A novel method to image thick samples

Research Slide Scanner VS200 SILA

Sponsored content: Widefield (WF) fluorescence microscopes provide high-resolution imaging for thin samples but are limited in their ability to image thicker samples due to background fluorescence causing image blur. Optical sectioning effectively overcomes this problem but has traditionally required a confocal system or deblurring software. With optical sectioning technology now on slide scanners, researchers can benefit from confocal capabilities for imaging thick samples — with significantly improved throughput.

Limitations of wide-field imaging

WF microscopy is a ubiquitous imaging technique that is highly effective for thin samples (<10 µm). In WF microscopy, light from both in-focus and out-of-focus planes are collected by the camera, which is not a problem for thin samples. When imaging thicker samples, this unavoidable background fluorescence causes image blur and poor contrast, obscuring structures of interest. Confocal microscopy and other scanning illumination techniques can overcome this problem. They work by shaping the illumination into a fixed pattern, resulting in an optically sectioned image. These systems can produce excellent images under favorable conditions. However, they come at a significant expense and rely on the delivery of well-defined and controlled illumination patterns into the sample.

A novel approach to illumination

To negate the effects of background fluorescence on image contrast and quality, well-defined and controlled illumination patterns are not always necessary. One technique known as HILO microscopy uses random speckle patterns to illuminate the fluorescent sample. Speckle patterns exhibit granular intensity with inherently high contrast, making fluorescence images obtained via speckle illumination appear granular. This granularity provides contrast and a direct indication of how in-focus the sample is.

During HILO imaging, two raw images are collected and processed. The first is a regular WF image with uniform illumination. A high-pass filter is applied on this image to extract the high-frequency information—the ‘HI’ part of HILO microscopy. This step eliminates low-frequency information, including both in-focus and out-of-focus information. The second image is captured using speckle illumination to recover the low-frequency in-focus image information—the ‘LO’ part of HILO microscopy. These images are processed using an algorithm that extracts in-focus information and eliminates background fluorescence. The two images are then fused together (Fig. 1) to obtain an image containing information from the full frequency range, with the out-of-focus light removed.

The SILA optical sectioning device is a high-throughput imaging solution for Slideview VS200 research slide scanners based on HILO microscopy. It is an add-on technology for widefield (WF) microscopes that eliminates out-of-focus light and produces sharp optical sectioning equal to confocal microscopy. The SILA device can easily be added to any VS200 system and brings benefits to a broad range of applications requiring the high-quality optical imaging of thicker samples.

Adjustable optical sectioning

Since the SILA device mathematically processes the traditional WF image and speckle image, it is possible to adjust the degree of optical sectioning using a single parameter, which we call sectioning thickness (ST). When the ST is set to high, images display information from a larger depth of field, giving the appearance of a WF image.

Figure 2 shows a brain section taken at ST5. At this high ST value, many out-of-focus areas remain. As the ST value reduces, the image displays information from a smaller depth of field. Therefore, when observing the brain section taken at ST2 and ST1, the in-focus information remains while out-of-focus elements have disappeared. The ability to optimize optical sectioning in this way enables visualization at different depths while removing background fluorescence. This feature of the SILA device for the VS200 scanner is comparable to confocal microscope systems, in which changing the pinhole size can achieve a similar effect.

Deconvolution capabilities

Before the development of the SILA device, VS200 scanners had an alternative solution for removing out-of-focus light from WF images. Using Trusight deconvolution software, it is possible to deconvolve images using 2D constrained iterative (CI) algorithms. This software-based approach works well at removing some of the out-of-focus light. Yet, it does not remove as much out-of-focus light nor provide the optical sectioning optimization as the combined software and hardware used in the SILA module. However, deconvolution does provide a viable intermediate option for viewing thinner samples where SILA optical sectioning is unavailable. The differences in image quality between conventional WF, software-deconvolved, and SILA images are demonstrated in figure 3.

Applications of SILA imaging

SILA optical sectioning technology can bring significant value to many research applications, especially in the imaging of fixed, thick cell layers and tissue samples. With sectioning optimization features, plus comparable blur elimination and image contrast to that of confocal microscopes, SILA delivers high-quality imaging with vastly improved throughput.

The ability to image thicker samples is beneficial in neuroscience imaging, where thick tissue sections are often required to preserve the morphology of the specimen. Imaging brain sections is notoriously challenging. It is particularly difficult because of the propensity of brain tissue to produce a light-scattering effect. Traditionally, confocal or two-photon microscopy systems would be required to capture a high-quality, high-contrast image, but these systems would take a substantial amount of time to image a large area such as a brain section. The automated slide scanning provided by the VS200 system with the SILA device makes imaging thick samples over a large area much faster, delivering high-quality images in a fraction of the time (Figure 4).

Organoid research is another area that benefits from SILA imaging, since organoid imaging presents similar challenges to neuroscience. Imaging organoids is difficult due to thick samples, while large areas need to be imaged so that organoid morphology can be properly interrogated. As a whole slide scanner, the VS200 system with the SILA module can provide the coverage necessary to image these large samples. Meanwhile, its automated imaging processing, sample-independent point spread function (PSF), and simple workflow results in rapid image acquisition. The SILA device also brings value to cancer research, spatial biology, botany, embryology, and many others that require high-quality imaging of thick samples over large areas.

Summary

SILA imaging can optimize optical sectioning, reduce sample blur, and improve image contrast comparable to sophisticated confocal laser scanning microscopes. Providing significantly improved throughput versus confocal systems, the VS200 slide scanner with the SILA device enables rapid imaging of large and traditionally difficult-to-image samples, bringing significant benefits to neuroscience, organoid research and more.