Two-Photon Microscopy

Talk Overview

This talk introduces two-photon microscopy which uses intense pulsed lasers to image deep into biological samples. It can be used for imaging thick tissue specimens or even imaging inside of live animals.

Questions

Explain why only red light is emitted through thin skin when a flashlight is shone on it.
What is clearing and what is it used for?
What are the advantages of using two infrared photons instead of one blue photon in two-photon microscopy to excite a green-light emitting molecule?
Why does two-photon microscopy not require a pinhole (select all that apply)?
1. Because the emitted light in two-photon microscopy does not scatter
2. Because there is no out-focus light in two-photon microscopy
3. Because two-photon microscopy gives rise to localized excitation
4. None of the above
What would be the result of using a pinhole in two-photon microscope?
1. The detected image would include less background signal
2. The detected image would be dimmer
3. None of the above
Do you agree or disagree with this statement? “In two-photon microscopy, one unique laser setting can be used to simultaneously excite both the Alexa Fluor 488 (green) and Alexa Fluor 568 (orange) dyes.” Explain why you agree or disagree.

Answers

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Because biological materials absorb light in most wavelengths, except in the red color. Therefore, more red light is let through the skin than the other wavelengths
Clearing removes scattering from biological samples. The solution uses vary and include improving “deep” imaging in thick fixed samples. The clearing solutions are either organic solvents, detergents, or urea.
Using two photons instead of one will increase penetration depth; Because biological molecules do not usually absorb light in the red spectrum, using red photons to excite fluorescent markers can decrease the image background; In addition, two red photons used simultaneously can excite a fluorescent marker that is usually excited in the blue wavelength (in one-photon microscopy). This increases the range of fluorescent markers that can be used in thick sections; Last, because two-photon microscopy involves localized excitation, it does not involve pinholes.
B and C
1. Incorrect: The emitted light in two-photon microscopy does scatter, but it does not matter. Because only one point emits light (the focus of the light beam), the total emission is measured.
2. Correct: Because there is no out-focus light in two-photon microscopy, the pinhole is not required in two-photon microscopy.
3. Correct: Two-photon microscopy gives rise to localized excitation, which means that only the focus point receives sufficient light (from two photons) to be excited and emit light. Therefore, the excitation photons cannot produce sufficient light to excite markers, which means that there is no background light to filter through a pinhole.
B
1. Incorrect: The detected image would include less background signal. There is no background signal in two-photon microscopy
2. Correct: The detected image would be dimmer. Because the total emitted light is measured in two-photon microscopy, and because the pinhole would filter out a significant portion of that emitted light, light would be lost if the pinhole were to be kept on.)
Agree. Because two-photon microscopy uses two simultaneous photons of shorter wavelengths to excite dyes, the excitation spectrum of different dyes may overlap.

Speaker Bio

Kurt Thorn

Kurt Thorn is an Assistant Professor of Biochemistry and Biophysics at UCSF and Director of the Nikon Imaging Center – a facility that provides cutting edge light microscopy equipment to UCSF researchers. Kurt can be followed on his blog at http://nic.ucsf.edu/blog/. Continue Reading