Label-Free Super-Resolution Imaging Techniques

Abstract

Biological and material samples contain nanoscale heterogeneities that are unresolvable with conventional microscopy techniques. Super-resolution fluorescence methods can break the optical diffraction limit to observe these features, but they require samples to be fluorescently labeled. Over the past decade, progress has been made toward developing super-resolution techniques that do not require the use of labels. These label-free techniques span a variety of different approaches, including structured illumination, transient absorption, infrared absorption, and coherent Raman spectroscopies. Many draw inspiration from widely successful fluorescence-based techniques such as stimulated emission depletion (STED) microscopy, photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM). In this review, we discuss the progress made in these fields along with the current challenges and prospects in reaching resolutions comparable to those achieved with fluorescence-based methods.

Keywords: chemical imaging; coherent Raman imaging; label-free; photothermal infrared; subdiffraction; super-resolution Raman microscopy.

Figure 1.. Super-resolution strategies and the importance of nanoscale imaging.

A. Photo-localization methods use photo-switchable tags to build a super-resolution image over time. B. Spatially-patterned methods use a depletion beam so that signal is limited to a subdiffraction-limited region. C. Tip-based methods use a physical tip only tens of nanometers in width so that signal detected is limited to a sub-diffraction-limited region. D. A typical membrane measured with a diffraction-limited technique gives an ensemble picture, while sub-diffraction-limited measurements localize heterogeneous components such as proteins, lipids, and cholesterol, as shown by their respective Raman signatures.

Figure 2.. Current resolution improvements and best resolution reported for super-resolution imaging techniques.

The size of each point represents the resolution fold improvement over the diffraction-limited version of the same technique. The striped ovals indicate near-field methods and the gradient ovals are far-field methods. Numbers refer to the reference number (, , , , , , , –56).

Figure 3.. One super-resolution strategy for transient absorption microscopy (TAM): demodulated pump-probe microscopy (PPM).

A. An energy level diagram for TAM. B. An image of single-walled carbon nanotubes, diffraction-limited pump-probe (left), and sub-diffraction-limited demodulated pump-probe (DPPM, right), with insets demonstrating the image improvement with DPPM. Panel B adapted with permission from Reference ; copyright 2021 American Chemical Society. C. The pulse trains (left) and beam diagrams (right) for DPPM.

Figure 4.. Super-resolution strategies for infrared spectroscopy.

A. A relative size comparison of the diffraction-limited focal spot for infrared (6 μm) and visible (532 nm) light for the same 0.8 NA objective. B. A super-resolution infrared image of polystyrene beads using photothermal IR (PTIR, inset) and the spectrum at one location. Panel B adapted with permission from Reference ; copyright 2017 American Chemical Society. C. An energy level diagram for fluorescence encoded IR spectroscopy (FEIR).

Figure 5.. Super-resolution strategies for Raman spectroscopy.

A. Higher-order coherent anti-stokes Raman spectroscopy (HO-CARS) uses eight-wave-mixing (EWM) instead of four-wave-mixing to improve resolution. CARS image of 1,4-diphenylbuta-1,3-diyne crystal in water (left), and EWM-CARS image of the same crystal (right). Panel A adapted with permission from Reference ; copyright 2020 Springer Nature. B. Femtosecond saturated stimulated Raman spectroscopy (SRS) uses an intense depletion beam offset from the Stokes beam to saturate a Raman transition and improve resolution. Edge profile of potassium yttrium tungsten (KYW) crystal using SRS and femtosecond saturated SRS demonstrating resolution improvement with depletion on. Panel B adapted with permission from Reference ; copyright 2019 AIP Publishing. C. Competing coherent Raman modes uses a strong depletion beam to drive an SRS process, resulting in a CARS process signal that only occurs in a super-resolution region. The power dependence shows the CARS signal and suppression efficiency as a function of depletion power. Panel C adapted with permission from Reference ; copyright 2019 OSA Publishing. D. Femtosecond vibrational coherence depletion spatially depletes coherence similar to STED, resulting in SRS signal that only occurs in a super-resolution region. Nonlinear dependence of Raman gain with depletion power for cyclohexane (left), and edge profile of diamond plate demonstrating resolution improvement with depletion on (right). Panel D (left) adapted with permission from Reference ; copyright 2016 American Chemical Society. Panel D (right) adapted with permission from Reference ; copyright 2020 John Wiley and Sons. E. SREF image without (left) and with (right) a STED beam to improve resolution. Panel E adapted with permission from Reference ; (CC BY 4.0).