Here is my train of thought please correct me if I missed or misintepreted any of the information: So epifluroscence uses excitation ligth to excite a sample however it is inefficent (specificlaly for deep tissue sample) because it doesnt focus on one focal point and out of focus ligth blurs otu the sample not allwoign for analysis for a specific point in the tissue. Confocal microscopy is much mroe better due to the pinhole which blocks out out-of-focus light to better analyze a tissue sample however the problem comes in the scattering of the ligth due to deep tissue penetration and since the confocal micrsocopy works with shorter wavelegnths, and as mentioned in the video previously less scattering and absorption occurs at infrared (longer wavelengths). Therefore, two photon microscopy is most opimal for focusing on a specific focal point in a deep tissue sample due to the use of higehr wavelengths leading to less scattering and focsu on one focal point without out of focus light coming in and overall excitation light is longer than confocal and epifluorescence microscopy techniques.
excellent video..i have a question please,i want to do a fluorescent exerience with confical and 1 EMCCD camera ..what is the difference between using 2 camera or 2 ...thank you
Thanks for the excellent explanation. I have a doubt : two photons are two different quanta, even if they arrive simultaneously, would they not excite two different dye molecules with the same energy and not the same molecule with twice the energy ?
From my understanding, that's the trick of 2 photon, at the focal plane, the density of the photons is so high that 2 photons will hit the same molecule. Outside of the focal plane, the likelihood of 2 photons hitting the same molecule goes down, therefore photo-bleaching is minimized outside of focal plane
Agreed: 1) Excellent explanation. 2) I had(?) the same doubt. It doesn't square with my understanding of how excited states happen ... the reaction of a molecule to being hit with half the energy needed to excite it would be: "hold my beer." It appears my understanding is incomplete because they are making it work.
22:40-22:45 this is not truly the reason, in my opinion. A more appropriate explanation would be that the two-photon cross-section is extremely low and thus high intensities are a necessity for inducing two-photon absorption. Two photons will always be present, but the absorption will not be stimulated unless there is a high density of photons, owing to the low cross-section. In fact, this cross-section will become orders of magnitude weaker for even higher multi-photon events.
Hellen Ishikawa Dear Dr. Ishikawa-Ankerhold, unfortunately we are not able to give out the powerpoint presentations. If there are particular slides you are interested in let us know and we'll see what we can do!
Hi Five-Sigma! You are right - the link doesn't seem to work anymore. I contacted the speaker and he suggested these two similar (but not the same) graphs: www.nature.com/icb/journal/v88/n4/fig_tab/icb2009116f1.html research.stowers-institute.org/microscopy/external/Technology/NLO/index.htm Sorry that we cannot find the exact one used in the talk!
So the goal is to measure photon from extraction rather than defining photon by compression however a photon compression engine will suffice most excellently for any security system weather it be for a spacecraft or for a human body.
Wonderful presentation, except the occasional tongue-clucking "sucking" sound (for instance, see 25:06, 26:19, 26:38, 26:50). This sound can be annoying for many audience. Thank you and I hope you take this suggestion positively.
10:10 was the moment the advantage of two photon microscopy clicked for me. 16:37 is another great point.
mvp
Much better than my university's newbie lecturer(surprisingly hired at US top 30 university) Wish he watch this and learn how to teach.
Here is my train of thought please correct me if I missed or misintepreted any of the information: So epifluroscence uses excitation ligth to excite a sample however it is inefficent (specificlaly for deep tissue sample) because it doesnt focus on one focal point and out of focus ligth blurs otu the sample not allwoign for analysis for a specific point in the tissue. Confocal microscopy is much mroe better due to the pinhole which blocks out out-of-focus light to better analyze a tissue sample however the problem comes in the scattering of the ligth due to deep tissue penetration and since the confocal micrsocopy works with shorter wavelegnths, and as mentioned in the video previously less scattering and absorption occurs at infrared (longer wavelengths). Therefore, two photon microscopy is most opimal for focusing on a specific focal point in a deep tissue sample due to the use of higehr wavelengths leading to less scattering and focsu on one focal point without out of focus light coming in and overall excitation light is longer than confocal and epifluorescence microscopy techniques.
Very complicated but well explained technique. Good job!
Excellent and easy-to-follow!
EXCELLET explanation!! no doubts left behind, very well organized and fulfilled with information! great job!
Thank you so much for this video!! I was 100% lost on how the multiphoton system worked.Very clear explanation.
Using the talk for preparing for exam. Really helpful! Thank you
Excellent explanations--didn't realize how much I wanted to use multi-photon microscopy until seeing this video!
Great presentation, very clear, thank you
Very well explained and helpful. Thank you!
SO clear and helpful- thank you!
excellent lecture! very clear and easy-to-follow
Thanks for this, the basic principles are clear for me now!
Still a great vid 10 years later
Extraordinarily explained 👍🏾
Great lecture. Thanks
Very good information.. Easy to understand.. Thanks
Great lecture!
Very thorough, thank you!
Easily comprehensible, thanks a lot :)
excellent video..i have a question please,i want to do a fluorescent exerience with confical and 1 EMCCD camera ..what is the difference between using 2 camera or 2 ...thank you
Thanks for a great talk!
10/10 In every category
Amazing stuff thank you.
Awesome, thank you!
wow - It's so nice to se another way for using second harmonic, that just PTK. dubling.
Super helpful and logical! thanks !
Thanks :D ... Good explanation.
Thanks for the excellent explanation. I have a doubt : two photons are two different quanta, even if they arrive simultaneously, would they not excite two different dye molecules with the same energy and not the same molecule with twice the energy ?
From my understanding, that's the trick of 2 photon, at the focal plane, the density of the photons is so high that 2 photons will hit the same molecule. Outside of the focal plane, the likelihood of 2 photons hitting the same molecule goes down, therefore photo-bleaching is minimized outside of focal plane
There is no real upper level (excited-state) corresponding to a single photon’s energy and therefore there will be no absorption.
Agreed: 1) Excellent explanation. 2) I had(?) the same doubt. It doesn't square with my understanding of how excited states happen ... the reaction of a molecule to being hit with half the energy needed to excite it would be: "hold my beer." It appears my understanding is incomplete because they are making it work.
So this is ionizing radiation at wavelengths far longer than those for x-rays?
22:40-22:45 this is not truly the reason, in my opinion. A more appropriate explanation would be that the two-photon cross-section is extremely low and thus high intensities are a necessity for inducing two-photon absorption. Two photons will always be present, but the absorption will not be stimulated unless there is a high density of photons, owing to the low cross-section. In fact, this cross-section will become orders of magnitude weaker for even higher multi-photon events.
thank you
Very good presentation
Brilliant! Thank you!
HI it will be possible to have the ppt presentation for didactic propose? It will be nice!
Thanks
Dr. Hellen Ishikawa-Ankerhold LMU Munchen
Hellen Ishikawa Dear Dr. Ishikawa-Ankerhold, unfortunately we are not able to give out the powerpoint presentations. If there are particular slides you are interested in let us know and we'll see what we can do!
Excellent
Thank you!
any idea where i can get that graph at 6:25? the link to it doesn't work
Hi Five-Sigma! You are right - the link doesn't seem to work anymore. I contacted the speaker and he suggested these two similar (but not the same) graphs:
www.nature.com/icb/journal/v88/n4/fig_tab/icb2009116f1.html
research.stowers-institute.org/microscopy/external/Technology/NLO/index.htm
Sorry that we cannot find the exact one used in the talk!
Thanks very much! Those graphs are great!
@@iBiologyTechniques the links seem broken now. Thanks a lot for the great lecture!
THANK YOU!!!
Who dislikes such videos
Are you from the future!?
Next time just present the source to your data and include user interactivity
good explanation, he speaks like sheldon cooper :)
So the goal is to measure photon from extraction rather than defining photon by compression however a photon compression engine will suffice most excellently for any security system weather it be for a spacecraft or for a human body.
Wonderful presentation, except the occasional tongue-clucking "sucking" sound (for instance, see 25:06, 26:19, 26:38, 26:50). This sound can be annoying for many audience. Thank you and I hope you take this suggestion positively.
This is violating Einstein's statement for photoelectric efect
why is he so annoyingly excited by this
lmaoooo
Why are you annoyed? It is a well-delivered and interesting presentation.