Multiphoton Microscopy Application Guide
Basics of Multiphoton Microscopy
Pulsed laser light sources are required for a group of applications known collectively as multiphoton microscopy. Given the extremely brief duration of each laser pulse that these sources are capable of generating (often in the picosecond to femtosecond range), it is possible to deliver multiple photons of light to a target very close to simultaneously in time and space. These photons can generate fluorescence from a fluorochrome in much the same way that “1photon” microscopy can (in laser-scanning confocal imaging, for example), but with the additional benefits of using a lower total amount of power to do so, and with the ability to penetrate tissue samples more deeply.
Tunable pulsed lasers are typically used to generate a multiphoton event and these lasers often range from 700-1100, -1350 or even -1500 nm. By directing extremely brief pulses into the sample, these lasers can effectively excite fluorochromes by striking the fluorescent molecules virtually simultaneously with multiple photons. In 2photon microscopy, a theoretical fluorochrome with an absorption/excitation peak at 470 nm can be excited by a 2p laser tuned to 940 nm or a 3p laser tuned to 1410 nm, as an example. The fluorescence generated by that excited fluorochrome would peak at the typical “1p” emission peak published for that fluorochrome and can be measured or imaged as such.
Filter Requirements for Multiphoton Microscopy
Excitation Filters
Because the lasers being used for these applications are tunable through a wide range (i.e. 700-1100 nm), excitation/clean-up filters are generally not used in the excitation/illumination path of the microscope.
Historically, there have been instances where a multiphoton laser appears to emit light in the “visible” range (i.e. 680 nm), as well as its more-typical “far-red/near-IR” range, and in those cases a longpass filter which can block “visible” and transmit “far-red/near-IR” has been used.
Dichroic Mirrors
As in laser-scanning confocal microscopy, the dichroic mirror used in multiphoton microscopy applications must adhere to fairly tight flatness characteristics to ensure coherence of the beam being reflected off of it. We generally recommend 0.5 waves/inch (0.5λ), though some applications and systems appear to require even “flatter” dichroics.
Since this application requires excitation wavelengths in the “far-red/near-IR” region, shortpass dichroics are most often used. These will reflect the “far-red/near-IR” light for effective multiphoton excitation, while transmitting the “visible” wavelength range corresponding to the emission properties of most fluorochromes.
Emission Filters
Barrier/emission filters for multiphoton are similar in their blocking requirements to those in confocal or TIRF (OD6 minimum, OD8 or more preferred), though given the nature of the pulsed lasers used in these applications, the highest order of blocking should occur within the “far-red/near-IR” spectral region corresponding to the laser being used.
Barrier/emission filters can be either bandpass or shortpass in nature, and often both are used in series to increase/improve blocking of the laser within the detection/imaging pathway. For instance, an ET750SP-2p8 shortpass barrier/emission filter can be used in the common emission/detection pathway before the fluorescence emerges from the microscope itself. Then, an additional bandpass barrier/emission filter (i.e. ET525/50m-2p) may be used to both limit the fluorescence being detected to a narrow band AND improve upon the blocking of the laser at the detector.
Additional Resources
Still Have Questions?
Our applications team works with multiphoton setups across most major commercial platforms. Tell us your instrument and fluorochromes and we'll point you in the right direction.
