{"id":188,"date":"2019-07-05T15:32:03","date_gmt":"2019-07-05T19:32:03","guid":{"rendered":"https:\/\/sites.bu.edu\/biomicroscopy\/?page_id=188"},"modified":"2019-07-15T09:43:06","modified_gmt":"2019-07-15T13:43:06","slug":"dai","status":"publish","type":"page","link":"https:\/\/sites.bu.edu\/biomicroscopy\/research\/dai\/","title":{"rendered":"Differential aberration imaging"},"content":{"rendered":"
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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.<\/figcaption><\/figure>\n

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 example, DAI was demonstrated with TPEF microscopy using as an aberrating element a deformable mirror provided by Boston Micromachines Corporation. More recently, instantaneous DAI has been achieved using temporal multiplexing with no moving parts.<\/p>\n

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GFP-labeled mouse olfactory bulb without and with DAI.<\/figcaption><\/figure>\n
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Near-instantaneous DAI with temporal multiplexing. Excitation laser beam is split into two alternating beams producing Gaussian (non-aberrated) and donut (aberrated) foci. Resulting alternating fluorescence is parsed using synchronized detection.<\/figcaption><\/figure>\n

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