Team:SUSTech Shenzhen/Hardware/Light

Playing with Light in Space & Time

Let there be light!

Multiple optic devices are designed for the various experimental requirements, such as stimulating neuron of Caenorhabditis elegans, training C. elegans and inducing C. elegans to move in a certain direction. They can regulate output temporally and spatially in an elegant and efficient way.

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Forming Pattern with Light

Biology background in our project: We need to use 395nm light to activate Calcium indicator GEM-GECO for neural activity 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 . Due to these proteins' selectivity to the wavelength of stimulating light, we need to "purify" the light from the projector.

Fig.1 A) Schematic of fluorescence microscope. B) The proteins' spectra in our system

Fluorescence microscope plays an important role in our experiment, because we need to use specific lights to stimulate worms. The Zeiss Company creates a perfect visual animation to show how the fluorescence microscope works, and you can find it on their webpage. However, typical fluorescence microscopes can only uniformly illuminate the specimen ( C. elegans) with the same light within entire visual field. Thus, we modify a consumer 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 a short response time and high light intensity, which will be an effective and promising tool for synthetic biology studies.

Fig.1 Playing a video clip with the modulator and observe the fluorescence :)

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Optical System

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

We use a EPSON CB-X03 LCD projector as the red & blue light sources for CoChR & Chrimson. The projector's 3 LCDs(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].

Fig. 2 The projector structure. A)Parts of projector: Super high pressure mercury lamp, 3LCDs, original lens. B) Light path inside projector

Firstly, we need to "purify" the light wavelengths 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).

Fig. 3 A) The spectrum of filters and mirrors that are used in our system. B) Filter ET630/20X for Chrimson, and Filter ET480/20x for CoChR. Both these are installed outside 3LCDs. C) Di-mirror and emission filter are installed inside microscope

Fig.4 Play movie on hand.

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 Dukane 3 inch/2.5 (we used to replace the original lens) is 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 computer controlled projector.

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System Integration

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).

Fig. 5 Apparatus to merge projector and microscope. Tube Lens Holder is used to hold new lens.

Fig.6 The design of lens holder and connection tube. The position of lens can be adjusted in thread.

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

Fig. 7 Projector holder and cloth to make dark environment

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Software Tool

Fig.8 Use Libreoffice for excitation.

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 multiple 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 Lima(Library for Image Acquisition). ColorMapping tests on Arch Linux.

Fig. 9 Help from 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.

Fig. 10 The design's flowchart of ColorMapping . Image display in user interface from camera by Lima with Andor SDK3. User can track worm with some parameter. Then software generate image and project into microscope from projector. Finally, camera captures these pattern.

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Generating Rhythm with Light

Fig. 10 Demo

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

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).

Fig. 11 The PCB of Arduino modulate Mercury Lamp. Arduino's power is USB, and connect a servo motor to rotate the shutter.

Fig. 12 Overview of Arduino modulate Mercury lamp(A) Arduino transfer the pulse command to servo. (B)The interview of Mercury Lamp. The servo fix at the original bottom. Optical fiber connect to beam expander. (C) The beam expander with filter connect to stereoscope

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

Playing worm with Light

Because C. elegans expresses channelrhodopsins CoChR, C. elegans can move following the red light point in plate. We use optical fiber, boards and mercury lamp to build it.

'Fig. 13 How to build the light source with optical fiber and board'

. You can found the result in behavioral experiments part

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Bill

Here are detail bill.

Parts	Unit	COST/unit (CNY)	COST (CNY)
EPSON	1	3960	3960
CNC Components	1	600	600
Precise 3D-print Component	1	213.22	213.22
Optical plate	1	230	230
Lifting columns(GCM-2211)	3	210	630
Arduino nano v3.0	1	13.50	13.50
Tower-Pro Servo	1	8	8
Filter 89402bs	1	3159.45	3159.45
Filter ET480/20X	1	2161.73	2161.73
Filter ET630/20X	1	2161.73	2161.73
Emission Filter 89402	1	2161.73	2161.73
		TOTAL (CNY)	15299.36
		TOTAL (USD)	2312.62

References

Made by from the elegans.Inc in SUSTech_Shenzhen.

Licensed under CC BY 4.0.

• ©2017 SUSTech_Shenzhen