Widefield and Confocal Fluorescence Microscopy

We just learned about electron microscopy, so what was the next major innovation in microscopy in the 20th century? That would be fluorescence microscopy, of both the widefield and confocal varieties. How does this work? What is fluorescence in the first place? What are fluorophores? What can we do with this technique? Let's get a closer look now.
Script by Kia Mackey
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[@monkeybusiness673]

I watched this kind of content as a student. Then I watched it as a PhD student, when I worked with this technique. Now, at a Post-Doc level, coming back to the setup...A screw it, let's watch that; I might actually learn something! EDIT: Holy crap...ACTUALLY learned something! Well done, Professor! Thanks a lot!

[@hopegate9620]

This was really clear and easy to understand, and somehow much better than the one and a half hour lecture I got on fluorescence confocal microscopy yesterday. Thank you. I'd also really like a video on atomic force microscopy as well as on other more recent types of microscopy like the 3D LIMON microscopy you showed here.

[@DrBovdin]

I did single molecule fluorescent imaging for my doctorate. Great to see the field promoted 😃👍🏻

[@ameliac504]

oooh

[@ninjuhdelic]

wow, never woulda thought a random question on my mind was answered by such a boss. so much succulent knowledge here. lit

[@vladimirkirichenko1972]

As always, thanks for saving the day Dave.

[@elinemarieholm4004]

Watching this for my exam preparation. Made me understand immunoflourescence and phenotyping in flow cytometry much better - THANK YOU.

[@masjos7103]

This was quite helpful, thanks!

[@radhikap2437]

This is so helpful. You are awesome!! Thanks a ton!

[@kinzspices3416]

Brilliant work Sir🥰🥰🥰🥰

[@valeriewarkins3704]

I've been using confocal in the lab as an undergraduate for 3 years and I feel like I finally understand, thank you!!

[@davyc412]

Nothing like taking a thick stack only to find a massive black square on your embryo when you're ready to shoot your overview. Moral of the story, take your overviews first.

[@sumwun2445]

I'm watching this cuz it's entertaining and helpful!

[@victor3431]

Very helpful!

[@Microscopy1]

Very good!

[@backstreetfan2887]

very nice, thank you

[@thatsalotofsodiumcoins1615]

I like your funny words magic man

[@alphabeta3528]

This is very cool

[@Hanny4God]

I love this

[@arfianwisnu1253]

Hi.. sorry, what do you mean with relaxation causes the emission of a photon? one may be confused with this term. relaxation in fluorescence is often understood as vibrational relaxation or non-radiative relaxation. I would say the electron returns to its ground state instead of relaxation to describe fluorescence process, even though one person may choose this term to explain fluorescence.

[@ProfessorDaveExplains]

Hmm, I always thought the term applied to any electron moving to a lower energy shell. Does it not? Fluorescence is just a subset of this phenomenon.

[@arfianwisnu1253]

@@ProfessorDaveExplains Actually you are correct. Relaxation is only electron movements, from higher energy state to lower ones; either it is non-radiative or radiative. But term of relaxation is mostly understood as vibrational relaxation, which is non-radiative. Some researchers may call the radiative relaxation as transition, although both are the same. For Master student or higher, your term is OK (hopefully they can distinguish), for Bachelor this may distract if you do not explain completely.

[@MrKhan-dc9gu]

Wow nice sir love from Pakistan

[@ujala6542]

Greeting to prof. Dave As you had explained the working /functiong of flouroscene microscopy In which I have a doubt that if there is no energy transition for a perticular part (may be due to the provided energy is less than the required energy OR MAY BE SOME OTHER REASONS) it will not be seen and the structure will be wrong So how one can know whether the final structure , which is seen by help of it, is correct or not ?

[@ujala6542]

Or may be the case where the wavelength, Which emits , are not belong to visible light

[@prince5063]

Hey Dave. I’ve been wondering if you have any videos about human psychology or explaining how emotions work. Please tell me if you do or are planning to make any. Thx for the channel.

[@prince5063]

Nvm, just found one lol

[@ProfessorDaveExplains]

Yep just that one for now but I’m planning a proper psychology series!

[@prince5063]

@@ProfessorDaveExplains Thank you, hoping to be a therapist in the future.

[@shahabbaloch8338]

Great work professor. these videos help us understand the reality and rejects religious myths

[@NathanW5555]

professor dave, how can they tell there's a coronavirus if it's impossible to tell if rna in a cell culture is of cellular or viral origin? what true scientific control have they to tell the difference? thanks for reading

[@ProfessorDaveExplains]

It absolutely is not impossible. We can sequence genomes. We know the organismal identity of any genetic sequence we are working with. This is trivial.

[@NathanW5555]

@@ProfessorDaveExplains thanks for taking the time to respond to my question, i do appreciate it, i thought the question of identifying viruses might relate to the above topic, similar to the use of luciferase to assay transcriptional activity of regulated activation sequences of DNA - pubmed.ncbi.nlm.nih.gov/2698191/ (as in the use of the property of fluorescence to give us information on things). i could ask you about 20 questions and i wouldn't want a focus on a trivial topic but i'll ask is there a method to cause bioluminescence of viruses to identify viruses or can fluorescence microscopy identify viruses? what's the control to distinguish which of the rna is cellular or viral? again, thanks very much for your response

[@ProfessorDaveExplains]

No problem, happy to help, though of course my utility is limited to very general questions such as your first one. I don't know much about the specifics of laboratory techniques as I'm not a microbiologist. Someone else wrote these microscopy scripts.

[@NathanW5555]

@@ProfessorDaveExplains Gentleman, thanks very much for your time and patience.

[@marquisecuffe4061]

@@ProfessorDaveExplains professor dave, you should make a video on how race realism conspiracy theories are pseudoscientific. thanks for reading.

[@alexandrevaliquette3883]

4:43 Why we need a dichroic mirror? Won't it be as efficient (less distortion and cheaper) to just aim the filtered light directly to the sample, in 45 degree angle for example. Then, the fluorescent light is emitted and filtered. Let me know if I won a Nobel Prize for that. I'll stack it with the other I have in the garage.

[@philosophyofreality1739]

Let's hope that we can use proton and neutron energy instead of electron for microscopy

[@mattparker9726]

Hey Prof. Dave, could you do a video on why there are firefly chemicals in the new vaccine for covid 19?

[@rickkwitkoski1976]

@Matt Parker HEY MATT!!! You are a complete and utter ignorant FOOL!! Are you talking about luciferase and luciferin? Yeah. CHEM ICK ALS!!! EVERY THING is a chemical bud!!! You are BREATHING chemicals right now! And you drink a toxic chemical every single day! Ever hear of dihydrogen monoxide?? What about the deadly chemical Pentahydroxyhexanal??? That is inFUSED in you right now! Luciferase is an enzyme. Enzymes are PROTEINS!!! And luciferin is a compound that has fluorescent properties. These are used in the DEVELOPMENT of many biologically active substances, including VACCINES! An assay method was developed using the reaction of this enzyme and its substrate to help determine the efficacy of biomolecules. They are not PART of the COVID-19 vaccines. JUST LIKE human tissue is NOT in the vaccines. Cell lines derived from aborted fetuses from 50 years ago (amazing that these cell lines are still with us), originally developed to help in CANCER research, are also used to develop various vaccines including the mRNA vaccines of Moderna and Pfizer Biontech. The cell lines are use to proof the efficacy of immune responses in human cells. Pull your dumbass out the conspiracy crap and actually learn some REAL SCIENCE!!! Dave didn't answer you because your piddly ass is not worth his time.

[@x00g40]

@@rickkwitkoski1976 hey Rick, take a break buddy

[@mariocesarsousa]

I believe i Will study it in biophysics.

[@DrBovdin]

You will be hard pressed avoiding it 😉

[@mariocesarsousa]

@@DrBovdin i really cant.....it is part of the curriculum. Unless i had chosen the teaching curriculum. Bacharelado 👈

[@DrBovdin]

@@mariocesarsousa that’s what I meant. You can’t avoid an integral part of one of the most powerful and most commonly used analysis tools we have in the field.

[@monkeybusiness673]

Look forward to it. It's one of the coolest things I ever encountered in biophysical chemistry!

[@sharkawy.]

because i tried to send you a present but i couldn't bec. i need your mail

[@angelaoso]

Do you have an instagram account?

[@ProfessorDaveExplains]

yep @daveexplains

[@gappythegoat5397]

e

[@sharkawy.]

can i have your mail