Laser scanning fluorescence microscopy with compensation for spatial dispersion of fast laser pulses

A laser scanning system for use with an optical microscope comprising:

• (a) a laser light source emitting a pulsed light beam;
• (b) a fluorescent light detector;
• (c) optical elements forming an optical path from the light source to a position from which the beam may be received by an objective lens of a microscope and focussed on a specimen and such that light reflected or emitted from the specimen is received by the objective lens and returned on the optical path, the optical elements including an acousto-optical deflector and a second deflector arranged to scan the laser light beam in an X and Y direction onto the specimen, a light separation element arranged in the optical path to direct the beam from the laser light source on the optical path to the objective lens and specimen and to direct fluorescent light emitted from the specimen at a different wavelength than the wavelength of the laser light source on a path to the fluorescent light detector, the acousto-optical deflector having an input face which receives the light beam from the laser light source and an exit face from which the light beam exits the acousto-optical deflector, a dispersive prism mounted to receive the beam exiting from the exit face of the acoustic-optical deflector and to converge spectral components of the beam exiting from the acousto-optical deflector caused by spatial dispersion of wavelengths in the light beam, and a mirror positioned to deflect the beam from the laser light source to the input face of the acousto-optical deflector and wherein the mirror and the acousto-optical deflector are mounted for adjustment relative to one another to allow the angle of incidence of the light beam on the input face of the acousto-optical deflector to be adjusted, thereby to allow the beam incident on the input face of the acousto-optical deflector to be at an angle to match the Bragg condition for maximum diffraction efficiency for the center wavelength of the light beam.

A laser scanning multi-photon microscope system comprising:

• (a) a laser light source emitting a pulsed light beam,
• (b) a microscope objective lens;
• (c) a reflected light detector;
• (d) a fluorescent light detector;
• (e) optical elements forming an optical path from the light source to a position from which the beam may be received by the objective lens of the microscope and focussed on a specimen and such that light reflected or emitted from the specimen is received by the objective lens and returned on the optical path, the optical elements including an acousto-optical deflector and a second deflector arranged to scan the laser light beam in an X and Y direction onto the specimen, and a light separation element arranged in the optical path to direct the beam from the laser light source on the optical path to the objective lens and specimen and to direct fluorescent light emitted from the specimen at a different wavelength than the wavelength of the laser light source on a path to the fluorescent light detector, the acousto-optical deflector having an input face which receives the light beam from the laser light source and an exit face from which the light beam exits the acousto-optical deflector, a dispersive prism mounted to receive the beam exiting from the exit face of the acoustic-optical deflector and to converge spectral components of the beam exiting from the acousto-optical deflector caused by spatial dispersion of wavelengths in the light beam, a mirror positioned to deflect the beam from the laser light source to the input face of the acousto-optical deflector and wherein the mirror and the acousto-optical deflector are mounted for adjustment relative to one another to allow the angle of incidence of the light beam on the input face of the acousto-optical deflector to be adjusted thereby to allow the beam incident on the input face of the acousto-optical deflector to be at an angle to match the Bragg condition for maximum diffraction efficiency, and a polarizing beam Splitter receiving the light from the source and transmitting polarized light of a first orientation on the optical path to the specimen, and wherein reflected light returned from the specimen is orthogonally polarized with respect to the first orientation and is reflected by the polarizing beam splitter to the reflected light detector.

A method of multi-photon fluorescence microscopy comprising:

• (a) passing a pulsed light beam from a laser source through an acousto-optical deflector to deflect the beam in a pattern over an objective lens of a microscope to focus the light on a specimen and excite multi-photon fluorescence emissions from a fluorophore in the specimen;
• (b) collecting fluorescent light emitted from the specimen and directing such fluorescent light to a fluorescent light detector;
• and
• (c) passing the pulsed light beam exiting from the acousto-optical deflector through a dispersive prism to converge spectral components of the beam exiting from the acousto-optical deflector caused by spatial dispersion of wavelengths in the light beam introduced by the pulse modulation of the light beam.

A method of multi-photon fluorescence microscopy comprising:

• (a) passing a pulsed light beam from a laser source through an acousto-optical deflector to deflect the beam in a pattern over an objective lens of a microscope to focus the light on a specimen and excite multi-photon fluorescence emissions from a fluorophore in the specimen;
• (b) collecting fluorescent light emitted from the specimen and directing such fluorescent light to a fluorescent light detector;
• and
• (c) reflecting the light beam with a mirror positioned adjacent to an input face of the acousto-optical deflector, and adjusting the relative position of the mirror and the input face of the acousto-optical deflector such that the beam is incident on the input face of the acousto-optical deflector at an angle which matches the Bragg condition for maximum diffractive efficiency at the center wavelength of the light beam from the laser source and such that the acousto-optical deflector attenuates both sides of the spectrum of the light beam pulses which are mismatched to the Bragg condition.