Trends in Cell Biology
ReviewSpecial Issue – Imaging Cell BiologyPhotoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging
Introduction
By allowing the visualization of cells and their constituents, genetically-encoded fusions of fluorescent proteins (FPs) have provided an important tool for dissecting the molecular control of cellular processes within cells, spurring a revolution in biology [1]. The advent of PA-FPs 2, 3, 4 offers new capabilities, such as pulse-chase labeling and single molecule localizations. These capabilities are achieved because PA-FPs undergo pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. That is, activated molecules switched on by photoactivation yield bright signals over dark backgrounds. This enables the spatial and temporal marking of specific structures and tracking of their signal in time over a “dark” background. Because PA-FPs are ‘switched on’, measurements are not impacted by freshly synthesized molecules that become fluorescent, as occurs in imaging with conventional FPs.
The optical highlighting capability of PA-FPs, together with their ability to be expressed as fusion proteins that retain complex biological functions, has led to the development of diverse and novel imaging strategies. For example, pulse-chase labeling of cellular compartments using PA-FPs has helped clarify protein transport pathways and the intricate connections between compartments. Furthermore, by summing the locations of single molecules determined by the optical on/off switching of sparse subsets of PA-FPs and determining the centroids of their fluorescence emission, a final super-resolution image can be obtained, revealing the complex distributions of molecules within subcellular structures with nanometer precision. The full potential of PA-FPs in conventional, diffraction-limited and super-resolution imaging is only beginning to be realized. Here, we discuss the diverse array of PA-FPs available to researchers and the new imaging techniques they make possible for unraveling long-standing biological questions.
Section snippets
Palette of PA-FPs
Over 20 different varieties of PA-FPs have so far been described. As discussed below, these fall into three broad classes based on whether, in response to an activating light pulse, the PA-FP i) irreversibly switches from a dark-to-bright fluorescent state, ii) irreversibly photoconverts from one fluorescent color to another or iii) reversibly photoconverts, enabling on/off switching capability. Within these classes, individual PA-FPs can be further differentiated based on particular optical
Imaging with PA-FPs
Photoactivation usually requires a separate excitation source than the one used for imaging (with ∼400 nm irradiation required in most cases). After photoactivation, imaging is similar to that used for conventional FPs. An ideal PA-FP should be readily photoactivatable/photoconvertible to generate a high level of contrast. Nevertheless, the particular features of PA-FPs make them preferable for specific types of applications. For example, in experiments using a PA-FP-tagged fusion protein,
Tracking cellular compartments and structures
Most biological structures within cells are part of larger steady-state systems, in which input and outflow pathways continuously circulate components. Insight into these pathways, a requirement for understanding how the structure can maintain itself, is often impossible using conventional FP imaging. This is because the ensemble of visible FP fusion proteins in a compartment is at steady state, with signal gain balanced by signal loss. PA-FPs offer a powerful approach for dissecting protein
PA-FP applications in single molecule super-resolution imaging
PA-FPs have become valuable tools in new super-resolution microscopy techniques that overcome the diffraction barrier 17, 48, 49. These techniques permit biologists to visualize the structures and processes of the cell at the molecular level. Key among these super-resolution techniques employing PA-FPs are photoactivated localization microscopy (PALM) [17] and fluorescence photoactivation localization microscopy (F-PALM) [48], herein both referred to as PALM. Another variation of a molecular
Concluding remarks
PA-FPs and their associated emerging techniques are heralding a new era in cell biology. These genetically expressed FPs permit the non-perturbative optical imaging of dynamic processes in living cells. For a cell biologist interested in using photoactivation or even super-resolution experiments, the large number of possible probes can seem overwhelming. Many PA-FPs have contributed to the cell biology community over several years. Many of these have been cited throughout this review and
Glossary
- Diffraction barrier
- Limits resolution when imaging with a conventional fluorescence microscope to approximately 0.61 of the wavelength of light divided by the numerical aperture of the objective lens.
- Point spread function (PSF)
- This defines the blurry, diffraction-limited spot given off by a single point source light emitter (i.e. PA-FP) when imaged through an objective lens. When the shape of the PSF is measured, its centroid can be determined with high precision providing ‘super-resolution
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2021, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :Photo-switching occurs when fluorophores undergo a change in their spectral properties in response to irradiation with light of a specific wavelength and intensity [33–35]. Photo-switchable FPs are categorized into three broad classes: irreversibly photo-switchable from a dark to a bright state (i.e., from non-fluorescent to fluorescent), irreversibly photo-switchable from one emission wavelength to another, and reversibly photo-switchable enabling bright-to-dark switching capability [34,36]. In the case of irreversibly photo-switching from a dark to a bright state, the peak wavelength of the excitation spectrum changes and, sometimes, can shift to a different excitation wavelength as the chromophore changes from the neutral (protonated) state to anionic (deprotonated) state in response to the irradiation of light of certain wavelength and intensity [36,37].