细胞成像支持 – 问题排查

您可点击此处访问相应的页面。在“细胞成像系统”部分下，找到并点击您的EVOS™成像系统的链接。之后您即可发现三个下载链接和若干说明。我们建议您至少每六个月检查一次更新，或者在您的系统出现任何软件方面的小问题时检查一次更新。

如果镜头一直在样品上移动，可能是对焦太快且丢失焦平面（如果是手动聚焦）的问题，或者是物镜校准的问题（如果使用自动聚焦）。最好使用系统自带的FL Auto校准载玻片校准物镜。首先检查看您的物镜是长工作距离（LWD）物镜或盖玻片矫正（CC）物镜。如果是盖玻片矫正（CC）物镜，则只能采用超短的工作距离，通过薄盖玻片进行成像，不能通过载玻片、微孔板或培养皿内的塑料底进行成像。如果使用高放大倍数或者油镜，利用肉眼观察，向上移动物镜接触到样品的底部，然后缓慢地朝远离样品的方向移动物镜，进一步聚焦。 

物镜镜头在与样品的刮擦中会受到严重的损毁。如果发生刮擦，检查物镜的损坏情况。

当在Z轴（上和下）聚焦得太高时，物镜会擦到容器支架适配器。这是EVOS™ FL Auto成像系统启动、仪器启动时移动载物台或切换物镜时遇到的特殊问题。盖玻片矫正物镜的镜筒顶部更加宽和平，尤其是在样品容器边缘成像时，这意味着它们更容易触碰容器支架适配器的边缘，在这些情况下，无法使用物镜在这些区域成像。如果物镜被容器支架适配器‘’卡‘’住了，慢慢的旋转容器支架适配器上的拇指螺丝，将其竖直提起离开载物台，然后再朝着载物台的中心向下移动物镜聚焦。最好的办法是在您的实验室设定一个关机流程，包括将物镜移动到最低的放大倍数，在每天关闭仪器之前向下调整镜头。

物镜镜头在与容器支架适配器的刮擦时会严重损毁。如果发生刮擦，取出物镜，仔细检查物镜——尤其是镜头——是否损坏。 

对于EVOS™成像系统而言： 

• 确认灯已打开（很容易检查：在载物台上放一块薄纸） 
• 确认样品不要太混浊；与校准载玻片或其他薄的单细胞样品载玻片进行对比。 
• 检查物镜，确认物镜转轮对齐并且物镜完全转动到它的位置。 
• 对于EVOS™FL成像系统：移动光立方的位置 
• 对于EVOS™FL Auto成像系统：检查所有的USB端口，确保显微镜与电脑连接。 
•  对于明场设置，检查聚光镜滑块位置，确保聚光镜滑块在预定的位置。 

First, examine unstained/unlabeled tissue under all filter sets to determine that this is not due to endogenous autofluorescence.This is particularly a problem with paraffin sections.If the control still shows this autofluorescence, it may be reduced by washing 3 x 10 min with 1 mg/mL sodium borohydride prior to blocking and labeling.

What may be happening is non-specific binding of the secondary antibody due to dye charge, for example, where the negatively-charged dye is attracted to positively-charged cellular components.To block this, use Image-iT™ FX Signal Enhancer (Cat Nos. I36933 and R37107), which blocks non-specific binding due to charge interactions between the dyes on conjugates and cellular components.

Some labels, including some antibodies, are of low-enough affinity that they can come off over time during storage of the labeled slides.To slow this off-rate, samples can be post-fixed with formaldehyde for 5–15 min, after the secondary antibody, to cross-link the secondary antibody in place.The sample should also be mounted in a hardening mounting medium, such as ProLong™ Diamond Antifade Mountant, as the hardening mountant slows diffusion of the secondary antibody.Finally, after the mountant has fully hardened, the slide can be stored cold, preferably at –20^°C, to further slow any dissociation.

When cells and tissues are treated with solvents such as xylene or acetone (for example during deparaffinization of tissue sections), it affects the F-actin in a way that prevents phalloidins from binding.Cryosections, which are not typically washed with organic solvents, can be used instead of paraffin, or anti-actin antibodies may be used.

All fluorescent dyes will fade, or “photobleach,” to at least some extent when exposed to strong light at the wavelengths they absorb.Here are some causes for photobleaching and ways to fix the problem:

Here is a good guide to choosing an antifade reagent.

First, make sure you are exciting and detecting the dye in the appropriate wavelengths.Next, try optimizing the dye concentration with your controls, as well as the dye staining time.Our manuals have some guidelines.If you are using a live-cell system, we also recommend washing out any unreacted dye for many of our products to reduce background fluorescence, or adding a background suppressor such as BackDrop™ Suppressor ReadyProbes™ Reagent.Check your instrument settings; some plate readers, for instance, may have a means of adjusting the gain setting to get the best signal-to-background.Finally, some dyes are better than others for degree of change upon ion detection.Contact Tech Support by sending an email to techsupport@thermofisher.com if you would like to discuss dye options.

Bubbles may be removed by one of two methods:

1. Place the amount of ProLong™ antifade reagent/mounting medium mixture you wish to use on your sample (plus a little excess) in a microcentrifuge tube.Close the cap and centrifuge this aliquot using a tabletop microcentrifuge (speed from 7, 000 to 13,000 rpm).Bubbles should move to the top and these bubbles may be aspirated using a pipettor/pipette tip. 
2. Unscrew the lid of the bottle/vial containing the ProLong™ antifade reagent/mounting medium mixture to make it loose, but do not remove the lid.Place the entire bottle/vial into a vacuum flask, using a faucet aspirator (faucet T-tube).Apply a vacuum (water running through the faucet) and allow vacuum aspiration to occur from 10 to 20 minutes to degas the mixture. 

To avoid the formation of bubbles on a sample or to remove bubbles:

1. Before pipetting the desired amount of ProLong™ antifade reagent/mounting medium mixture for mounting, set the pipettor for a slight excess volume.When pipetting up the mixture, do not pipette up the complete amount, but lift up the pipette tip from the bottle with the pipettor not yet up to full volume.This prevents the aspiration of bubbles into the pipette tip. 
2. Bubbles trapped during application of the coverslip: When placing your coverslip onto your drop of ProLong™ antifade reagent/mounting medium mixture, place the coverslip at a slight angle then, gently lower the coverslip.If the coverslip is lowered flat onto the sample, or lowered too quickly, bubbles can be trapped.
3. Bubbles trapped in tissue: One problem with tissue sections, particularly cryosections is that air can get trapped within and under the section.Upon mounting, bubbles are not observed but as the mountant hardens, it compresses the sample slightly, forcing air out of the section.This leads to microscopic bubbles forming over the section, trapped within the mountant.To avoid this, degas the tissue sample prior to mounting.Place the sections submerged in buffer or blocking solution, into a vacuum chamber and expose the sample to the vacuum.This will degas the sections and buffer.Remove the sample from this degassed buffer and mount. 
4. If ProLong™ antifiade reagent–mounted samples have already cured but have bubbles, you can un-mount your sample by placing the slides into PBS (Coplin jar or a Petri dish filled with PBS).The ProLong™ antifiade reagent will swell and the coverslip will slide off or can be gently removed manually.You can then re-mount with a new aliquot of ProLong™ antifade reagent/mounting medium mixture.