Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores

It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP

Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8

Atomically Resolved Observation of Continuous Interfaces between As-grown MoS2 Monolayer and WS2/MoS2 Heterobilayer on SiO2

Van der Waals (vdW) heterostructures synthesized through the chemical vapor deposition (CVD) method allow creation and tuning of intriguing electronic and optical properties of two- dimensional (2D) materials, the knowledge of which is critical for a wide range of potential applications. Here we report our scanning tunneling microscopy/spectroscopy (STM/STS) study of as-grown MoS2 monolayer and WS2/MoS2 heterobilayer on SiO2. The heterobilayer appears smoother than the MoS2 monolayer, with root mean square (RMS) roughness of 0.230 +- 0.021 nm in the former and 0.329 +- 0.033 nm in the latter. For the first time, to our knowledge, we directly observed a continuous interface between the MoS2 monolayer and the top layer of the heterobilayer with atomic resolution. This finding contrasts to the previously reported open edges in the top layer of the heterobilayer. Our STS results and density functional theory (DFT) calculations revealed the band gaps of the heterobilayer and the MoS2 monolayer

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer

Vibrational Imaging of Glucose Uptake Activity in Live Cells and Tissues by Stimulated Raman Scattering

Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution

Live-Cell Bioorthogonal Chemical Imaging: Stimulated Raman Scattering Microscopy of Vibrational Probes

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of “omics”. As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenografts, and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells

Super-multiplex vibrational imaging

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure–function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a ‘colour barrier’, owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems

Live-cell imaging of alkyne-tagged small biomolecules by stimulated Raman scattering

Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors