}} to estimate position of the lens. Lens helps us to shrink the projector's image to microscope(Fig. 5). Finally, we can control every pixel of image on specimen from projector.

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The design of mechanics

We need to install a new lens on projector with adjustable component, and fix all device with microscope stably.

Lens Holder and Connection Tube are design(Solidworks Documents) by CNC and 3D-Print(Fig. 6).

To build dark environment, we also build a dark room with cloth(Fig. 7).

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The design of software

To control the ouput of light source, a software is required. However, a very easy method is using a slide(Demo Slide), such as LibreOffice(Test on 5.4.2.2.0+ in Arch Linux) or Microsoft Office. The image and time pattern is configured in slide, including color, intensity and pulse time etc.

We also develop a more powerful and hackable open source software suite called ColorMapping to track and activate multi C. elegans or cell independently in one view. User can modify multi color, intensity, time and locations of light alternately in GUI. Everyone is welcome to join us to develop ColorMapping in GitHub, which still is developing. ColorMapping, contains camera part, projector part, user&calculation part. It is coded by Python 2 Language, and third-party packages, PyGraph, PyQt5 and [http://lima.blissgarden.org/camera/andor3/doc/index.html?highlight=andor3 Lima(Library for Image Acquisition)]. ColorMapping tests on Arch Linux.

{{SUSTech_Image | filename=T--SUSTech_Shenzhen--LightModulator_andor.png | width=209px| caption=Fig. 9 Community with Andor Company}} To drive Andor Zyla (5.2), we apply Andor SDK3 from Andor Company firstly. All data are matrix during data transfer and calculation. Camera image lives in user interface. In camera part, exposure time and other parameters can be adjusted.

An important algorithm is how to project a special area in special color. The key is coordinate system transformation between camera view(2560*2160) and projector(1024*768). To simplify the question, only the left projector edge and 4 ROIs(region of interesting) are drew. Every point(X_{projector}, y_projector) in the image will be generated from following formula(Fig. 10). \left[\begin{matrix}x_{projector}\\y_{projector}\end{matrix}\right]= {\frac{1024}{L}}(\left[\begin{matrix}x_1\\y_1\end{matrix}\right]-\left[\begin{matrix}x_0\\y_0\end{matrix}\right])

In ROIs Setting, every ROI can define the color(Blue or Red) and intensity(0~255). 4 ROIs can move by mouse in real time, so the projector can response immediately. Generated image is showed a full-screen window in another extended desktop. Projector just show on extended desktop.

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Modulate Mercury Lamp

Biology background: To reduce the phototoxicity during train C. elegans, pulse of light is required rather than constant light^[2].

A simple and effective device output pulse of a certain wavelength of light. Arduino (A popular single-board microcontrollers) connect a servo, which is fixed in the shutter on mercury lamp by a 3D-print connector. When the servo motor rotates the shutter, on time and off time of the pulse is custom by Arduino . Wavelength of light is changed by replacing filter before beam expander(Fig. 12).

The 3D-print connector and Arduino code can download here. The result can be found in behavior result part.

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Bill

Here are detail bill.

References

1. ↑ Lisa C. Schild and Dominique A. Glauser , Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans, 2015, GENETICS.
2. ↑ Venkatachalam, V., & Cohen, A. E. (2014). Imaging GFP-Based Reporters in Neurons with Multiwavelength Optogenetic Control. Biophysical Journal, 107(7), 1554–1563. http://doi.org/10.1016/j.bpj.2014.08.020