A Comprehensive Guide to Super Resolution Microscopy Techniques

Super resolution microscopy techniques have revolutionized the field of biological imaging by allowing researchers to visualize structures and processes at resolutions beyond the diffraction limit of conventional light microscopy. These techniques can be broadly categorized into two groups: super-resolved ensemble microscopy techniques and super-resolved single fluorophore microscopy techniques.

Super-Resolved Ensemble Microscopy Techniques

Stimulated Emission Depletion (STED) Microscopy

STED microscopy is a super-resolved ensemble microscopy technique that uses a doughnut-shaped beam of light to selectively deplete the excited state of fluorophores, resulting in a smaller effective excitation volume and higher resolution. The resolution of STED microscopy is determined by the intensity of the depletion beam, which can be described by the following equation:

Resolution = λ / (2n sin(α) √(1 + I_depletion / I_saturation))

Where:
– λ is the wavelength of the excitation light
– n is the refractive index of the sample
– α is the numerical aperture of the objective lens
– I_depletion is the intensity of the depletion beam
– I_saturation is the saturation intensity of the fluorophore

The typical resolution achievable with STED microscopy ranges from 20 to 50 nanometers, depending on the specific experimental conditions and the choice of fluorophore.

Structured Illumination Microscopy (SIM)

Structured Illumination Microscopy (SIM) is another super-resolved ensemble microscopy technique that uses patterned illumination to create moiré fringes, which allow for the resolution of finer details. The resolution of SIM is determined by the spatial frequency of the illumination pattern, which can be described by the following equation:

Resolution = λ / (2n sin(α) √(1 + 2 cos(θ)))

Where:
– λ is the wavelength of the excitation light
– n is the refractive index of the sample
– α is the numerical aperture of the objective lens
– θ is the angle between the illumination pattern and the sample

The typical resolution achievable with SIM ranges from 80 to 100 nanometers, depending on the specific experimental conditions and the choice of fluorophore.

Super-Resolved Single Fluorophore Microscopy Techniques

Single-Molecule Localization Microscopy (SMLM)

Single-Molecule Localization Microscopy (SMLM) techniques, such as direct stochastic optical reconstruction microscopy (dSTORM) and photoactivated localization microscopy (PALM), rely on the precise localization of individual fluorescent molecules to build up an overall structure. The localization precision in SMLM techniques can be calculated using the Cramér-Rao lower bound (CRLB) formula, which is given by:

Localization precision = √(σ^2 + a^2 / (12N) + 8πσ^4b^2 / (a^2N^2))

Where:
– σ is the standard deviation of the Gaussian point spread function (PSF)
– a is the pixel size
– N is the number of photons detected from the fluorophore
– b is the background noise level

The typical localization precision achievable with SMLM techniques ranges from 10 to 20 nanometers, depending on the specific experimental conditions and the choice of fluorophore.

DNA-PAINT

DNA-PAINT is another SMLM technique that uses transient binding of fluorescently labeled oligonucleotides to specific target sites to achieve high-resolution imaging. The resolution in DNA-PAINT is determined by the binding kinetics of the oligonucleotides, which can be described by the following equation:

Resolution = √(D * t_bind / N)

Where:
– D is the diffusion coefficient of the oligonucleotides
– t_bind is the binding time of the oligonucleotides
– N is the number of binding events

The typical resolution achievable with DNA-PAINT ranges from 10 to 20 nanometers, depending on the specific experimental conditions and the choice of oligonucleotides.

Emerging Super Resolution Microscopy Techniques

Expansion Microscopy (ExM)

Expansion Microscopy (ExM) is an emerging super resolution microscopy technique that involves physically expanding the sample to increase the effective resolution. The resolution in ExM is determined by the expansion factor, which can be described by the following equation:

Resolution = Original resolution / Expansion factor

The typical expansion factor in ExM ranges from 4 to 10, resulting in a resolution improvement of 4 to 10 times compared to the original sample.

MINFLUX

MINFLUX is another emerging super resolution microscopy technique that uses a feedback loop to precisely localize individual fluorophores, allowing for even higher resolution imaging. The resolution in MINFLUX is determined by the intensity of the excitation beam, which can be described by the following equation:

Resolution = λ / (2n sin(α) √(1 + I_excitation / I_saturation))

Where:
– λ is the wavelength of the excitation light
– n is the refractive index of the sample
– α is the numerical aperture of the objective lens
– I_excitation is the intensity of the excitation beam
– I_saturation is the saturation intensity of the fluorophore

The typical resolution achievable with MINFLUX ranges from 1 to 10 nanometers, depending on the specific experimental conditions and the choice of fluorophore.

Quantifying Super Resolution Microscopy Performance

To assess the performance of super resolution microscopy techniques, it is important to consider various quantifiable metrics, such as the localization precision, the signal-to-noise ratio (SNR), and the resolution.

The SNR can be calculated as the ratio of the signal intensity to the standard deviation of the background noise, and is often used to assess the quality of the image. The resolution can be estimated using the full-width at half-maximum (FWHM) of the point spread function (PSF), which describes the spatial distribution of the fluorescence signal from a point source.

For example, in a STED microscopy experiment, the SNR can be calculated as:

SNR = Signal intensity / Standard deviation of background noise

And the resolution can be estimated using the FWHM of the PSF:

Resolution = FWHM of PSF

By considering these quantifiable metrics, researchers can better understand the performance of super resolution microscopy techniques and make informed decisions about the most appropriate technique for their specific research needs.

References:
– A guide to choosing the right super-resolution microscopy technique. Journal of Biological Chemistry. 2021.
– Super-resolution Microscopy. Feinberg School of Medicine, Northwestern University.
– Getting started with Super-Resolution Microscopy. ONI Bio. 2023.
– Quantitatively mapping local quality of super-resolution microscopy using rolling Fourier ring correlation. Nature Communications. 2023.
– Stimulated Emission Depletion (STED) Microscopy. Leica Microsystems.
– Structured Illumination Microscopy (SIM). Nikon Instruments.
– Single-Molecule Localization Microscopy (SMLM). Olympus Life Science.
– DNA-PAINT: A Powerful Tool for Super-Resolution Microscopy. Molecular Probes.
– Expansion Microscopy (ExM). Caltech.
– MINFLUX: A New Approach to Super-Resolution Microscopy. Max Planck Institute.