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Differential aberration imaging

When a nonlinear microscope such as a two-photon excited fluorescence (TPEF) or second-harmonic generation (SHG) microscope is used to image thick tissue, the scattering of the excitation beam can provoke a loss of signal and an increase in out-of-focus background. We have alleviated the problem of increased background by developing a nonlinear microscope based on a novel contrast mechanism called differential aberration imaging (DAI. The principle of DAI is to introduce a switchable aberrating element in the excitation beam path. When the aberrating element is switched off, the microscope operates in a conventional manner, producing images that contain both signal and background. When the aberrating element is switched on, the extraneous aberrations in the excitation beam provoke a severe quenching of the signal, and the microscope reveals only background. A simple subtraction of an aberrated from an unaberrated image then results in an essentially background-free image that contains only signal. This technique works for any scanning microscope based on nonlinear contrast. For wexample, DAI was demonstrated with TPEF microscopy. In our case, the aberrating element was a deformable mirror provided by Boston Micromachines Corporation.

Publications Related to this Research Area

Enhanced background rejection in thick tissue with differential-aberration two-photon microscopy

A. Leray, K. Lillis and J. Mertz,

Biophysical Journal

When a two-photon excited fluorescence (TPEF) microscope is used to image deep inside tissue, out-of-focus background can arise from both ballistic and nonballistic excitation. We propose a solution to largely reject TPEF background in thick tissue. Our technique is based on differential-aberration imaging with a deformable mirror. By introducing extraneous aberrations in the excitation beam path, we preferentially quench in-focus TPEF signal while leaving out-of-focus TPEF background largely unchanged. A simple subtraction of an aberrated, from an unaberrated, TPEF image then removes background while preserving signal. Our differential aberration (DA) technique is simple, robust, and can readily be implemented with standard TPEF microscopes with essentially no loss in temporal resolution when using a line-by-line DA protocol. We analyze the performance of various induced aberration patterns, and demonstrate the effectiveness of DA-TPEF by imaging GFP-labeled sensory neurons in a mouse olfactory bulb and CA1 pyramidal cells in a hippocampus slice.

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Rejection of two-photon fluorescence background in thick tissue by differential aberration imaging

A. Leray and J. Mertz,

Optics Express

We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. An added benefit of our technique is that it leads to somewhat improved image resolution.

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DAI principle

DAI involves inserting a switchable aberrating element (e.g. a deformable mirror) into the illumination path of a TPEF microscope. When the aberrator is off, the microscope produces a standard TPEF image that contains both signal and background. When the aberrator is on, the focus becomes blurred and the microscope produces a background-only image. A subtraction of the aberrated from the non-aberrated image leads to a signal-only image.

Standard TPEF image of GFP-labeled mouse olfactory bulb (without DAI)

TPEF image of GFP-labeled mouse olfactory bulb with DAI