Confocal microscopy is a standard approach for obtaining volumetric images of a sample with high axial and lateral resolution, especially when dealing with scattering samples. Unfortunately, a confocal microscope is quite expensive compared to traditional microscopes. In addition, the point scanning in confocal microscopy leads to slow imaging speed and photobleaching due to the high dose of laser energy. In this paper, we demonstrate how the advances in machine learning can be exploited to teach a traditional wide-field microscope, one that’s available in every lab, into producing 3D volumetric images like a confocal microscope. The key idea is to obtain multiple images with different focus settings using a wide-field microscope and use a 3D generative adversarial network (GAN) based neural network to learn the mapping between the blurry low-contrast image stacks obtained using a wide-field microscope and the sharp, high-contrast image stacks obtained using a confocal microscope. After training the network with widefield-confocal stack pairs, the network can reliably and accurately reconstruct 3D volumetric images that rival confocal images in terms of its lateral resolution, z-sectioning and image contrast. Our experimental results demonstrate generalization ability to handle unseen data, stability in the reconstruction results, high spatial resolution even when imaging thick (∼40 microns) highly-scattering samples. We believe that such learning-based microscopes have the potential to bring confocal imaging quality to every lab that has a wide-field microscope.
Confocal microscopy provides optical sectioning that is invaluable for many applications, most notably imaging into thick samples. However, the high cost of commercial confocal microscopes limits uses to specialized research and clinical settings. We present a minimalistic line-scanning confocal microscope costing less than $6,500 with optical performance comparable to a commercial laser scanning system. The optical sectioning and imaging performance are shown through measurement of the axial line-spread function and imaging of biological samples of varying thickness. Comparison is made to commercial widefield and confocal microscopes. The low cost of goods and optical sectioning capability of this microscope will allow the use of confocal microscopy in additional research and educational settings.
more » « less- NSF-PAR ID:
- 10369384
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Optics Letters
- Volume:
- 47
- Issue:
- 16
- ISSN:
- 0146-9592; OPLEDP
- Format(s):
- Medium: X Size: Article No. 4191
- Size(s):
- Article No. 4191
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
more » « less
Abstract Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.
-
more » « less
Recent advancements in image-scanning microscopy have significantly enriched super-resolution biological research, providing deeper insights into cellular structures and processes. However, current image-scanning techniques often require complex instrumentation and alignment, constraining their broader applicability in cell biological discovery and convenient, cost-effective integration into commonly used frameworks like epi-fluorescence microscopes. Here, we introduce three-dimensional multifocal scanning microscopy (3D-MSM) for super-resolution imaging of cells and tissue with substantially reduced instrumental complexity. This method harnesses the inherent 3D movement of specimens to achieve stationary, multi-focal excitation and super-resolution microscopy through a standard epi-fluorescence platform. We validated the system using a range of phantom, single-cell, and tissue specimens. The combined strengths of structured illumination, confocal detection, and epi-fluorescence setup result in two-fold resolution improvement in all three dimensions, effective optical sectioning, scalable volume acquisition, and compatibility with general imaging and sample protocols. We anticipate that 3D-MSM will pave a promising path for future super-resolution investigations in cell and tissue biology.
-
more » « less
There has been recent interest in the development of fluorescence microscopes that provide high-speed volumetric imaging for life-science applications. For example, multi-z confocal microscopy enables simultaneous optically-sectioned imaging at multiple depths over relatively large fields of view. However, to date, multi-z microscopy has been hampered by limited spatial resolution owing to its initial design. Here we present a variant of multi-z microscopy that recovers the full spatial resolution of a conventional confocal microscope while retaining the simplicity and ease of use of our initial design. By introducing a diffractive optical element in the illumination path of our microscope, we engineer the excitation beam into multiple tightly focused spots that are conjugated to axially distributed confocal pinholes. We discuss the performance of this multi-z microscope in terms of resolution and detectability and demonstrate its versatility by performing
in-vivo imaging of beating cardiomyocytes in engineered heart tissues and neuronal activity inc. elegans and zebrafish brains. -
more » « less
Abstract Laser scanning microscopes can be miniaturized for in vivo imaging by substituting optical microelectromechanical system (MEMS) devices in place of larger components. The emergence of multifunctional active optical devices can support further miniaturization beyond direct component replacement because those active devices enable diffraction-limited performance using simpler optical system designs. In this paper, we propose a catadioptric microscope objective lens that features an integrated MEMS device for performing biaxial scanning, axial focus adjustment, and control of spherical aberration. The MEMS-in-the-lens architecture incorporates a reflective MEMS scanner between a low-numerical-aperture back lens group and an aplanatic hyperhemisphere front refractive element to support high-numerical-aperture imaging. We implemented this new optical system using a recently developed hybrid polymer/silicon MEMS three-dimensional scan mirror that features an annular aperture that allows it to be coaxially aligned within the objective lens without the need for a beam splitter. The optical performance of the active catadioptric system is simulated and imaging of hard targets and human cheek cells is demonstrated with a confocal microscope that is based on the new objective lens design.
