At 06:40, you mentioned that for the 405nm, the BP450/50 filter is suitable but the range of this filter becomes 425 to 475 based on your example of BP500/30. Bur 405nm is not in the range; 425 to 475.
Thanks for the comment- you are correct. The range of this filter would be 425-475nm. In this example, you have both the excitation and emission spectra on the graph. The dotted line is the excitation and the solid is the emission. The 405nm laser cuts through near the peak of the excitation, which makes it the appropriate laser to use for this. The emission ranges from 400-500nm, with the peak around 420nm. So, yes, while the peak is outside of the filter range, the filter does capture a good portion of the emission spectra and thus can be used to detect the fluorescence from this fluorochrome. For emission, you do not need to capture to peak to detect fluorescence.
Image at 05:01 : Is it a standard peak range correspondingly to the markers (e.g., FITC will always peak at around 530/30) or is it customized according to the experience? [No medical background]. Also, if it is standard, does this chart have a name? Please share link if possible.
Yes that’s correct! This is a excitation/emission plot for a fluorochrome. Most major flow antibody companies will have a spectra viewer. Here is an example: www.thermofisher.com/order/fluorescence-spectraviewer#!/
It depends on the setup of the instrument. In an instrument with spatially separated lasers, yes the cells do pass through one laser at a time sequentially.
I love your explanation! Thank you so much for creating this video!
Really good, clear explanation, thank you so much for your work!
Thanks :)
At 06:40, you mentioned that for the 405nm, the BP450/50 filter is suitable but the range of this filter becomes 425 to 475 based on your example of BP500/30. Bur 405nm is not in the range; 425 to 475.
Thanks for the comment- you are correct. The range of this filter would be 425-475nm. In this example, you have both the excitation and emission spectra on the graph. The dotted line is the excitation and the solid is the emission. The 405nm laser cuts through near the peak of the excitation, which makes it the appropriate laser to use for this. The emission ranges from 400-500nm, with the peak around 420nm. So, yes, while the peak is outside of the filter range, the filter does capture a good portion of the emission spectra and thus can be used to detect the fluorescence from this fluorochrome. For emission, you do not need to capture to peak to detect fluorescence.
@@ajarieger_flow Okay got it. Thank you so much...
Very good videos!
Thanks!!
Image at 05:01 : Is it a standard peak range correspondingly to the markers (e.g., FITC will always peak at around 530/30) or is it customized according to the experience? [No medical background]. Also, if it is standard, does this chart have a name? Please share link if possible.
Yes that’s correct! This is a excitation/emission plot for a fluorochrome. Most major flow antibody companies will have a spectra viewer. Here is an example: www.thermofisher.com/order/fluorescence-spectraviewer#!/
In a multi-laser setup does a cell pass through each laser sequentially in a way that only one laser is exciting a particular cell at a time?
It depends on the setup of the instrument. In an instrument with spatially separated lasers, yes the cells do pass through one laser at a time sequentially.
This was great
Thanks!