Abstract
Multiphoton excitation fluorescence microscopy is the preferred method for in vivo deep tissue imaging. Many biological applications demand both high imaging speed and the ability to resolve multiple fluorophores. One of the successful methods to improve imaging speed in a highly turbid specimen is multifocal multiphoton microscopy (MMM) based on use of multi-anode photomultiplier tubes (MAPMT). This approach improves imaging speed by using multiple foci for parallelized excitation without sacrificing signal to noise ratio (SNR) due to the scattering of emission photons. In this work, we demonstrate that the MAPMT based MMM can be extended with spectral resolved imaging capability. Instead of generating multiple excitation foci in a 2D grid pattern, a linear array of foci is generated. This leaves one axis of the 2D MAPMT available for spectral dispersion and detection. The spectral-resolved MMM can detect several emission signals simultaneously with high imaging speed optimized for high-throughput, high-contents applications. The new procedure is illustrated using imaging data from the kidney, peripheral nerve regeneration and dendritic morphological data from the brain.
Highlights
Multiphoton excitation fluorescence microscopy has inherent 3D resolution due to the nonlinear dependence of excitation efficiency on incident light flux [1,2,3]
This system has been demonstrated to successfully perform spectral-resolved imaging in ex vivo tissue applications and in vivo deep in the mouse brain with comparable signal to noise ratio (SNR) as single focus systems but with much higher acquisition speed. This spectral-resolved multifocal multiphoton microscopy (MMM) based on multianode photomultiplier tube (MAPMT) is an interesting addition to other previously developed spectral-resolved MMMs
The use of the MAPMT with single-photon counting circuitry can achieve much higher frame rate compared with the system using imaging cameras [26], especially ones with long dead-time streak cameras [23,24,25]; streak cameras have the advantage of providing lifetime-resolved imaging capability at the same time
Summary
Multiphoton excitation fluorescence microscopy has inherent 3D resolution due to the nonlinear dependence of excitation efficiency on incident light flux [1,2,3]. This difficulty was recently partly circumvented by introducing descanned MMM using a detection scheme based on using a multianode photomultiplier tube (MAPMT). With this approach, the scattered emission photons are much more effectively collected by the large area anodes that correspond to tens of micron size areas on the specimen plane providing much better immunity to SNR degradation due to emission photon scattering [15]. The emission signals from the foci are collected by the same objective lens, travel along the same beam path as the excitation light, and are descanned by the same scanning mirrors. The deleterious effect of emission photon scattering can be effectively suppressed
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