Photon-by-photon imaging: Microscopy's new frontier

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Photon-by-photon imaging is a new technique for microscopy that uses a laser scanning microscope to build images one photon at a time. This technique allows for much higher resolution and sensitivity than traditional confocal laser scanning microscopy. The advent of fast and compact detector arrays has made photon-by-photon imaging possible. These arrays replace the typical single-element detector of traditional confocal laser scanning microscopes, enabling new and unique capabilities.

Photon-by-Photon Imaging: Advancing Microscopy with Unparalleled Resolution and Information

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Microscopy has revolutionized our understanding of the world around us, allowing us to peer into the intricate details of cells, tissues, and molecules. Traditional microscopes, however, have limitations in terms of resolution and the amount of information they can provide.

Enter photon-by-photon imaging, a groundbreaking technique that promises to transform the field of microscopy. This innovative approach uses fast and compact detector arrays to capture not just the intensity of light, but also its spatial distribution. This enables the creation of small images for each scan point, resulting in significantly richer and more informative data.

Photon-by-Photon Imaging-Enabled Image Scanning Microscopy: Unlocking Super-Resolution and Beyond

One of the key applications of photon-by-photon imaging is image scanning microscopy (ISM). ISM computationally constructs a single image from a multidimensional dataset, yielding superior signal-to-noise ratio, optical sectioning, and spatial resolution compared to conventional confocal microscopes.

The beauty of ISM lies in its ability to surpass the Abbe diffraction limit, a fundamental barrier in microscopy that limits the resolution of images. By exploiting spatial information, ISM can achieve a lateral resolution that is up to twice better than what is possible with traditional techniques.

Fluorescence Lifetime Imaging: Unveiling Dynamic Processes with Photon-by-Photon Imaging

Fluorescence lifetime imaging (FLIM) is another powerful technique that can be seamlessly integrated with ISM. FLIM measures the time it takes for a fluorescent molecule to emit light after absorbing it. This information provides insights into the molecular environment and dynamics, making it a valuable tool for studying biological processes.

The combination of ISM and FLIM, known as FLISM, offers the best of both worlds. It not only enhances the spatial resolution of images but also enables the extraction of fluorescence lifetime information, providing a more comprehensive understanding of the sample.

Photon-by-Photon Imaging Enabled STED Microscopy and Multispecies Imaging: Expanding the Horizons

Photon-by-photon imaging can be further enhanced by combining it with stimulated emission depletion (STED) microscopy. STED uses a second laser beam to selectively deplete fluorescence, improving lateral resolution even further. This technique, known as separation by lifetime tuning (SPLIT), allows for the acquisition of images with exceptional resolution and contrast.

Multispecies imaging is another exciting application of photon-by-photon imaging. By utilizing the unique fluorescence lifetime values of different dyes, it becomes possible to distinguish between multiple species within a single sample using a single detector. This capability opens up new avenues for studying complex biological systems.

The Future of Microscopy: Compact, Effective, and Accessible Photon-by-Photon Imaging Systems

The compact and effective design of photon-by-photon imaging systems makes them highly accessible and user-friendly. The integration of data acquisition and processing on a single field-programmable-gate-array (FPGA) board simplifies the microscope architecture and streamlines the imaging process.

This ease of use, combined with the wealth of information provided by photon-by-photon imaging, makes it a promising tool for a wide range of applications in biology, chemistry, and materials science.

Wrapping Up: Photon-by-Photon Imaging – A Revolution in Microscopy

Photon-by-photon imaging is a transformative technology that is revolutionizing the field of microscopy. By capturing both spatial and temporal information, it enables the acquisition of images with unprecedented resolution, contrast, and dynamic range.

The integration of ISM, FLIM, STED, and multispecies imaging techniques further expands the capabilities of photon-by-photon imaging, making it a versatile and powerful tool for exploring the intricacies of life at the molecular and cellular level.

As this technology continues to advance, we can expect even more groundbreaking discoveries and insights into the world around us..

FAQ’s

1. What is photon-by-photon imaging?

Photon-by-photon imaging is a revolutionary microscope technique that captures not just the intensity of light, but also its spatial distribution. This enables the creation of small images for each scan point, resulting in significantly richer and more informative data.

2. What are the advantages of photon-by-photon imaging over traditional microscopy techniques?

Photon-by-photon imaging offers several advantages over traditional microscopy techniques, including improved resolution, higher signal-to-noise ratio, and the ability to extract additional information such as fluorescence lifetime.

3. How does image scanning microscopy (ISM) work?

Image scanning microscopy (ISM) computationally constructs a single image from a multidimensional dataset, yielding superior signal-to-noise ratio, optical sectioning, and spatial resolution compared to conventional confocal microscopes. ISM can also surpass the Abbe diffraction limit, achieving a lateral resolution that is up to twice better than what is possible with traditional techniques.

4. What is fluorescence lifetime imaging (FLIM) and how is it used with ISM?

Fluorescence lifetime imaging (FLIM) measures the time it takes for a fluorescent molecule to emit light after absorbing it. This information provides insights into the molecular environment and dynamics, making it a valuable tool for studying biological processes. FLISM combines ISM and FLIM, offering both enhanced spatial resolution and the ability to extract fluorescence lifetime information.

5. What are the applications of photon-by-photon imaging?

Photon-by-photon imaging has a wide range of applications in biology, chemistry, and materials science. It is used to study biological processes, such as cell division and protein interactions, as well as to investigate the structure and properties of materials.

Links to additional Resources:

https://www.nature.com/articles/s41420-022-01035-x https://www.sciencedirect.com/science/article/abs/pii/S0960982222006650 https://www.osapublishing.org/oe/fulltext.cfm?uri=oe-30-19-34210&id=497055.