Multi-photon Microscopy

Location: Thomas Building, DE-512
Contact phone: (206) 667-4205
Contact e-mail: imaging@fredhutch.org

Two-photon microscopy uses non-linear fluorescence excitation with pulsed infra-red or near infra-red lasers, combined with sensitive non-descanned detectors to image fluorescence deeper inside biological specimens compared to conventional confocal microscopy.

In conventional fluorescence microscopy, short wavelength visible light is used to excite fluorescence of a fluorophore, which then emits light of a slightly longer wavelength. For example, blue light at 488 nm is used to excite fluorescein or green fluorescent protein, which then emit green light centered around 530 nm. In two photon microscopy, two longer wavelength photons in the near infra-red (NIR) are absorbed quasi-simultaneously, providing the same energy as a single shorter wavelength photon, while the emission wavelength remains the same. To achieve quasi-simultaneous absorption of two photons, extremely high illumination intensities are needed. This is achieved with special ultrafast NIR pulsed lasers. Since the illumination intensity required for the stimulation of fluorescence is reached only at the focal spot, other regions of the sample will not fluoresce, and therefore optical sectioning is achieved without need for a pinhole or deconvolution software. The increased imaging depth of two photon microscopy is due to the lower scatttering of the NIR excitation light, and also to the more efficient detection of scattered emitted light, compared to confocal microscopy. An added benefit of two photon imaging is better viability for certain types of specimens, due to lower toxicity of longer wavelength light.

Multi-photon microscopy uses the same principle, but in this case three (or more) photons are absorbed simultaneously. The imaging resource provides two multi-photon systems: The Zeiss LSM 780 NLO, based on an inverted Zeiss Observer Z1 stand, combines conventional confocal with multi-photon imaging. A dedicated Zeiss LSM 7 MP in vivo multi-photon system is also available in the animal facility for in vivo imaging of mouse model systems. Both systems use state of the art multi-photon lasers and high sensitivity non-descanned detectors.

Equipment for two-photon and multi-photon microscopy:

(For technical details, follow the links to individual microscopes)

Zeiss LSM 780 NLO. Laser scanning confocal and multi-photon microscope system. Recommended uses: Tissue sections, brain sections, biopsies, fly and worm embryos, adult worms, F-techniques (FRAP/FRET/FCS), deep imaging of thick specimens; live imaging, imaging of unconventional fluorescent dyes, separation of spectrally close dyes.

Zeiss LSM 7 MP in vivo multi-photon microscope. This dedicated multi-photon microscope is located in, and managed by the animal facility and is primarily for the in vivo imaging of small animals. For access, please contact Comparative Medicine. For training and assistance with the microscope, please contact Scientific Imaging.

Scheduling time for instruments, training, and support:

To schedule time on any of our microscopes, or to schedule staff assistance or training, please use iLab. The resource is open to all from 9:00 AM - 5:00 PM, Monday through Friday. After hours access can be granted to experienced users and requires training by the resource's staff. Please contact Scientific Imaging for further details.

How to access your data:

Data acquired on Scientific Imaging instruments is transferred to the si folder in the user's fred account. If you do not have a fred account, please submit the computing account application form. Once you have obtained a fred account, please contact Scientific Imaging so that we can create an si link for you. External users without access to fred can obtain their data via ftp, or can download to their own data storage device from one of our dedicated computers. Please note that no portable drives are allowed on instrument computers.