请问：单颗粒样品收集需要多少个Square？

Question 1

单颗粒样品收集需要多少个Square？

How much Squares are you need in CryoEM SPA？

A：

结构生物学博士后：2-3个。

Postdoc in structural biology : 2-3 squares.

新进结构生物学研究生：😮那我为啥要做那么多！！

New Structural Biology Graduate Student: 😮 Then why do I have to do so many!!

应用场景 Application scenarios

数据收集条件 Colletion

• 数据收集相机 Camera ：K2

• 数据收集模式 Model：TEM

• 放大倍数 Magnification：165Kx

• Pixel size：0.82Å

• 照片像素尺寸 Size：4K x 4K

目标数据量 Data

• 总颗粒数：30万

Particles : 300,000

• 目标照片数量：3000张

Micrograph : 3000

• 每张照片颗粒数：100

Particles in one micrograph : 100

对样品的要求 Sample

• 冰层厚度：50nm

Ice thickess : 50nm

• 样品浓度：15mg/ml(500kDa)理论计算

Concentration : 15mg/ml(500kDa)

是的，你其实不用做那么多，实际上2-3个square就够用了。

Actually, you don't really have to do so many, 2-3 square is enough.

我们简单做个计算，我们以Apoferritin为例，我们希望有30万个颗粒，现在我们用165K倍成像，每张照片的边长大约是0.33um，Apoferritin颗粒的直径大约为12nm，每张照片中大约有100个不重叠的好颗粒，所以我们需要3000张照片。

孔径孔距为1.2/1.3um的ANTcryo载网，每个Square有625个孔，扣除bar附近的厚冰区，剩余部分大约有300-400个孔。每个孔可以收集4张照片，每个square可以收集1200-1600张照片，所以我们需要2-3个square。

Let's do a simple calculation. Taking Apoferritin as an example, we want to have 300,000 particles. We are imaging at a magnification of 165,000x, and each photo has an approximate side length of 0.33um. The diameter of Apoferritin particles is about 12nm, and there are approximately 100 non-overlapping good particles in each micrograph. Therefore, we will need 3,000 micrographs.

For the ANTcryo grid with an aperture hole size of 1.2/1.3um, each square has 625 holes. After excluding the thick ice regions near the bar, the remaining area has around 300-400 holes. Each hole can collect 4 photos, and each square can collect 1,200-1,600 photos. Therefore, we will need 2-3 squares.

每个Square有300-400的孔，可以收集1200~1600张照片

Each Square has 300-400 holes and can collect 1200~1600 photos

Question 2

每张照片有100个颗粒，应该用多少浓度的样品去制样呢？

There are 100 particles in each photo, how much concentration of sample should be used to prepare the sample?

A：

我们可以计算下，照片中的样品浓度是多少：

Let's calculate the concentration of the sample in the photo:

• 电镜照片边长 Micrograph side length：

4K x 0.82Å=328nm≈0.33um

• 冰层厚度 Thickness：50nm

• 一张照片所拍到的样品体积 :

The volume of the sample in a micrograph：

V=0.33um∗0.33um∗50nm≈5.4*10-¹⁵ml

• 一张照片有100个Apoferritin颗粒:

100 Apoferritin particles in one Micrograph:

（M=500kDa=5*10⁵g/mol）

• 照片中的样品浓度：

Calculation results:

通过上述的计算，每张照片100个颗粒，样品浓度应该在15mg/ml左右。但是实验中我们在使用Vitrobot制样时，一般1.5mg/ml的Apoferritin就可以获得上述的照片了，1.5mg/ml的样品冷冻后变成了15mg/ml，样品大约被浓缩了10倍。这被认为是气液界面对样品的富集作用，如果采用可以降低气液界面影响的VitroJet制备样品，相应的需要提高制备样品的浓度。

如此，我们了解了，想获得目标的数据量，不必要求载网上有很多Square，只要有2-3个square是好的，就可以达到目标了。我们加油干吧！！

According to the above calculation, there are 100 particles each micrograph, and the sample concentration is about 15mg/ml. However, when Vitrobot was used to prepare samples in the experiment, the micrograph could be obtained with 1.5mg/ml Apoferritin. After vitrification, the 1.5mg/ml sample became 15mg/ml, so the sample was about 10 times concentrated. This is considered to be the enrichment effect of the air-water interface on the sample. If the sample is prepared by VitroJet, which can reduce the influence of the air-water interface, the concentration of the prepared sample should be increased accordingly.

In this way, we understand that if we want to get the target amount of data, as long as 2-3 square is good, we can reach the goal. Let's work hard!!

END

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