Microscopy is one of the oldest scientific fields, and the microscope has had many improvements over the ages. Traditional microscopy techniques have proven instrumental in research, but technologies such as confocal microscopy tend to be limited in their resolutions by the diffraction properties of light. A couple decades ago, scientists overcame the diffraction limitation through structuring illumination patterns and by establishing algorithms that enabled greater resolutions, with a technique known as super-resolution microscopy.

Certain companies have combined super-resolution techniques with confocal microscopy to enhance analysis quality, including Leica Microsystems with its HyVolution 2 system, Nikon’s N-SIM S, N-SIM E and N-STORM microscopes, and Olympus Scientific Solutions’ FLUOVIEW FV3000 with FV-OSR and the IXplore SpinSR10.

IBO had the opportunity to discuss the integration of confocal and super-resolution microscopy in systems such as the IXplore SpinSR10, with Lauren Alvarenga, product manager, Life Science Microscopy, at Olympus’ Scientific Solutions Group.

Olympus’ FV3000 has capabilities of 2D-6D (x,y,z,t,λ,p) macro to micro imaging of cells, tissues and small organisms, facilitating research in a variety of areas, including cell biology, cancer research, electrophysiology, neuroscience and stem cell applications. The SpinSR10, a spinning disk confocal super-resolution microscope, is intended for live-cell imaging with 120 nm resolution. With its integration of speed, lessened phototoxicity and stability when conducting time-lapse experiments, researchers are able to use the microscope to focus on the vibrant fluctuations that take place inside cells, according to the company.

“The combination of these techniques makes super resolution more accessible to a broader range of research scientists,” Ms. Alvarenga told IBO. “Since confocal microscopy experiments are commonplace, the integration of super resolution allows researchers to get more out of existing experimental protocols. Super-resolution systems built upon spinning disk confocal microscopes have further benefits associated with cell health, bringing super resolution within reach for live-cell imaging experiments.”

Garbage in, garbage out’ is a popular phrase among microscopists, as samples must be high enough quality to achieve the desired results.”

The combination of confocal and super-resolution techniques promotes a more efficient and cost-effective option, as the multi-modal SpinSR10 can acquire images with widefield, confocal and super resolution on one microscope, as Ms. Alvarenga explained. “We have done this in a way that is completely motorized and software controlled, making super resolution as simple as a single click and with existing samples,” she continued. “This removes the traditional barriers associated with super- resolution microscopy; there is no need to modify sample protocols or purchase an additional system.”

An issue that may come up during analysis using integrated microscopy is clear samples. “While Olympus has no restrictions on sample preparation, super-resolution microscopy requires a high enough signal-to-noise ratio to be able to process the images in a reliable manner,” Ms. Alvarenga noted. “‘Garbage in, garbage out’ is a popular phrase among microscopists, as samples must be high enough quality to achieve the desired results. That said, recent developments from Olympus have allowed us to collect 3X more photons than previous models for brighter images and higher signal-to-noise ratios.”

Key to analyzing data made available by the integrated microscopes is software, and the company’s Olympus Super Resolution software module is widely applicable. As Ms. Alvarenga stated, “Proprietary algorithms, like Olympus Super Resolution, handle high spatial frequency data in different ways than deconvolution alone, which translates into clear, accurate data below the diffraction limit.” The software is ideal for colocalization analysis, and can acquire wavelengths sequentially or simultaneously at a 120 nm resolution, which is almost double the resolution of widefield microscopy, according to her.

The integration of techniques has enabled a new level of analysis in a variety of applications, and its growing prevalence points towards a trend that will prove to be invaluable, as health research and analysis becomes more personalized in applications such as precision medicine and immunotherapy. “Super-resolution microscopy is more accessible than ever before,” Ms. Alvarenga said. “It is no longer necessary to purchase an entirely new microscope to access super resolution, especially as trends towards differentiating algorithms begin to emerge.”

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