h****g
发帖数: 439 | 1 这是个困扰我很久的问题。还有，为啥我们用rosa yfp 比较多，为啥不用rosa gfp呢？
还有，为啥rosa rfp 的老鼠能用肉眼看到红色，为啥yfp的小鼠没有颜色呢？
还有，为啥yfp的信号要用PFA固定保存呢？
望达人解惑！ | s******y
发帖数: 28562 | 2 1. the color of YFP: it is green-yellow, so if you don't have a control
color (like the real green), it will looks green. Plus, part of its spectra
will go through the green filter.
2. RFP is activated by green light, which is abundant in natural light.
YFP is activated by blue light, which is NOT abundant in natural light.
In order to see bright fluorescent of YFP you will need to use special
activation light source.
3. Conservation of the fluorescent light: it is important to keep the
structur
【在 h****g 的大作中提到】
: 这是个困扰我很久的问题。还有，为啥我们用rosa yfp 比较多，为啥不用rosa gfp呢？
: 还有，为啥rosa rfp 的老鼠能用肉眼看到红色，为啥yfp的小鼠没有颜色呢？
: 还有，为啥yfp的信号要用PFA固定保存呢？
: 望达人解惑！
| h****g
发帖数: 439 | 3 sunnyday真是《十万个为什么》啊
还有个问题，我们实验室的荧光显微镜上，有一个蓝光channel，一个绿光channel，这
两个channel都能看YFP信号和FITC信号。 这两个channel有啥区别
spectra
【在 s******y 的大作中提到】
: 1. the color of YFP: it is green-yellow, so if you don't have a control
: color (like the real green), it will looks green. Plus, part of its spectra
: will go through the green filter.
: 2. RFP is activated by green light, which is abundant in natural light.
: YFP is activated by blue light, which is NOT abundant in natural light.
: In order to see bright fluorescent of YFP you will need to use special
: activation light source.
: 3. Conservation of the fluorescent light: it is important to keep the
: structur
| b*****l
发帖数: 9499 | 4 是 band through 的 cube 还是简单的 band splitter？
use
【在 h****g 的大作中提到】
: sunnyday真是《十万个为什么》啊
: 还有个问题，我们实验室的荧光显微镜上，有一个蓝光channel，一个绿光channel，这
: 两个channel都能看YFP信号和FITC信号。 这两个channel有啥区别
:
: spectra
| s******y
发帖数: 28562 | 5 In most microscopes, the "color" label of the channel means the excitation
light permitted by the filter cube (or other spectra dividing instrument)
inside. Therefore, the "blue" channel should be used to visualize GFP, YFP
or FITC, and the "green" channel should be used for TRITC or RFP.
Most likely you guys put in two filters with similar cutting ranges,
and arbitrarily label them as "blue" and "green" but it is not really "blue"
and "green". So don't take these label too serious. If you reall
【在 h****g 的大作中提到】
: sunnyday真是《十万个为什么》啊
: 还有个问题，我们实验室的荧光显微镜上，有一个蓝光channel，一个绿光channel，这
: 两个channel都能看YFP信号和FITC信号。 这两个channel有啥区别
:
: spectra
| b*****l
发帖数: 9499 | 6 r u sure?
in my lab, blue is labeled as DAPI, green FITC, and red RHOD.
blue"
，这
【在 s******y 的大作中提到】
: In most microscopes, the "color" label of the channel means the excitation
: light permitted by the filter cube (or other spectra dividing instrument)
: inside. Therefore, the "blue" channel should be used to visualize GFP, YFP
: or FITC, and the "green" channel should be used for TRITC or RFP.
: Most likely you guys put in two filters with similar cutting ranges,
: and arbitrarily label them as "blue" and "green" but it is not really "blue"
: and "green". So don't take these label too serious. If you reall
| s******y
发帖数: 28562 | 7 Well, people label whatever they like for those channel and there is no
universal way for it. The microscope industry generally lable the channel as
the excitation light because it is more appropriate, but the laboratory may
have their own mind and can easily change the label to whatever they like.
【在 b*****l 的大作中提到】
: r u sure?
: in my lab, blue is labeled as DAPI, green FITC, and red RHOD.
:
: blue"
: ，这
| p*****r
发帖数: 805 | 8 我想Sunnyday的意思是，mercury lamp的光通过excitation出来的光的颜色（肉眼看到
的，objectives照到样品上的颜色，不是eye piece里看到的颜色），如果是“蓝色“
，那么通常是GFP或者FITC的filter（eye piece里应该看到绿色的fluorescence)；如
果是“绿色“，那么往往有可能是TRITC或者RFP的filter(eye piece里应该看到红色的
fluorescence）。
【在 b*****l 的大作中提到】
: r u sure?
: in my lab, blue is labeled as DAPI, green FITC, and red RHOD.
:
: blue"
: ，这
| b*****l
发帖数: 9499 | 9 这个我当然知道。我是说，大家通常 label channel 时是按啥规范？
我从未见过按 excitation 来 label channel 的。。。俺的显微镜自带的 label 最短
也就是 DAPI 了。要是按 Sunnyday 的意思，岂不是还要附带一个 label 上面写明 UV？
查了一下几家公司，有显微镜公司，有卖 antibody 的公司，还有说明书，都是按
emission 来 label 的。
大家的实验室都是怎么 label 的？
【在 p*****r 的大作中提到】
: 我想Sunnyday的意思是，mercury lamp的光通过excitation出来的光的颜色（肉眼看到
: 的，objectives照到样品上的颜色，不是eye piece里看到的颜色），如果是“蓝色“
: ，那么通常是GFP或者FITC的filter（eye piece里应该看到绿色的fluorescence)；如
: 果是“绿色“，那么往往有可能是TRITC或者RFP的filter(eye piece里应该看到红色的
: fluorescence）。
| b*****l
发帖数: 9499 | 10 应该有约定俗成的业界标准的，否则交流起来岂不要乱套误事了。
as the color of the excitation light because it is more appropriate,
to whatever they like.
【在 s******y 的大作中提到】
: Well, people label whatever they like for those channel and there is no
: universal way for it. The microscope industry generally lable the channel as
: the excitation light because it is more appropriate, but the laboratory may
: have their own mind and can easily change the label to whatever they like.
| s******y
发帖数: 28562 | 11 In my understanding, the most professional way is to label it with the
exitation wavelength, simply because the excitation is strictly controlled
by the sysytem, but the emission is not. The emission wavelength is mainly
controled by the chromophore whichever you use.
However, for the convenience of most user, the labeling of the channel are
often changed to its target chromophores, for example, DAPI, FITC,and TRITC,
which is also appropriate.
But it is not exactly appropriate to label it as "em
【在 b*****l 的大作中提到】
: 这个我当然知道。我是说，大家通常 label channel 时是按啥规范？
: 我从未见过按 excitation 来 label channel 的。。。俺的显微镜自带的 label 最短
: 也就是 DAPI 了。要是按 Sunnyday 的意思，岂不是还要附带一个 label 上面写明 UV？
: 查了一下几家公司，有显微镜公司，有卖 antibody 的公司，还有说明书，都是按
: emission 来 label 的。
: 大家的实验室都是怎么 label 的？
| b*****l
发帖数: 9499 | 12 我估计你用的是激光。对于一般的灯，excitation wave lenth 也是一个分布，也就是
说，光源不是单色的。
AF488 被 label 成 FITC channel 啊。一个 channel 被 label 成 FITC，是说用来看
FITC 的，这个应该是业界标准。AF488 就是按照 FITC 的标准设计的。
同意你说的 label 成颜色是不对的。
贴个 cube 的图片，你可以看到 cube 上 label 成 FITC 的的确是用来看 FITC 的。
TRITC,
blue","
most
because
最短
明
【在 s******y 的大作中提到】
: In my understanding, the most professional way is to label it with the
: exitation wavelength, simply because the excitation is strictly controlled
: by the sysytem, but the emission is not. The emission wavelength is mainly
: controled by the chromophore whichever you use.
: However, for the convenience of most user, the labeling of the channel are
: often changed to its target chromophores, for example, DAPI, FITC,and TRITC,
: which is also appropriate.
: But it is not exactly appropriate to label it as "em
| s*****j
发帖数: 6435 | 13 excitation max of YFP is 514nm, which is not blue.
spectra
【在 s******y 的大作中提到】
: 1. the color of YFP: it is green-yellow, so if you don't have a control
: color (like the real green), it will looks green. Plus, part of its spectra
: will go through the green filter.
: 2. RFP is activated by green light, which is abundant in natural light.
: YFP is activated by blue light, which is NOT abundant in natural light.
: In order to see bright fluorescent of YFP you will need to use special
: activation light source.
: 3. Conservation of the fluorescent light: it is important to keep the
: structur
| s*****j
发帖数: 6435 | 14 as I know, Nikon, Olympus, Zeiss, Leica all use emission filter color to
label cube.
blue"
【在 s******y 的大作中提到】
: In most microscopes, the "color" label of the channel means the excitation
: light permitted by the filter cube (or other spectra dividing instrument)
: inside. Therefore, the "blue" channel should be used to visualize GFP, YFP
: or FITC, and the "green" channel should be used for TRITC or RFP.
: Most likely you guys put in two filters with similar cutting ranges,
: and arbitrarily label them as "blue" and "green" but it is not really "blue"
: and "green". So don't take these label too serious. If you reall
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