[Enlarge image]In multifocal scanning microscopy (MSM), the fixed multifocal excitation patterns generated by a microlens array (MLA) are interlaced for simultaneous multicolor imaging. The interlaced coordinates from each color are used to reconstruct super-resolution images.
Traditional super-resolution techniques such as structured-illumination microscopy and image-scanning microscopy have significantly advanced cellular imaging with effective resolution improvement at moderate acquisition speed.1,2,3 Yet those techniques often involve complex setups and specialized equipment, limiting their accessibility. Furthermore, many super-resolution microscopies can limit applications to single-cell analyses due to high numerical-aperture and magnification needs. To address these challenges, we recently introduced multifocal scanning microscopy (MSM), a novel super-resolution microscopy technique that enables simultaneous multicolor acquisition with minimal complexity.4,5
MSM uses a stationary multifocal excitation pattern that leverages specimen motion from a motorized stage controller, enabling super-resolution imaging across larger fields without being bound by magnification limits. This makes MSM suitable for analyzing complex biological samples such as tissues or biofilms, while maintaining the benefits of super-resolution imaging.
In our demonstrations, MSM achieved twofold resolution improvement and enhanced optical sectioning on standard epifluorescence platforms. We demonstrated the versatility and effectiveness of this approach through experiments on various phantoms and biological samples. The simultaneous multicolor capability of MSM is particularly advantageous in biological research, as it allows the capture of multiple cellular components in a single acquisition without increasing the cost or complexity of the instrumentation or adding a temporal delay between acquiring multiple spectral channels.
MSM can also lend its accessibility and ease of integration into existing microscopies. For those without access to highly specialized equipment, the simplified setup of MSM enables super-resolution imaging without the need for expensive modifications or complex alignments. Further, its high compatibility with standard epifluorescence microscopes makes robust performance available for a wider range of researchers. This makes MSM an appealing option for live-cell imaging where multicolor acquisition is crucial for understanding cellular dynamics and their interactions.
We believe our proposed MSM uniquely bridges the opposing concepts of super-resolution and large-field-of-view imaging. This makes MSM a practical and versatile tool, enhancing the capabilities of super-resolution microscopy by making it accessible and applicable for advanced studies of both individual cells and larger tissue samples in multicolor.
Researchers
Kyungduck Yoon and Shu Jia, Georgia Institute of Technology, USA
References
1. C.J.R. Sheppard. Optik 80, 53 (1988).
2. C.B. Müller et al. Phys. Rev. Lett. 104, 198101 (2010).
3. I. Gregor et al. Curr. Opin. Chem. Biol. 51, 74 (2019).
4. K. Yoon et al. ACS Photonics 10, 3035 (2023).
5. K. Tadesse et al. Opt. Express 31, 38550 (2023).