User:Shantanu Ghosh/Notebook/Two photon microscope project

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Two-photon microscopy

Two-photon excitation (2PE) microscopy is a form of far-field fluorescence microscopy technique with advantages over conventional confocal microscopy, because of deeper tissue penetration, efficient fluorescence detection and little photodamage. 2PE occurs when two photons of low energy can excite a fluorophore in a quantum event, resulting in the emission of a fluorescence photon, at a higher energy than either of the two excitatory photons. Since the probability of the near-simultaneous absorption of two photons is extremely low, a high energy beam is typically required, usually supplied by a modelocked femtosecond laser.

2PE microscopy was pioneered by Winfried Denk at the Webb Lab at Cornell University.

We are fabricating a two-photon microscope to investigate neuronal plasticity in the auditory cortex.


The purpose of the project is to build a simple and compact imaging system for our laboratory, and to keep the total cost of construction as low as possible.

The femtosecond laser for our lab's multiphoton imaging facility is built around a 5%-at. Brewster cut Ytterbium-doped crystal (Yb:KYW), as it is particularly suited for lasing at ~1030 nm, pumped by a 980 nm diode laser at 3.5 W (compared to the more conventional Ti:Sapphire laser, but expensive and difficult to maintain, which has dominated the scene since its invention in the early 1990s). Ytterbium has a high stimulated emission cross-section (3×10-20 cm2) and a very long fluorescence lifetime (0.6 ms) when used within a KYW crystal; this makes it a relatively forgiving material when designing a femtosecond laser. In addition, the wavelengths ~1030 nm are particularly interesting for the excitation of several popular voltage-sensitive dyes, especially Oregon Green 514 and Calcium Green-1.

 Check a very nice spectrum viewer by Molecular Probes where you can compare spectra of various fluorophores. 
  • Remember
 Anything with significant absorption around 516 nm (half of 1033 nm) will fluoresce to some extent
 through two-photon absorption, although the conversion from absolute one-photon to two-photon cross-sections
 is not very strictly determined. The two photon absorption should also work with YFP, citrine, mOrange, mCherry, 
 dsRed, etc., all of which can be excited with the one laser at the same time. However, the 1033 nm wavelength is 
 too long for CFP or the original wild-type GFP.

  • This is a work in progress, and the pages are under constant flux. Comments and suggestions are welcome. If you wish to apply as an intern, email me at

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