Abstract
Multiphoton microscopy is a powerful, non-invasive technique to image biological specimens. One current limitation of multiphoton microscopy is resolution as many of the biological molecules and structures investigated by research groups are similar in size or smaller than the diffraction limit. To date, the combination of multiphoton and super-resolution imaging has proved technically challenging for biology focused laboratories to implement. Here we validate that the commercial super-resolution Airyscan detector from ZEISS, which is based on image scanning microscopy, can be integrated under warranty with a pulsed multi-photon laser to enable multiphoton microscopy with super-resolution. We demonstrate its biological application in two different imaging modalities, second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), to measure the fibre thicknesses of collagen and elastin molecules surpassing the diffraction limit by a factor of 1.7±0.3x and 1.4±0.3x respectively, in human heart and lung tissues, and 3-dimensional in vitro models. We show that enhanced resolution and signal-to-noise of SHG using the Airyscan compared to traditional GaAs detectors allows for automated and precise measurement of collagen fibres using texture analysis in biological tissues.
Highlights
Multiphoton microscopy has become the method of choice for imaging live, intact biological tissues due to the advantages of depth penetration and reduced photodamage, as a result of employing a near infrared femtosecond laser to generate observable nonlinear signals in the visible range [1,2,3,4]
We report here that the Airyscan when compared to standard MP microscopy for second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) surpasses the diffraction limit by a factor of 1.7±0.3x and 1.4±0.3x, respectively, when measuring the fibre thicknesses of collagen and elastin molecules in human tissues and 3-dimentional in vitro models
Samples with a high content of fibrillar collagen are ideal for the demonstration of SR-SHG imaging since the typical fiber size of fibrillar collagen is 50-200nm, which is below the diffraction limit of conventional SHG
Summary
Multiphoton microscopy has become the method of choice for imaging live, intact biological tissues due to the advantages of depth penetration and reduced photodamage, as a result of employing a near infrared femtosecond laser to generate observable nonlinear signals in the visible range [1,2,3,4]. Multiphoton excitation occurs when two (or more) photons arrive simultaneously at a fluorophore, and their energy sum satisfies the transition energy required to promote the fluorophore from a ground to an excited state. Such two-photon excitation fluorescence (TPEF) can be generated from a range of applications including exogenous probes (e.g. Hoechst), transfected proteins (e.g. Green fluorescent protein) or endogenous. Commercial solution for super resolution multiphoton microscopy by Canadian Foundation for Innovation (CFI) #31080. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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