Advanced Microscopy Techniques for Molecular Biophysics
Abstract
:1. Introduction
2. Microspectrophotometry
2.1. General Description
2.2. Theory
- (a)
- SR, the spectral radiance of the lamp, expressed in W cm−2 sr−1 nm−1, i.e., the radiant flux per unit area, solid angle and spectral bandwidth;
- (b)
- SB, the spectral bandwidth, expressed in nm, for each measuring wavelength;
- (c)
- OF, the optical flux of the optical system, i.e., the ability of the optical system to transfer light energy. OF is expressed in cm² sr and is a purely geometric quantity applied to the volume through which the light is transferred. In wide-field microscopy (WFM) the OF is about 10−3 cm2 sr, and the smallest optical flux giving a reliable measurement value is about 10−8 cm2 sr.
- (d)
- TR, the total transmittance of the optical system, is a measure of the percentage of light that remains after loss by absorption, reflection and diffraction of optical components (i.e., lens, mirrors, ...);
- (e)
- ILS, the interactions of the light with the sample, is related to the absorption cross section of absorbing molecules and their number, and it is a measure of the percentage of light that remains after the sample absorption.
2.3. A Working Set-Up
2.4. Experimental Example 1: MSP on Retinal and Extra-Retinal Photoreceptors of Teleost Fish
2.5. Experimental Example 2: Which Is the Prey?
3. Super-Resolution Localization Microscopy
3.1. General Description
3.2. Theory
3.3. A Working Set-Up
3.4. Experimental Example 1: Single-Molecule Tracking and Imaging
3.5. Experimental Example 2: STED or Confocal Microscopy, Which Is the Best?
4. Holotomography
4.1. General Description
4.2. Theory
4.3. A Working Set-Up
4.4. Experimental Example 1: Quantitative Measurement of Lipid Contents in Individual Microalgal Cells
4.5. Experimental Example 2: The 3D Structure of Euglena Gracilis Photoreceptor
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Lateral Resolution (nm) | Axial Resolution (nm) | Methods of Illumination | Methods of Image Generation | Fluorescent Probe | Five Aspects of Microscopy | Merits | Limitations | |
---|---|---|---|---|---|---|---|---|
WFM | 250 | 700 | Lamp | Direct visualization | Conventional | Perfect for living cell Very easy to use | Low axial and lateral resolution | |
CLSM | 180–250 | 500 | Pinhole | Scanning excitation beam | Conventional | High axial resolution 3D imaging capability Reduced photobleaching | Limited field of view Slow processing | |
2D-STED | 40–60 | 600 | Hardware shaped excitation beam | Scanning excitation beam | Photo switchable | Resolution directly improved with PSF to ~50 nm 2 colour imaging (more if combined with other advanced techniques) No post-processing Lateral resolution 20–70 nm 20 micron depth penetration | Photobleaching, Phototoxic Requires very stable dyes Difficult for live cells Requires complex alignment Low dynamic range | |
SIM | 100–125 | 2D 500–700 3D 250–350 | Patterned wide field | Multiple images combining Fourier space | Conventional | Live cell imaging Multicolour imaging No special sample preparation Simple to use Fast | Require sensitive cameras Photobleaching/toxicity Subject to artifacts Limited sample thickness | |
PALM/STORM | 10–50 | 500–700 | Stochastic fluorophore excitation | Multiple single molecules frames | Photoactivable | Single molecule sensitivity Highest potential resolution Multichannel imaging Low illumination power | Specific fluorophores required Shallow depth-limited Long acquisition times Impractical for live cell |
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Barsanti, L.; Birindelli, L.; Sbrana, F.; Lombardi, G.; Gualtieri, P. Advanced Microscopy Techniques for Molecular Biophysics. Int. J. Mol. Sci. 2023, 24, 9973. https://doi.org/10.3390/ijms24129973
Barsanti L, Birindelli L, Sbrana F, Lombardi G, Gualtieri P. Advanced Microscopy Techniques for Molecular Biophysics. International Journal of Molecular Sciences. 2023; 24(12):9973. https://doi.org/10.3390/ijms24129973
Chicago/Turabian StyleBarsanti, Laura, Lorenzo Birindelli, Francesca Sbrana, Giovanni Lombardi, and Paolo Gualtieri. 2023. "Advanced Microscopy Techniques for Molecular Biophysics" International Journal of Molecular Sciences 24, no. 12: 9973. https://doi.org/10.3390/ijms24129973