Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Nov;19(6):1103-9.
doi: 10.1007/s10895-009-0510-y. Epub 2009 Jul 10.

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

Affiliations
Free PMC article

Peak Multiphoton Excitation of mCherry Using an Optical Parametric Oscillator (OPO)

Tegy J Vadakkan et al. J Fluoresc. .
Free PMC article

Abstract

mCherry is a red fluorescent protein which is bright, photostable, and has a low molecular weight. It is an attractive choice for multiphoton fluorescence imaging; however, the multiphoton excitation spectrum of mCherry is not known. In this paper we report the two photon excitation spectrum of mCherry measured up to 1190 nm in the near infrared (NIR) region. Skin tissues of transgenic mice that express mCherry were used in the experiments. mCherry in the tissues was excited with a Titanium:Sapphire laser and an optical parametric oscillator pumped by the Titanium:Sapphire laser. We found that the peak excitation of mCherry occurs at 1160 nm.

Figures

Fig. 1
Fig. 1
Shown are representative images of skin tissue collected from Tg(Flk1::myr-mCherry) mice and excited with a Ti:Sapphire laser at various two-photon excitation wavelengths. An IR-improved prototype objective lens from Carl Zeiss (32×/0.85 NA W) was used as the objective
Fig. 2
Fig. 2
Normalized fluorescence intensity of mCherry is plotted against two-photon excitation wavelengths in the range from 1000 nm to 1080 nm
Fig. 3
Fig. 3
The output from a Titanium:Sapphire laser (Chameleon, Coherent Laser Group, Santa Clara, California) was reflected using a silver mirror to enter an OPO (Mira OPO, Coherent Laser Group, Santa Clara, California). The output of the OPO was reflected using a Protected Gold (PG1) mirror (Thorlabs). A polarized half wave plate (HWP) was used to adjust the power of the laser beam. The beam that exited the HWP was reflected by PG2 to enter a modified acousto-optic modulator (AOM). The AOM was modified to block the beam when the laser scanning microscope (LSM) is not scanning. The crystal in the AOM was removed so that the beam is just blocked by the shutter when the LSM is not scanning. The telescope, T was used to reduce the beam waist before it entered the LSM scan head after being reflected by PG3, PG4, and PG5. The mirrors PG3, PG4, and PG5 were adjusted such that the laser beam filled the back aperture of the objective lens and the power at the specimen was maximum. The beam was aligned using a 633 nm laser line as the reference
Fig. 4
Fig. 4
The power at the output of the OPO between PG1 and HWP was measured using a power meter (S212A, Thermopile Sensor, Thorlabs). The power was measured on fifteen different days and averaged over the number of days
Fig. 5
Fig. 5
The attenuated power at the specimen was measured using a power meter (S212A, Thermopile Sensor, Thorlabs). The power was measured on eight different days and averaged over the number of days. An IR-improved prototype objective lens from Carl Zeiss (32×/0.85 NA W) was used as the objective
Fig. 6
Fig. 6
The mean pulse width of the laser beam immediately after the telescope was measured using a Carpe autocorrelator. The average power at the specimen was 20 mW for the solid line and 27 mW for the dotted line. The change in the pulse width in the range from 1120 nm to 1150 nm and from 1150 nm to 1190 nm was less than 100 fs
Fig. 7
Fig. 7
Excitation fingerprinting of mCherry using an OPO pumped by a Titanium:Sapphire laser. The images were taken in two sets from 1120 nm to 1150 nm in steps of 10 nm, and from 1150 nm to 1190 nm in steps of 10 nm. An IR-improved prototype objective lens from Carl Zeiss (32×/0.85 NA W) was used as the objective. The mCherry signal was collected using an HQ620/60 m-2p filter (Chroma Technology Corp.)
Fig. 8
Fig. 8
Normalized excitation spectrum of mCherry in the range from 1120 nm to 1190 nm. mCherry in the tissue was excited with an OPO pumped by a Titanium:Sapphire laser

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

Publication types

Substances

LinkOut - more resources

Feedback