Multiphoton Imaging With and Without a Dispersion-Compensating Mirror Pair
Multiphoton microscopy is recognized as the premier method for obtaining high-resolution, three-dimensional images from within thick biological samples. Compared to confocal microscopy, multiphoton imaging features more efficient fluorescence detection and reduced photodamage. Furthermore, the use of near-infrared excitation allows for improved imaging depth due to a reduction in scattering losses at the surface.
Since multiphoton microscopy requires simultaneous absorption of two or more photons by the fluorophore, mode-locked pulsed lasers with high repetition rates and ultrashort pulses are employed. These lasers provide the high peak powers necessary to generate sufficient multiphoton absorption in the focal plane while preventing damage to the sample by keeping the average power absorbed low.
However, the standard optical components found in most imaging systems tend to broaden ultrashort laser pulses to the point where the quality of the image will be affected. This pulse broadening occurs because of the wavelength dependence of the refractive index of optical components through which the light travels; shorter wavelengths have higher indices of refraction in glass than longer wavelengths, causing shorter wavelengths to travel slower.
The dispersion of the laser pulse is corrected using dielectric mirrors with specialized coatings that are specifically designed so that longer wavelengths experience a larger group delay than shorter wavelengths, thereby negating pulse broadening caused by other optical elements within the imaging system. The two-photon images of a mouse kidney shown below demonstrate the benefits of using the Dispersion-Compensating Mirror Set for increasing image quality.
|Figure 1. Pseudocolored multiphoton images of a mouse kidney obtained without (frame a) and with (frame b) the use of a Dispersion-Compensating Mirror Set. The glomeruli and convoluted tubules are stained with Alexa Fluor 488 (green) while the nuclei are stained with DAPI (blue). By introducing the mirror pair into the setup, the signal to noise was increased by a factor of approximately 38, thereby providing a higher quality image of the mouse kidney.|
The figure to the right shows an images of a mouse kidney specimen that were taken prior to and after inserting the Dispersion-Compensating Mirror Set into the setup. In the specimen (Molecular Probes®, Invitrogen Corp.), the glomeruli and convoluted tubules were labeled with Alexa Fluor 488 (green), while the cell nuclei were labeled with DAPI (blue).
Simultaneous excitation of both fluorophores was accomplished using a Ti:Sapphire oscillator that produces ultrashort (<6 fs) pulses with a 1 GHz repetition rate. Two-photon fluorescence images were obtained with Thorlabs' in-house multiphoton microscope equipped with a 40X objective (Olympus, NA = 0.50).
As shown in the figure, the introduction of a dispersion-compensating mirror set into the experimental setup prior to the imaging optics dramatically improves the quality of the image. Figure 1a shows an image of a mouse kidney specimen that was taken without the use of the mirror set. The group delay dispersion (GDD) attributed to the optical elements in the multiphoton microscope is ~4200 fs2 at 800 nm. Figure 1b shows the same image acquired by using the dispersion-compensating mirror set to negate the GDD of the imaging optics. An intensity analysis of Fig. 1 indicates that the signal-to-noise ratio increased by a factor of approximately 38 (16 dB).