Multi-Photon Objective

Inventors: Dejan Vucinic, Thomas Bartol, Terrence Sejnowski
Potential Uses: Deep Tissue and Neuron Imaging
Hybrid reflecting micoscope objectives for deep-tissue functional two-photon imaging

Most microscope objectives commonly used for two-photon imaging of neurons are optimized for high image quality under widefield illumination. The constraints that guide the design of such objectives are not optimal for imaging using nonlinear excitation. To approach the physical limits on the measurement of small and fast fluorescence changes we designed an objective, compatible with existing microscopes, optimized specifically for the needs of deep-tissue functional two-photon imaging: very high epifluorescence collection efficiency, negligible dispersion of femtosecond pulses, low magnification and long working distance. Our design has separate imaging and nonimaging pathways for incident and emitted light, making use of the optical sectioning intrinsic in nonlinear fluorescence excitation, which relaxes a number of design constraints. We showed through modeling that a twofold to fourfold improvement in fluorescence collection efficiency over traditional objective designs is easily achievable while maintaining nearly diffraction-limited imaging performance within a 200-micron field of view and a 1mm working distance.

Wider commercial availability of such hybrid objectives would enable longer physiological recordings with less tissue damage, as well as the observation of faster and smaller fluorescence changes.