生活荧光成像的隔离脊椎动物的感光细胞的制备
Summary
一个方法是为单身的生活，从不同的脊椎动物物种的荧光成像的感光细胞的准备。该方法可用于图像的内源性荧光基团，如NADH或维生素A，的荧光，或外源性补充钙敏感的荧光染料，<sup> 2 +</sup>或其他因素。
Abstract
在脊椎动物视网膜，phototransduction，光转换为电信号，进行了由杆和锥感光细胞1-4。棒感光器负责在昏暗的光线，在明亮的光线锥的视野。 Phototransduction的感光细胞外，一个专门的车厢，其中包含一个视觉色素，主光探测器的高浓度段的地方。视色素是一个生色，11 – 顺视网膜附着的蛋白质，视蛋白组成。视觉色素吸收光子异构从11生色团- 独联体国家全反式 。这种光致异构化带来了一个启动一个级联反应，最终在膜电位的变化，为电信号的传导光刺激的视觉色素的构象变化。光刺激的细胞恢复涉及的失活，由光激活的中间体和膜电位的重建。的Ca 2 +调节几个phototransduction涉及的酶的活性，减少光刺激后，其浓度。在这样的Ca 2 +在细胞光刺激，其背景光的适应复苏起着重要作用。
恢复过程的另一个重要组成部分，已在光检测摧毁其11的光致异构化的视觉色素的再生–独联体生色全反式5-7。这种再生开始的全反式视网膜的光敏色素的释放，留下的APO -蛋白视蛋白。被释放的全反式视网膜利用NADPH的全反式视黄醇，视蛋白反应迅速减少，结合新鲜的11 –顺视网膜带来改革的视觉色素外段。全反式视黄醇，然后转移到邻近的细胞外段和专门载体Interphotoreceptor维甲酸结合蛋白（IRBP）。
单一的感光细胞的荧光成像可用于他们的生理和细胞生物学研究。的Ca 2 +敏感的荧光染料，可用于详细研究外段的Ca 2 +的变化和反应之间的相互作用 ，以光 8-12以及内心的Ca 2段中的作用的Ca 2 +存储+动态平衡13,14 。荧光染料，也可用于测量镁2 +浓度，pH值，以及水和膜车厢 16示踪剂。最后，全反式视黄醇（维生素A）的内在荧光可以用来监测其形成的动力学和17日至19日在单一的感光细胞去除。
Protocol
1。 Sylgard盖菜，试验室，和刀片的制备 35毫米猎鹰的Petri Sylgard弹性体涂层的菜是一个孤立的视网膜上获得单的感光细胞需要适当切碎。根据供应商的说明和少量浇到每道菜，其底部覆盖一层弹性体准备。更换的菜后，涂层覆盖，并将其储存。在几天的时间弹性体变硬的菜都准备好了。 隔离光感受器需要坚持的实验室的底部，让他们固定在成像实验的过程中。这是通过涂层与聚- L -?…
Discussion
如果未取得健康孤立的细胞，但问题在于与隔离或视网膜的健康，或者其斩。通常情况下，取出后，眼睛的玻璃体前，视网膜随时升空色素上皮细胞。如果没有，尝试从外围眼罩开始剥离它关闭。如果它仍然是难以分开，一个可能性是，动物还没有足够的时间内，暗适应，或太亮了红灯。确保适当的暗适应的动物，和昏暗的红灯。在视网膜上的杆光感受器的存在，可以很容易地通过检查孤立的视?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
支持内授予EY014850。
Materials
| Name | Company | Catalogue number | Comments |
|---|---|---|---|
| Dark room (100-150 ft2) | |||
| Red lights19 | Online stores | ||
| Infrared light sources and infrared image viewers | FJW Optical Systems, Inc. | ||
| Dissecting microscope19 | Outfitted with infrared viewers | ||
| Epifluorescence microscope enclosed in a light-tight cage19 | |||
| Dissecting tools (scissors, forceps, blade holder) | Roboz or Fine Science Tools | ||
| Sylgard elastomer | Essex (Charlotte, NC) | Sylgard 184 elastomer kit | |
| Poly-L-ornithine (0.01%) | Sigma-Aldrich | P4957 | |
| Poly-L-lysine (0.1%) | Sigma-Aldrich | P8920 | Dilute to 0.01% |
| Experimental chambers | Warner Instruments (Hamden, CT) | D3512P | |
| Petri dishes, plastic pipettes | Fisher Scientific |
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Tags
Living Isolated Vertebrate Photoreceptor CellsFluorescence ImagingPhototransductionRod PhotoreceptorsCone PhotoreceptorsOuter SegmentVisual PigmentChromophoreOpsinPhoton AbsorptionPhotoisomerizationConformational ChangeCascade Of ReactionsMembrane PotentialElectrical SignalRecovery ProcessDeactivation Of IntermediatesCa2+ ModulationAdaptation To Background LightRegeneration Of Visual Pigment
Cite This Article
Boyer, N. P., Chen, C., Koutalos, Y. Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging. J. Vis. Exp. (52), e2789, doi:10.3791/2789 (2011).