Abstract
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.
🔬 Techniques
✨ Fluorophores
🧪 Sample Preparation
🏭 Microscope Brands
🔎 Objectives
💻 Software Details
🏛️ Research Organizations (ROR)
Affiliated research institutions:
📊 Figures
Fig. 1.
Schematic of an EDOF microscope. The vertical dashed line indicates the location of the intermediate image plane.
Fig. 2.
Standard versus EDOF imaging of 1 u03bcm diameter fluorescent beads embedded in PDMS. Images taken with a 20 u00d7 0.5 NA objective; R P = 2.21 mm , R DM = 2 mm , and the EDOF = 69 u03bcm [ Eq. (1) ] ...
Fig. 3.
High-speed images of 4 u03bcm diameter beads. (a) Standard and (b) extended DOFs taken at 472 fps in a reduced field-of-view with a 40 u00d7 0.8 NA objective; R P = 1.44 mm , R DM = 1.5 mm , and the E...
Fig. 4.
EOTF ( u03ba u22a5 ; D ) for a 20 u00d7 0.5 NA objective for focal sweep ranges D of (a) 0, (b) 5, (c) 10, and (d) 20 u03bcm. The EOTF calculated using Eq. (6) (orange) is a good approximation to the ...
Fig. 5.
Images of tissue paper labeled with a highlighter marker, taken with a 20 u00d7 0.5 NA objective; the EDOF = 69 u03bcm with 26 DM frames. (a) Raw EDOF image has poor contrast and few distinguishable f...
Fig. 6.
Images of GCaMP-labeled mouse brain neurons taken with a 20 u00d7 0.5 NA objective; the EDOF = 69 u03bcm with 26 DM frames. (a) Standard image shows an out-of-focus neuron in the marked area. (b) With...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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