Name: automated charting of the visual space of housefly compound eyes
Uploaded: 2022-03-31T14:20:00
Description: Watch this Scientific Journal Video about Automated Charting of the Visual Space of Housefly Compound Eyes at JoVE.com

这项研究得到了空军科学研究办公室/欧洲航空航天研究与发展办公室AFOSR / EOARD的财政支持（授予D.G.S.FA9550-15-1-0068）。我们感谢普里莫兹·皮里赫博士的许多有益讨论，并感谢科汉·萨图、海因·莱尔图维尔和奥斯卡·林孔·卡德尼奥的协助。

该协议符合大学的昆虫护理指南。
1. 家蝇的准备，麝香
<ol><li>从实验室饲养的种群中收集苍蝇。将苍蝇放入黄铜支架中（图1）。<ol>
<li>从约束管的上部切开6毫米（见材料表）。管子的新上部外径为4 mm，内径为2.5 mm（图1A）。将活苍蝇放在管内，用棉花密封管子以防止损坏苍蝇，并推动苍蝇，使头...

<ol>
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家蝇眼睛视觉轴的空间分布可以用复眼的伪病头现象和光依赖性瞳孔机制引起的反射变化来绘制。因此，将研究的苍蝇安装在测角系统中，该系统允许使用配备数码相机的显微镜设置检查局部刻面图案，所有这些都在计算机控制之下。图像分析产生眼图。遇到的一个基本困难是，如果在测量开始时没有仔细定位眼睛，即使测角装置的微小旋转，眼睛和伪瞳孔的观察位置也会发生很大变化。通过将?...

昆虫视觉系统的紧凑性及其所有者的敏捷性，展示了高度发达的视觉信息处理，引起了来自科学和非科学背景的人们的兴趣。昆虫复眼已被公认为强大的光学设备，可实现敏锐和多功能的视觉能力1，2。例如，苍蝇以其对移动物体的快速反应而闻名，蜜蜂以拥有色觉和偏振视觉2而闻名。
节肢动物的复眼由许多解剖学上相似的单元组成，即ommatidia，每个单元都由刻面透镜覆盖。在双翅目（苍蝇）中，小平面透镜（统称为角膜）的组装通常近似于一个半球。每个奥马蒂迪姆从半宽为1°的小立体角对入射光进行采样。两只眼睛一起的卵形体大约是全立体角，但卵形的视觉轴分布不均匀。某些眼部区域具有高密度的视觉轴，这创造了一个具有高空间敏锐度的区域，俗称中央凹。然后，眼睛的其余部分具有较粗糙的空间分辨率3，4，5，6，7，8，9。
对复眼光学组织的定量分析对于视觉信息神经处理的详细研究至关重要。研究昆虫大脑10的神经网络通常需要了解视轴的空间分布。此外，复眼还激发了多项技术创新。许多生产受生物启发的人造眼睛的举措都是建立在对真实复眼的现有定量研究的基础上的，11，12，13。例如，基于昆虫复眼模型11，14，15，16，17设计了具有高空间分辨率的半导体传感器。然而，迄今为止开发的设备尚未实现现有昆虫眼睛的实际特征。昆虫复眼及其空间组织的准确表示将需要来自自然眼睛的详细和可靠的数据，而这些数据尚未广泛获得。
数据匮乏的主要原因是绘制眼睛空间特征的可用程序极其繁琐。这促使人们尝试建立一种更加自动化的眼睛映射程序。在昆虫复眼自动分析的第一次尝试中，Douglass和Wehling18 开发了一种扫描程序，用于绘制角膜中小平面尺寸，并证明了其对几种苍蝇物种的可行性。在这里，我们通过开发不仅扫描角膜刻面的方法，而且还评估这些小平面所属的角膜的视觉轴来扩展他们的方法。我们以家蝇眼为例，以举例说明所涉及的程序。
扫描昆虫眼睛的实验装置是：部分光学，即带有相机和照明光学元件的显微镜;部分是机械的，即用于旋转所研究昆虫的测角仪系统;和部分计算，即使用仪器和程序的软件驱动程序来执行测量和分析。开发的方法包括一系列计算程序，从捕获图像，选择相机通道和设置图像处理阈值到 通过 从凸面反射的亮点识别单个小平面位置。傅里叶变换方法在图像分析中至关重要，无论是检测单个小平面还是分析小平面模式。
本文的结构如下。我们首先介绍实验装置和伪病蛀现象 - 用于识别活人眼睛中光感受器的视觉轴的光学标记19，20，21。随后，概述了扫描过程和图像分析中使用的算法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digital Camera</td>
			<td>PointGrey</td>
			<td>BFLY-U3-23S6C-C</td>
			<td>Acquision of amplified images and digital communication with PC</td>
		</tr>
		<tr>
			<td>High power star LED</td>
			<td>Velleman</td>
			<td>LH3WW</td>
			<td>Light source for observation and imaging the compound eye</td>
		</tr>
		<tr>
			<td>Holder for the investigated fly</td>
			<td>University of Groningen</td>
			<td></td>
			<td>Different designs were manufactured by the university workshop</td>
		</tr>
		<tr>
			<td>Linear motor</td>
			<td>ELERO</td>
			<td>ELERO Junior 1, version C</td>
			<td>Actuates the upper microscope up and down. (Load 300N, Stroke speed 15mm/s, nominal current 1.2A)</td>
		</tr>
		<tr>
			<td>Low temperature melting wax</td>
			<td>various</td>
			<td></td>
			<td>The low-temperature melting point wax serves to immobilize the fly and fix it to the holder</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any alternative microscope brand will do; the preferred objective is a 5x</td>
		</tr>
		<tr>
			<td>Motor and LED Controller</td>
			<td>University of Groningen</td>
			<td>Z-o1</td>
			<td>Designed and built by the University of Groningen and based on Arduino and Adafruit technologies.</td>
		</tr>
		<tr>
			<td>Motorized Stage</td>
			<td>Standa (Vilnius, Lithuania)</td>
			<td>8MT175-50XYZ-8MR191-28</td>
			<td>A 6 axis motorized stage modified to have 5 degrees of freedom.</td>
		</tr>
		<tr>
			<td>Optical components</td>
			<td>LINUS</td>
			<td></td>
			<td>Several diagrams and lenses forming an epi-illumination system (see Stavenga, Journal of Experimental Biology 205, 1077-1085, 2002)</td>
		</tr>
		<tr>
			<td>PC running MATLAB</td>
			<td>University of Groningen</td>
			<td></td>
			<td>The PC is able to process the images of the PointGrey camera, control the LED intensity, and send control commants to the motor cotrollers of the system</td>
		</tr>
		<tr>
			<td>Power Supply (36V, 3.34A)</td>
			<td>Standa (Vilnius, Lithuania)</td>
			<td>PUP120-17</td>
			<td>Dedicated power supply for the STANDA motor controllers</td>
		</tr>
		<tr>
			<td>Soldering iron</td>
			<td>various</td>
			<td></td>
			<td>Used for melting the wax</td>
		</tr>
		<tr>
			<td>Stepper and DC Motor Controller</td>
			<td>Standa (Vilnius, Lithuania)</td>
			<td>8SMC4-USB-B9-B9</td>
			<td>Dedicated controllers for the STANDA motorized stage capable of communicating with MATLAB</td>
		</tr>
		<tr>
			<td>Finntip-61</td>
			<td>Finnpipette Ky, Helsinki</td>
			<td>FINNTIP-61, 200-1000&#956;L</td>
			<td>PIPETTE TIPS FOR FINNPIPETTES, 400/BOX. It is used to restrain the fly</td>
		</tr>
		<tr>
			<td>Carving Pen Shaping/Thread Burning Tool</td>
			<td>Max Wax</td>
			<td></td>
			<td>The tip of the carving pen is designed to transfer wax to the head of fly</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks, Natick, MA, USA</td>
			<td>main program plus Image Acquisition, Image Analysis, and Instrument Control toolboxes.</td>
			<td>Programming language used to implement the algorithms</td>
		</tr>
	</tbody>
</table>

automated charting of the visual space of housefly compound eyes

本文描述了昆虫复眼视觉轴的空间组织的自动测量，该视觉轴由数千个称为ommatidia的视觉单元组成。每个ommatidium从一个小立体角对光学信息进行采样，其近似高斯分布灵敏度（半宽在1°的数量级上）以视轴为中心。欧马蒂迪亚一起从近乎全景的视野中收集视觉信息。因此，视觉轴的空间分布决定了眼睛的空间分辨率。了解复眼的光学组织及其视敏度对于视觉信息的神经处理的定量研究至关重要。在这里，我们提出了一种自动化程序，用于绘制复眼的视觉轴，使用固有的 体内 光学现象，伪丘皮尔和光感受器细胞的瞳孔机制。我们概述了扫描昆虫眼睛的光学机械设置，并使用从家蝇 Musca家蝇获得的实验结果来说明测量过程中的步骤。

动物和光学刺激 实验是在格罗宁根大学进化遗传学系维持的文化中获得的家蝇（Musca sera）上进行的。在测量之前，通过将苍蝇用低熔点蜡粘合在合适的管子中来固定苍蝇。随后，苍蝇被安装在电动测角仪的载物台上。两个旋转平台的中心与微观设置24的焦点重合。落射照明光束由光源提供，光源将光线聚焦在 通过 半镜在苍蝇眼上成像的光膜上。因?...

Watch this Scientific Journal Video about Automated Charting of the Visual Space of Housefly Compound Eyes at JoVE.com

Automated Charting of the Visual Space of Housefly Compound Eyes

这里的协议描述了家蝇眼睛视觉轴的空间组织的测量，由自动装置映射，使用伪丘皮尔现象和光感受器细胞的瞳孔机制。

家蝇复眼视觉空间自动制图

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

该系统和协议旨在通过最少的人为干预进行映射来分析苍蝇的物理系统。通过系统和协议，我们可以精确地知道苍蝇大小的视觉空间是如何组织的。该系统的优点是映射的可重复性和速度。复眼的研究是动物视觉研究的重要组成部分，并激发了一些产生人造眼的技术创新。我们已经为如何构建和测试用于扫描复眼的自动设备树立了榜样。需要特别注意将各部分组合在一起的算法开发细节。从实验室饲养的种群中收集一只苍蝇开始。通过从上部切割六毫米来准备约束管，使管子的外径为4毫米，上部的内径为2.5毫米。将苍蝇放在切好的管内，用棉花密封管子，以防止损坏苍蝇。然后推动苍蝇，使头部从管中突出，身体被限制在管中。使用蜂蜡固定头部，同时眼睛保持未覆盖。完成后，切割管子以达到10毫米的长度。然后将装有苍蝇的塑料管放在黄铜支架中，苍蝇的一只眼睛指向上方，支架放在桌面上。按照手稿中所述调整显微镜上管子的方向，以在设置允许的方位角和仰角范围内扫描整个眼睛。通过在方位角旋转台上安装一个对准销来设置显微镜，以便可以调整尖端的X-Y位置以与电动载物台上的方位角轴重合。使用配备5X物镜的显微镜进行观察时，使用Z轴操纵杆聚焦在尖端上。接下来，将方位角轴的X-Y调整与显微镜的光轴对齐，并使用X和Y轴操纵杆确保仰角和方位角旋转轴与居中销预先对齐。操纵方位角和高程操纵杆以检查引脚是否相对于两个自由度居中。如果居中良好，引脚尖端在方位角和立面旋转期间保持在同一位置。将苍蝇与立面台的零度对齐并安装，并在方位角级上固定。然后用显微镜观察苍蝇的眼睛。打开照明LED后，调整苍蝇的水平位置以对齐伪癡蚜的中心。然后使用支架的旋转螺钉改变伪蜱的垂直位置，使深伪蜱在仰角轴的水平上聚焦。接下来，通过将深伪蜃蚣相对于方位角和高程轴在视野中居中来排列它。设置准备就绪后，将视图切换到安装在显微镜上的数码相机，并运行宽限系统的软件初始化，其中包括初始化电机控制器和Arduino LED控制器。为此，请打开 MATLAB 版本 2020a 或更高版本并运行 MATLAB 脚本。在计算机屏幕上，确认苍蝇的伪傀儡位于投影图像的中心。使用 Z 轴操纵杆将焦点带到角膜假癥的水平。焦点对齐后，运行自动聚焦算法以获得角膜水平的清晰图像。然后通过调整电动Z轴载物台将焦点返回到深伪木偶水平。存储深假癡蝶和角膜假痂起之间的距离。接下来，使用自动对中算法微调伪癡脂对中，然后将焦点带回角膜伪癡脂水平。重新运行自动对焦算法，并将电动载物台归零到其当前位置。扫描眼睛时，运行扫描算法以五度步长沿轨迹对眼睛图像进行采样，同时执行自动对中和自动对焦算法。采样后，关闭 LED 和电机控制器。稍后，通过应用图像处理算法来处理图像。在苍蝇眼光学的研究中，眼睛表面水平的图像显示了活化状态下的刻面反射和颜料颗粒反射。在眼睛曲率中心水平拍摄的图像说明了感光细胞以梯形模式排列的反射，其远端位于小平面透镜的焦平面附近。将两个连续的图像相关联，以确定小平面图案平移的变化。在眼睛上扫描期间拍摄的图像与小平面质心一起显示。在方位角旋转五度后，此处说明了后续图像。质心过程无法识别所有刻面。由轻微的表面不规则性或灰尘规格引起的低局部反射率导致错误的质心。通过计算快速傅里叶变换解决了该错误。第一个谐波环定义了由蓝色、红色和绿色线条指示的三个方向。谐波沿三个方向的逆变换产生了灰色带。家蝇的右眼从正面到侧面扫描了24个步骤。该图将分面的组装显示为沃罗诺伊图。在扫描开始时，应特别注意在测角系统旋转中心调整飞眼的深伪丘比。在这里，我们应用落射照明显微镜。该方法可以直接扩展到荧光显微镜，以研究没有反射假蜉蚓的昆虫。对眼睛视觉轴分布的定量知识将允许了解物理系统如何针对某些任务（例如狩猎，交配或检测捕食者）进行优化。

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Dissecting the Non-human Primate Brain in Stereotaxic Space

剖析在非人类灵长类动物脑立体定向空间

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Using the optokinetic response to study visual function of zebrafish

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

轨迹数据分析行人时空活性研究

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

使用自动成像系统测量 iPSC 的汇合度。

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...