Super-resolution microscopy uses a type of microscope that allows observation of samples beyond the diffraction limit typically associated with conventional optical microscopes. The spatial resolution of an ordinary optical microscope is generally calculated using Rayleigh’s resolution formula:
δ= 0.61 × λ / NA
According to this formula, in the visible light range (400 nm to 700 nm), the resolution limit is approximately 162 nm to 284 nm.*1
To surpass this resolution limit, there are several types of super-resolution microscopy have been developed, and the following are explanations of the mechanisms behind different types.
*1: Calculated with an NA (numerical aperture) of 1.5 for the objective lens.
Structured illumination microscopy is a method that obtains super-resolution images by observing Moiré patterns generated when a patterned excitation light called “structured illumination” is applied to the sample and then reconstructing those Moiré images.
Moiré refers to patterns that occur when overlapping periodic motifs. When the sample is illuminated with structured light containing periodic stripe patterns, Moiré interference arises between these patterns and the sample’s fine structures (see Figure 1). Since Moiré images contain information about the fine structures, capturing multiple images with varying structured illumination angles and phases allows computational reconstruction of fine structures, resulting in images with approximately twice the resolution of the original image.
Figure 1: Example of Moiré pattern
The localization method is a method for generating super-resolution images. It involves randomly illuminating fluorescent molecules and acquiring their centroid positions at the single-molecule level. By overlaying and reconstructing these centroid positions, super-resolution images are obtained.
In conventional optical microscopes, when fluorescent molecules are closer together than the microscope’s resolution limit, their emitted light appears overlapped due to diffraction, making it impossible to distinguish them. However, if we can observe closely spaced molecules individually, we can determine their coordinates within the sample by calculating the centroid positions of their emission points.
In the localization method, the sample is labeled with special fluorescent molecules capable of switching between ON and OFF states. These molecules are randomly activated to emit fluorescence while being repeatedly captured by a camera. The centroid positions of the emission points are then determined from the acquired images, and by performing computational operations on these images, a single super-resolution image is generated (see Figure 2).
There are two approaches within the localization method: PALM (photoactivated localization microscopy) and STORM (stochastic optical reconstruction microscopy). PALM uses fluorescent proteins as labels, while STORM employs synthetic dyes.
Figure 2: Process of super-resolution image generation using localization method
STED (stimulated emission depletion) is a method to obtain super-resolution images by combining two lasers: one for exciting fluorescent molecules and another for deactivating them.
When using lasers to excite fluorescent molecules, the spot size of the laser is determined by its wavelength. If you want to observe fluorescent molecules smaller than the laser spot size, it is necessary to reduce this spot size. However, since the laser spot size is determined by the wavelength, it cannot be physically made smaller (see Figure 3, top).
In STED microscopy, a doughnut-shaped STED light is applied to the fluorescent molecules excited by the excitation laser. This selectively de-excites the excitation light in the peripheral region of the laser spot, allowing only the small central area to emit fluorescence (see Figure 3, bottom). As a result, it becomes possible to detect only the fluorescence within the laser spot size, improving resolution.
While STED microscopy can enhance the XY resolution in the two-dimensional plane using the doughnut-shaped STED light, it does not improve resolution in the Z direction.
Figure 3: Differences between conventional laser microscopy and STED (stimulated emission depletion) method
Method | Advantages | Disadvantages |
---|---|---|
SIM |
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PALM/STORM |
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STED |
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The ORCA-Quest 2 qCMOS camera, with its small pixel size, allows us to obtain higher-resolution images compared to the ORCA-Fusion Digital CMOS camera.
Data courtesy of Steven Coleman (Visitech international Ltd.)
Time lapse super-resolution image of HeLa cells (localization method)
Time lapse STORM image of African green monkey kidney cells (BSC-1)
The ORCA-Quest 2 qCMOS camera achieves the ultimate in quantitative imaging with extremely low noise performance. Its 0.3 electrons rms readout noise enables photon counting. Compared to a traditional qCMOS camera, the ORCA-Quest 2 qCMOS camera achieves faster readout speeds in extremely low noise scan modes and improved sensitivity in the ultraviolet region.
In super-resolution microscopy using localization methods, a high-sensitivity camera is used as the detector. To improve the accuracy of determining the centroid position of fluorescent signals for super-resolution image generation, it is crucial to have a small pixel size and minimal noise. The ORCA-Quest 2 qCMOS camera, with its small pixel size and exceptionally low readout noise, is an optimal choice for cameras used in localization methods.
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