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− | 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 | + | 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. |
{{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Light_modulator_whole.jpg | width=2345px |caption=''}} | {{SUSTech_Image_Center_fill-width | filename=T--SUSTech_Shenzhen--Light_modulator_whole.jpg | width=2345px |caption=''}} | ||
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− | '''Biology background in our project''': We need 395nm light to activate Calcium indicator | + | '''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 <ref>Lisa C. Schild and Dominique A. Glauser , Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans, 2015, GENETICS.</ref>. Due to these proteins' selectivity to the wavelength of stimulating light, we need to "purify" the light from the projector. |
{{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Schematic_fluorescencemicroscope.png | caption=<B>Fig.1 A) Schematic of fluorescence microscope. B) The proteins' spectra in our system</B>}} | {{SUSTech_Image_Center_8 | filename=T--SUSTech_Shenzhen--Schematic_fluorescencemicroscope.png | caption=<B>Fig.1 A) Schematic of fluorescence microscope. B) The proteins' spectra in our system</B>}} | ||
− | 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 [http://zeiss-campus.magnet.fsu.edu/tutorials/basics/axioobserver/indexflash.html | + | 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 [http://zeiss-campus.magnet.fsu.edu/tutorials/basics/axioobserver/indexflash.html 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. |
<html><div class="row"><div class="col-md-8 col-md-offset-2"><div class="thumbnail"><video class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/4e/T--SUSTech_Shenzhen--microproj.mp4" autoplay="1" loop="1"></video></div><div class="caption"><b>Fig.1 Playing a video clip with the modulator and observe the fluorescence :)</b></div></div></div></html> | <html><div class="row"><div class="col-md-8 col-md-offset-2"><div class="thumbnail"><video class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/4e/T--SUSTech_Shenzhen--microproj.mp4" autoplay="1" loop="1"></video></div><div class="caption"><b>Fig.1 Playing a video clip with the modulator and observe the fluorescence :)</b></div></div></div></html> | ||
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GEM-GECO is activated by 395nm light from [https://micro-manager.org/wiki/LumencorSpectraLumencor LumencorSpectra LED Illuminator]. | GEM-GECO is activated by 395nm light from [https://micro-manager.org/wiki/LumencorSpectraLumencor LumencorSpectra LED Illuminator]. | ||
− | We use | + | We use a [http://www.epson.com.cn/products/projectors/239/CB-X03/ 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]. |
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− | 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 [https://www.chroma.com/products/parts/et630-20x Chroma ET630/20X] and blue filter [https://www.chroma.com/products/parts/et480-20x Chroma ET480/20X] outside 3LCDs. (Fig. 3B) | + | 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 [https://www.chroma.com/products/parts/et630-20x Chroma ET630/20X] and blue filter [https://www.chroma.com/products/parts/et480-20x Chroma ET480/20X] outside 3LCDs. (Fig. 3B) |
According to the principle of fluorescence microscope, corresponding Di-Mirror [https://www.chroma.com/products/parts/89402bs 89402bs] and emission filter [https://www.chroma.com/products/parts/89402m 89402m] are installed inside the microscope filter wheel(Fig. 3C). You can see all filters we use in our project (Fig. 3). | According to the principle of fluorescence microscope, corresponding Di-Mirror [https://www.chroma.com/products/parts/89402bs 89402bs] and emission filter [https://www.chroma.com/products/parts/89402m 89402m] are installed inside the microscope filter wheel(Fig. 3C). You can see all filters we use in our project (Fig. 3). | ||
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{{SUSTech_Image | filename=T--SUSTech_Shenzhen--projectorintomicroscope.jpg|width=200px| caption=Fig.4 Play movie on hand.}} | {{SUSTech_Image | filename=T--SUSTech_Shenzhen--projectorintomicroscope.jpg|width=200px| caption=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 [http://www.ebay.ph/itm/141444870051 Dukane 3 inch/2.5] | + | 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] (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:{{SUSTech_Shenzhen/bmath|equ=<nowiki> \frac{1}{f_{len}}=\frac{1}{D_{len-LCDs}}+\frac{1}{d_{len-adapter}} </nowiki>}} 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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To control the ouput of light source, a software is required. However, a very easy method is using a slide([https://static.igem.org/mediawiki/2017/f/f1/T--SUSTech_Shenzhen--slidestimulation.zip Demo Slide]), such as [https://www.libreoffice.org/en 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. | To control the ouput of light source, a software is required. However, a very easy method is using a slide([https://static.igem.org/mediawiki/2017/f/f1/T--SUSTech_Shenzhen--slidestimulation.zip Demo Slide]), such as [https://www.libreoffice.org/en 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 | + | We also develop a more '''powerful''' and '''hackable''' open source software suite called <b><i>ColorMapping</i></b> to track and activate multiple <i>C. elegans</i> 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 [https://github.com/JiangXL/ColorMapping 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}'''} | {{SUSTech_Image | filename=T--SUSTech_Shenzhen--LightModulator_andor.png | width=209px| caption='''Fig. 9 Community with Andor Company}'''} |
Revision as of 01:49, 2 November 2017
Playing with Light in Space & Time
Let there be light!
Contents
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.
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 [1]. Due to these proteins' selectivity to the wavelength of stimulating light, we need to "purify" the light from the projector.
Optical System
GEM-GECO is activated by 395nm light from LumencorSpectra LED Illuminator.
We use a [http://www.epson.com.cn/products/projectors/239/CB-X03/ 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].
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).
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).
To build dark environment, we also build a dark room with cloth(Fig. 7).
Software Tool
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 [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.
Generating Rhythm with Light
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.
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.
. You can found the result in behavioral experiments partBill
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
- ↑ Lisa C. Schild and Dominique A. Glauser , Dual Color Neural Activation and Behavior Control with Chrimson and CoChR in Caenorhabditis elegans, 2015, GENETICS.
- ↑ 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