Abstract
Nonlinear microscopy techniques crucially rely on efficient signal detection. Here, we present a ring of large-core optical fibers for epi-collection of fluorescence photons that are not transmitted through the objective and thus normally wasted. Theoretical treatments indicated that such a supplementary fiber-optic light collection system (SUFICS) can provide an up to 4-fold signal gain. In typical in vivo imaging experiments, the fiber-ring channel was brighter than the objective channel down to 800 microm depth, thus providing a gain >2. Moreover, SUFICS reduced noise levels in calcium imaging experiments by about 23%. We recommend SUFICS as a generally applicable, effective add-on to nonlinear microscopes for enhancing fluorescence signals.
Highlights
Nonlinear optical microscopy is the current gold standard for high-resolution in vivo imaging deep inside biological tissue [1, 2]
Using in vivo two-photon imaging of mouse neocortex we show that this more than 2-fold signal gain is maintained throughout the cortical depths and that it improves the signal-to-noise ratio (SNR) of calcium measurements from neuronal populations
In this study we introduced supplementary fiber-optic light collection system (SUFICS) as a simple, inexpensive, and generally applicable method to enhance signal collection in nonlinear microscopy
Summary
Nonlinear optical microscopy is the current gold standard for high-resolution in vivo imaging deep inside biological tissue [1, 2]. An up to 3-fold gain in fluorescence can be achieved for tissue slices by additional fluorescence collection through a high-NA condensor in the trans-illumination pathway [9, 10]. The latter method cannot be utilized, for in vivo imaging experiments because photon collection from intact tissue is restricted to the 'epi'-hemisphere that is oriented towards the objective. Using in vivo two-photon imaging of mouse neocortex we show that this more than 2-fold signal gain (total collected fluorescence divided by fluorescence in objective channel) is maintained throughout the cortical depths and that it improves the SNR of calcium measurements from neuronal populations
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