Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

Abstract

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task.

Fig. 1

(Color online) (a), (b) Hybrid objectives for multiphoton imaging. (a) All-reflecting design (HR) consisting of image-forming mirrors M1 and M2 arranged in a Cassegrain configuration, and a nonimaging mirror M3 that increases the efficiency of epifluorescence collection. Dotted red and solid green rays illustrate the paths of excitation pulses and fluorescence photons, respectively. (b) Slightly higher collection efficiencies are achievable in principle by using a refractive imaging portion without a central obstruction. (c) Relaxed optical requirements for the nonimaging mirror allow part of it to be removed to permit access to tissue with electrodes while incurring a minimal loss of collection efficiency. (d) Virtual test setup for comparison of epifluorescence collection efficiency. Emitted photons were scattered in tissue and propagated through the objectives and three apertures (A1, objective mount; A2, filter cube; A3, detector mount) placed to mimic the arrangement in the Nikon Eclipse E600FN upright microscope.

Fig. 2

Multiphoton imaging resolution of the HR design detailed in Table 1 is limited by the sum of all spatial aberrations of excitation pulses delivered through the imaging pathway, plotted here as the fraction of encircled energy delivered within a given distance from the centroid of the focal spot. A slight worsening of the resolution is evident only in the corners of the 200 μm field of view.

Fig. 3

Comparison of epifluorescence collection efficiency as a function of imaging depth in a semi-infinite turbid slab. HR design with the parameters in Table 1. Dashed curves indicate that the configuration of the HR objective was modified by defocusing the nonimaging reflector to maximize the collection efficiency. The outlined values at negative imaging depths refer to a nonscattering sample. The “thin slab” configuration refers to a 200 μm thick slab placed in a recording chamber with a reflecting bottom. The crossing at 400 μm is an artifact of objective selection.