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Fast volumetric imaging

We present a wide-field fluorescence microscopy add-on that provides a fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror with an update rate of 20 kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.

Significantly increased contrast can be obtained by combining EDOF imaging with targeted illumination. The latter strategy involves delivering illumination only to in-focus structure during the focal sweep, using illumination masks controlled by a fast DMD.


Publications Related to this Research Area

Video-rate volumetric neuronal imaging using 3D targeted illumination

S. Xiao, H.-A. Tseng, H. Gritton, X. Han, J. Mertz,

Scientific Reports

Fast volumetric microscopy is required to monitor large-scale neural ensembles with high spatio-temporal resolution. Widefield fluorescence microscopy can image large 2D fields of view at high resolution and speed while remaining simple and costeffective. A focal sweep add-on can further extend the capacity of widefield microscopy by enabling extended-depth-of-field (EDOF) imaging, but suffers from an inability to reject out-of-focus fluorescence background. Here, by using a digital micromirror device to target only in-focus sample features, we perform EDOF imaging with greatly enhanced contrast and signal-to-noise ratio, while reducing the light dosage delivered to the sample. Image quality is further improved by the application of a robust deconvolution algorithm. We demonstrate the advantages of our technique for in vivo calcium imaging in the mouse brain.

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Axial localization with modulated-illumination extended-depth-of-field microscopy

W. J. Shain, N. A. Vickers, J. Li, X. Han, T. Bifano, J. Mertz ,

Biomedical optics Express

High-speed volumetric imaging represents a challenge in microscopy applications. We demonstrate a technique for acquiring volumetric images based on the extended depth of field microscopy with a fast focal scan and modulated illumination. By combining two frames with different illumination ramps, we can perform local depth ranging of the sample at speeds of up to half the camera frame rate. Our technique is light efficient, provides diffraction-limited resolution, enables axial localization that is largely independent of sample size, and can be operated with any standard widefield microscope based on fluorescence or darkfield contrast as a simple add-on. We demonstrate the accuracy of axial localization and applications of the technique to various dynamic extended samples, including in-vivo mouse brain.

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Dual fluorescence-absorption deconvolution applied to extended-depth-of-field microscopy

W. J. Shain, N. A. Vickers, A. Negash, T. Bifano, A. Sentenac, J. Mertz,

Optics Letters

Fast imaging over large volumes can be obtained in a simple manner with extended-depth-of-field (EDOF) microscopy. A standard technique of Wiener deconvolution can correct for the blurring inherent in EDOF images. We compare Wiener deconvolution with an alternative, parameter-free technique based on the dual reconstruction of fluorescence and absorption layers in a sample. This alternative technique provides significantly enhanced reconstruction contrast owing to a quadratic positivity constraint that intrinsically favors sparse solutions. We demonstrate the advantages of this technique with mouse neuronal images acquired in vivo. .

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Extended depth-of-field microscopy with a high-speed deformable mirror

W. J. Shain, N. A. Vickers, B. B. Goldberg, T. Bifano, J. Mertz,

Optics Letters

We present a wide-field fluorescence microscopy add-on that provides a fast, light-efficient extended depth-of-field (EDOF) using a deformable mirror with an update rate of 20 kHz. Out-of-focus contributions in the raw EDOF images are suppressed with a deconvolution algorithm derived directly from the microscope 3D optical transfer function. Demonstrations of the benefits of EDOF microscopy are shown with GCaMP-labeled mouse brain tissue.

view on publisher's web-site

PSF vs EPSF

Targeted illumination EDOF

In-vivo mouse brain imaging. Comparison of EDOF imaging with conventional and targeted illumination.