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Super-resolution microscopy
What is super-resolution microscopy?
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.
SIM: Structured illumination microscopy
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
PALM/STORM: Localization method
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
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
Comparison of the methods
| Method | Advantages | Disadvantages |
|---|---|---|
| SIM |
|
|
| PALM/STORM |
|
|
| STED |
|
|
Example images
Super-resolution images using SIM
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.
Super-resolution image obtained with ORCA-Quest qCMOS camera
qCMOS camera / 4.6 μm pixel size / Super-resolution system: VT-iSIM
Super-resolution image obtained with ORCA-Fusion Digital CMOS camera
Gen Ⅲ sCMOS camera / 6.5 μm pixel size / Super-resolution system: VT-iSIM
Data courtesy of Steven Coleman (Visitech international Ltd.)
Super-resolution images using localization method
Time lapse super-resolution image of HeLa cells (localization method)
Time lapse STORM image of African green monkey kidney cells (BSC-1)
Recommended products
Regardless of technique, to image below the limit of optical resolution requires precision in the optical system and the use of computational methods to extract the super-resolved image. When errors enter the optical system and/or noise dominates the signal in the image detection, these methods underperform, given less certainty in the quality of the super resolution data. For this reason, the ORCA-Quest2, our flagship quantitative CMOS camera, is the camera of choice. The ultra-quiet noise performance of the ORCA-Quest2 is not just a number on a data sheet. In these experiments this performance reduces errors in the raw image data, leading to more confidence, better information and prettier pictures in the computational results.
The ultimate camera, evolved
With 5x faster photon number resolving capabilities, the ORCA-Quest 2 remains the apex camera for ultra low-light, quantitative imaging.
Highlights:
- Ideal for ultra low light and replacing EM-CCDS
- Exclusive quantitative CMOS for pioneering imaging
- Ultra-low noise enables single photon resolution
- High sensitivity does not require speed compromise
This ORCA is ideal for:
- TIRF
- Computational/super-resolution microscopy
- Genetically encoded voltage imaging
- Luminescence
- Quantum computing
- UV applications
Small pixels, big benefit
The ORCA-Fire is a unique back-thinned sCMOS that is optimized for fast, low-mag, large field of view, low-light quantitative imaging.
Highlights:
- Ideal for low light with high pixel volume and speed
- Nyquist sampling at 40x and below
- High sensitivity even at fast speeds
- Advanced bidirectional lightsheet readout modes
This ORCA is ideal for:
- Lightsheet
- Simultaneous multi-wavelength imaging
- High-throughput widefield fluorescence
- Genetically encoded voltage imaging
- Tissue mapping
Uncompromising performance
The ORCA-FusionBT sCMOS camera is the perfect synthesis of sensitivity, speed, resoution and overall quantiative, low-light performance.
Highlights:
- Ideal for very low light applications at 60x and 100x
- Superior SNR from maximized QE, minimized noise
- Three speed/noise modes for use-specific imaging
- Exclusive, high QE, back-thinned sCMOS sensor
This ORCA is ideal for:
- Spinning disk confocal
- Lightsheet
- Optogenetics
- Structured illumination microscopy
- Single-molecule localization microscopy
Our photomultiplier tubes (PMTs) empower super-resolution microscopy by delivering unparalleled sensitivity, wide dynamic range and low-noise performance, leading to higher image contrast. Their fast time response allows researchers to image faster, enabling precise analysis of real time processes. By delivering higher sensitivity and faster imaging speeds, Hamamatsu’s PMTs enhance the contrast and speed of data measurements, making them ideal for advanced biomedical research and pushing the boundaries of what's achievable in super-resolution microscopy.
The LCOS-SLM (Liquid Crystal on Silicon - Spatial Light Modulator) is a device that allows electrical control of the phase of laser light. It consists of a structure where a liquid crystal is sandwiched between a CMOS chip with pixel electrodes arranged in a two-dimensional pattern and transparent electrodes deposited on a glass substrate. Digital images output from a PC are converted to analog signals by a dedicated driving circuit and applied with voltage to the pixel electrodes on the CMOS chip.
The LCOS-SLM takes standard laser light, and shapes it into precise patterns of excitation light, crucial for reconstructing high-resolution images, and handle multiple wavelengths for multicolor imaging. The LCOS-SLM optimizes contrast and image clarity, essential for revealing intricate details at the nanoscale. With exceptional phase stability and flexibility, Hamamatsu's LCOS-SLM empowers researchers to push the limits of spatial resolution, facilitating discoveries in cell biology and molecular structures that drive innovation in life sciences and medical research.
Supercontinuum white light lasers, such as the SuperK series from NKT Photonics, are ideal for super-resolution microscopy due to their broadband tunability, high spectral power density (SPD), pulsed nature, and single-mode output. These lasers provide bright, diffraction-limited light across a wide spectrum, enabling precise and efficient localization of fluorescent molecules. The pulsed and diffraction-limited nature of the SuperK lasers allows for high temporal and spatial resolution, making them perfect for advanced microscopy techniques.
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