Multi-Color Three Dimensional Imaging Using Multifocus Microscopy

This technology advances imaging techniques of biomicroscopy using an innovative type of a wide-field Multi-Focus microscope to enable fast, high-resolution 3D imaging.

Today, many three-dimensional (3D) biological specimens and processes are studied in two-dimensions (2D) due to the slow speed of the light microscope in recording a 3D image. This technology advances imaging techniques of biomicroscopy using an innovative type of a wide-field Multi-Focus microscope to enable fast, high-resolution 3D imaging. The system includes a novel grating design and chromatic correction scheme that is appended to the camera port of a high-resolution epifluorescence microscope to produce an instant focal stack of high-resolution 2D images simultaneously displayed on a single camera. In addition, the 3D microscope is designed to minimize photobleaching and phototoxicity while enabling high-speed imaging of weak fluorescent samples such as single fluorophores and to acquire multiple focal planes without aberrations to avoid loss in resolution and contrast.

The concept of Multi-Focus imaging uses optical manipulation to produce an entire focal series instantaneously. The Multi-Focus system developed is based on a binary phase plate with a specially designed pattern placed in the Fourier plane, a plane with special optical properties, which allows the grating to split up the emission light from the sample into a set of diffraction orders. Each diffraction order forms a separate image of the sample. The resulting series of images is recorded as a 2D array on a single, large-chip electron-multiplying charge-coupled device camera.

Publications:

The publications and supplemental materials contain guides to recreate the MFM setup.

• Fast multicolor 3D imaging using aberration-corrected multifocus microscopy Nature Methods. 2013;10(1):60-3. https://doi.org/10.1038/nmeth.2277
• MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously Optics Express Vol. 23, Issue 6, pp. 7734-7754 (2015). https://doi.org/10.1364/OE.23.007734 
• Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging Biomedical Optics Express. 2016 Feb 16;7(3):855-69. https://doi.org/10.1364/BOE.7.000855

Advantages:

• A powerful tool for fast and sensitive fluorescence microscopy of live samples
• The light efficiency of the imaging path is better than 50 percent
• The current design allows imaging of a volume of 33x33x18 microns at 31Hz

Applications:

• Basic life science research
• Fluorescence biomicroscopy
• Single-particle tracking in live cells
• Neuronal imaging
• Development imaging of small organisms

News:

Microscope expert develops powerful new tools for biologists - 2019

Issued Patent: 

United States 9,477,091

How to get it:

• Free for Non-Profit Research. Please contact Innovation Management at innovation@janelia.hhmi.org.
• Commercial Licenses available (Commercial Licensing Managed by David Fung, UCSF)

For inquiries, please reference:

Janelia 2012-006