Resonance-Scanning Confocal Microscopes for Large Area Scans

Table of Content

Introduction
The RS-G4 Features
Applications
The RS-G4 Advantage

Introduction

Fluorescence imaging has been a key tool in biological research applications, covering an increasing range of methods. Confocal imaging is an extensively used technique that involves removing out-of-focus light from a sample, which ultimately leads to an increased signal-to-noise ratio and better resolution.

Traditional confocal microscopes are point-scanning microscopes, with which a diffraction-limited laser spot can be scanned over a sample. Each point in this sample emits light, which then travels via a pinhole, removing the out-of-focus light.

The final image obtained is built point-by-point. Although this method generates high-resolution images, it can only cover a small area of the sample, limiting the size of the field of view it can cover.

A resonant-scanning galvanometer used by resonance-scanning confocal microscopes scans the full aperture of the objective lens to focus the light to the sample. This objective lens is then swept across the field of view at high frequency.

Light is then collected after standard confocal imaging with a pinhole aperture and emission filters to enable selective depth of image control. Based on the size of the field of view, the scanning speed of the resonance scanners can provide several 10 Hz frame rates.

However, both resonant-scanning and conventional confocal microscopes have a major disadvantage: they are limited to imaging a comparatively small region. While larger regions can be imaged, this can take a much longer time. The ability to only image a small field of view has become increasingly limiting to biological researchers.

Research is advancing beyond the scope of single cells, making the use of confocal microscopes for imaging whole tissues and larger groups of cells neccessary. There is also a requirement for techniques that enable high-resolution confocal imaging of large areas of tissues.

The RS-G4 Features

The Caliber I.D RS-G4 is a large-format resonant-scanning confocal microscope (Figure 1) specifically designed to provide multiple laser lines (405/488/561/640/785) for sequential, single, or simultaneous image acquisition of fluorescent labels. The RS-G4 can collect high-quality confocal images of huge biological samples of up to 80 x 120 mm in a much shorter period of time than traditional confocal microscopes (Figure 2).

Figure 1. Caliber I.D. RS-G4

Figure 2. Comparison of the speed of conventional confocal imaging vs. the RS-G4.

A combination of proprietary software algorithms and innovative hardware design contribute to the extraordinary speed of the RS-G4. Mosaics are large-format images (Figure 3) that are produced by organizing a sequence of mosaic strips that are acquired continuously.

Each mosaic strip is obtained along one axis of the sample, and a set of mosaic strips are obtained across the sample with a high-precision x-y stage to shift the sample while the images are being acquired by the resonant scanner.

Figure 3. Mouse Brain – Neurons activated during stress test. Image courtesy of Boston Childern’s Hospital – D. Ehlinger and K. Commons.

Caliber I.D.’s proprietary “image stitching” in the RS-G4 allows it to assemble and align the various mosaic strips at pixel-level resolution in order to generate the entire large-format mosaic (Figure 4).

Figure 4. Strip mosaic movie - Camilla Leaf Stem. Image courtesy of The University of Chicago - C. Labno and V. Bindokas.

Applications

Acquiring high-quality confocal images of large regions is an important ability for many application areas. For instance, in neurological studies, it is now possible to image the whole brain slices at high resolution, enabling comprehensive cellular level characterizations as well as gross morphological visualization (Figure 3). It is also possible to image whole organs, such as kidneys (Figure 5).

Figure 5. Mouse Kidney. Image courtesy of Caliber I.D.

Whole model organisms, such as C. elegans and Zebra fish can be imaged at high-resolution in developmental biology can be imaged at high resolution at various stages of the developmental cycle, providing a better understanding of the developmental process, which was otherwise not possible with traditional high-resolution imaging or low-resolution macroscopic imaging of smaller sub-regions of the organism.

The RS-G4 is also used in plant biology. The capacity to image gross morphological structures as well as the ability to zoom into the same image to view high-resolution cellular structures creates potential for new studies that were not possible with existing imaging techniques.

The RS-G4 Advantage

Certain computing challenges are faced during high-resolution, large-format confocal imaging of samples, which can range up to several square centimeters. A key area of concern is how to view a large format, high-resolution data set. The proprietary software package in the RS-G4 has been purpose built to resolve these issues.

The RS-G4, equipped with advanced memory management capabilities and image-visualization, is suitable to visualize such large data sets. For instance, users can detect large-scale morphologies and structures by visualizing a large-area view of the sample at a lower resolution and then instantly zooming into an area of interest to visualize high-resolution structures inside that region.

Morphogenesis, for example, can now be visualized at both the micro and macro scale, with the same confocal clarity. There is a full correlation available to interact, from the macro view to zoom into the preferred micro cellular detail for the fluorescent images captured.

In addition to performing high-resolution fluorescent imaging with visible wavelength lasers, the RS-G4 delivers an infrared laser (785 nm) to allow in-vivo reflectance imaging of a biological specimen with a penetration depth of up to 250 µm, based on the cellular density.

This provides unique imaging capabilities to capture images within adult or juvenile model specimens. Figure 6 shows a coronal section of marmoset brain.

Figure 6. Marmoset Brain – Coronal section of marmoset brain, stained for rabies virus in green and neuronal somata in red. 20 x 20 mm area scan. Capture time for simultaneous imaging of both 488 and 635 nm channels equaled 4 minutes. Image courtesy of University of Pittsburgh Center for Biologic Imaging – A. Rose, P. Strick and S. Watkins.

Conclusion

Standard confocal microscopy has restricted field of view, which has been shown to be a limiting factor for many researchers in their studies. In contrast, the RS-G4 provides large-format, high-speed confocal imaging without affecting the resolution and image quality. This feature will provide greater clarity, precision, and efficiency for many laboratories.

This information has been sourced, reviewed and adapted from materials provided by Caliber I.D.

For more information on this source, please visit Caliber I.D.

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