Miniaturized Chemical Tags for Optical Imaging

Abstract

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g., single aryl fluorophores, Raman-active triple bonds and isotopes) and embed them into building blocks (e.g., amino acids, nucleobases, sugars) to construct native-like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging.

Keywords: Bioconjugates; Fluorophores; Probes; Raman Spectroscopy.

Figure 1

Miniaturized chemical labels for optical imaging. Representative chemical structures of miniaturized tags for imaging four major targets, namely amino acids, nucleic acids, metabolites and subcellular organelles. Examples include fluorescent amino acids and nucleobases as well as Raman‐active molecules for metabolite and organelle imaging. Inset: Representative chemical structures of small fluorophores with emission in the UV/Visible spectrum. Copyrights (2011–2021) from American Chemical Society, Wiley‐VCH, The Royal Society of Chemistry and Springer Nature under a creative commons license.

Figure 2

Small and tunable organic fluorophores. A) Structures of single benzene carbocycle (top) and benzodiazole (bottom) fluorophores spanning blue to NIR regions of the visible spectrum. B) Dual‐site coumarin probe for the simultaneous detection of cysteine and its metabolite SO₂ and confocal microscopy fluorescence images of zebrafish upon incubation with the probe. Reproduced with permission. ^[9d] Copyright 2017 American Chemical Society. C) Structure of C₃‐NBD ceramide and confocal microscopy fluorescence images of A549 cells upon incubation with the probe (red), LysoTracker (magenta) and ER (endoplasmic reticulum) Tracker Green (green). Reproduced with permission. ^[15a] Copyright 2019 Wiley‐VCH. D) Chemical structure of the mitochondria‐targeting probe mitoSBF and colocalization with Mitotracker Green (green) in HepG2 cells. Reproduced with permission. Copyright 2019 The Royal Society of Chemistry.

Figure 3

Miniaturized fluorescent nucleobases. A) Chemical structures of native nucleobases and representative analogues with their corresponding absorbance–emission wavelengths. B) Time‐lapse imaging of mitosis in U2‐OS cells after incorporation of the dCmBdp nucleotide. Scale bar: 20 μm. Adapted with permission. ^[33a] Copyright 2018 American Chemical Society. C) Fluorescence spectra of single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA) containing the nucleobase dioxT flanked by the bases AT (excitation: 330 nm). Adapted with permission. Copyright 2019 Wiley‐VCH. D) Simultaneous detection of photoproducts and oxidative DNA damage in HEK (human embryonic kidney) cells exposed to UVC radiation. Top panel: Fluorescence image of DNA labeled with the multilabeling reaction. Bottom panel: Image analysis where green color features DNA molecules, red highlights the photoproduct labels, and blue corresponds to oxidative damage sites. Adapted with permission. Copyright 2019 The Royal Society of Chemistry.

Figure 4

Small fluorescent amino acids for site‐specific peptide labeling. A) Chemical structure of the Trp‐BODIPY‐containing fluorogenic peptide Apo‐15 for in vivo imaging of apoptotic cells (green) in mouse lungs using intravital fluorescence microscopy. Adapted with permission. Copyright 2020 Springer Nature under a creative commons license. B) Antimicrobial peptides including red emitting Phe‐BODIPY amino acids as fluorogenic probes for the detection of fungal pathogens in human urine. Comparative detection of GFP‐labeled Candida albicans in urine samples using the GFP (green) and fluorogenic peptide (red) readouts. Confocal fluorescence microscopy of peptide‐stained Candida albicans cells. Adapted with permission. Copyright 2022 Wiley‐VCH. C) Representative examples of fluorescent amino acids with featured applications and absorbance‐emission wavelengths.

Figure 5

Miniaturized Raman labeling of biometabolites. A) Generic Raman spectrum of mammalian cells highlighting miniaturized Raman labels that produce characteristic peaks in the cell silent region (1800–2600 cm⁻¹). B) Examples of vibrational Raman tags (highlighted in red) for imaging small molecule metabolites and examples of biological applications. C) SRS imaging of 3‐OPG in U‐87MG tumor xenograft tissue. The uptake of 3‐OPG is higher in proliferating areas than in the necrotic areas. Reproduced with permission. Copyright 2015 Wiley‐VCH. D) Real‐time uptake of aminopropargyl sucrose into live BY2 cells using SRS microscopy. Reproduced with permission. Copyright 2021 Wiley‐VCH. E) Three‐color multiplex imaging of EU‐¹³C₂ (RNA), EdU‐¹³C (DNA) and 17‐ODYA (fatty acids) using an alkyne isotopic editing strategy. Reproduced with permission. Copyright 2014 American Chemical Society.

Figure 6

Multiplexed Raman imaging and sensing in mammalian cells. A) Raman spectra of the polyyne “Carbow” collection of multiplexable Raman labels. SRS images of cellular mitochondria, lysosomes and plasma membranes labeled with polyynes containing organelle‐targeting motifs. Reproduced with permission. Copyright 2018 Springer Nature B) 8‐color multiplexed cell imaging using fluorescence and epr‐SRS probes. Fluorescence: NucBlue for nucleus, Alexa‐488‐WGA for plasma membrane (PM), and MitoTracker Orange for mitochondria. SRS: Label‐free detection at 2945 cm⁻¹ for proteins, MARS2238‐Azide with EdU for DNA, MARS2210‐NHS labeled antibody for α‐tubulin, MARS2184‐PEG2‐Alkyne with L‐azidohomoalanine (AHA) for nascent proteins and MARS2155‐NHS labeled antibody for fibrillarin. Reproduced with permission. Copyright 2021 Springer Nature under a creative commons license. C) Ratiometric SRS imaging of mitochondrial pH using Mitokyne. SRS images acquired at 2230 cm⁻¹ (green, protonated Mitokyne) and 2216 cm⁻¹ (cyan, deprotonated Mitokyne). The ratio 2230 cm⁻¹/2216 cm⁻¹ is featured. Reproduced with permission. Copyright 2021 American Chemical Society under a creative commons license.

Figure 7

Activatable Raman probes for smart sensing. A) Structures and reactions of alkyne precursors photo‐DIBO, 1‐cyclo, 2‐cyclo and 3‐cyclo as model systems for UV‐activated alkyne generation. Spontaneous Raman and hyperspectral SRS spectra of precursors and products (10 mM). 3‐cyclo (5) did not produce an activatable signal following UV irradiation, and the hyperspectral SRS spectra of (5) and (6) were shown to overlap. Reproduced with permission. Copyright 2022 American Chemical Society. B) Activatable Raman probes based on isotope edited xanthenes for multiple enzyme targets: gGlu‐9CN‐JCP (red) for γ‐glutamyl transpeptidase (GGT), Leu‐9C¹⁵N‐JCP (green) for leucine aminopeptidase (LAP), EP‐9¹³CN‐JCP (blue) for dipeptidyl peptidase‐4 (DPP‐4) and β‐Gal‐9¹³C¹⁵N‐JCP (yellow) for β‐galactosidase (βGal). Simultaneous detection of enzyme activities in live A549 cells in epr‐SRS images (where the background image at 2250 cm⁻¹ was subtracted in each case), together with the normalized SRS spectra of all 4 probes. Reproduced with permission under ACS Editors’ Choice. Copyright 2020 American Chemical Society.