Flexible nonlinear laser scanning microscope for noninvasive three-dimensional detection

A flexible nonlinear laser scanning microscope for noninvasive three-dimensional detection comprising:

• at least one radiation source which generates an excitation beam of pulsed laser radiation for initiating a secondary radiation;
• a measuring head having at least one detector system and focusing optics for focusing the excitation beam in a measurement object, collecting the secondary radiation emitted from the object, and directing the secondary radiation to the at least one detector system;
• flexible transmission optics for transmitting the excitation beam from the at least one radiation source to the measuring head and enabling the measuring head to be freely pivotable and rotatable in space and flexibly positionable for carrying out upright microscopy, inverted microscopy, and microscopy at any solid angle;
• at least one controllable tilt mirror for deflecting and aligning at least one excitation beam of the at least one radiation source, the controllable tilt mirror serving to align the at least one excitation beam in the measuring head through the transmission optics in order to keep the excitation beam arranged concentrically to an aperture-limited optical element of the focusing optics of the measuring head in any position of the measuring head;
• a beamsplitter for coupling a test beam split from the excitation beam, the beamsplitter being disposed in the measuring head in front of the focusing optics;
• a spatially resolving photodetector for determining a beam position of the excitation beam, the photodetector being disposed in a position of the test beam conjugate to a target position of the excitation beam in relation to the aperture-limited optical element of the measuring head, wherein a center alignment of the test beam on the photodetector corresponds to a normal orientation of the excitation beam in the target position;
• and
• a control unit for driving the at least one controllable tilt minor depending on a determined deviation of the test beam from its center alignment on the photodetector to achieve a stabilization of the excitation beam in the target position irrespective of a magnitude of a position-dependent impairment of the transmission optics,
• wherein the at least one detector system comprises TCSPC detectors for MPT imaging, and a trigger diode using a back reflection of another beamsplitter for coupling out measurement signals.