Coloring Cell Complexity: The Case for an Expansive Fluorophore Palette.

It is reasonable to wonder: do we need more fluorophores for imaging cells? As demonstrated by Lavis and colleagues in this issue of ACS Central Science, custom-designed fluorophores can offer improved spectral properties and enhanced functionality.[1] Their work describes the rational design of a new fluorogenic, green-emitting rhodamine for high-resolution fluorescence microscopy. In this essay, I will put the Lavis work into context, while making the argument that an expansive color palette enables researchers to investigate and make fundamental discoveries on the molecular complexity contained in living cells.

This is an exciting era for studying living systems, with modern microscopes facilitating the precision mapping of molecules inside living cells. Micrographs expose hidden details including cell and nuclear boundaries, structural elements, various organelles, and protein localization. Live cell imaging further reveals protein trafficking, binding interactions, signaling cascades, enzyme activity, cell division, and even cell death. However, these molecular features and dynamic events would be largely invisible without fluorescent molecules to highlight them.

Chemists play a central role in creating the fluorescent molecules used to light up cell biology. The most useful fluorophores for imaging are bright, photostable, and compatible with common imaging systems. Additionally, the fluorophore should offer a high signal-to-noise ratio, which can be imparted by fluorogenicity. New fluorophores are often derived from scaffolds first described over 100 years ago, such as rhodamine. For example, synthetic chemists recently developed novel, far-red emitting rhodamines with superior brightness by substituting the xanthene’s bridging oxygen with silicon[2] or phosphorus.[3] Another approach, pioneered by Lavis’s group, is to replace the amines with azetidines—a small change that can improve the quantum yield.[4]

Rhodamines exist in equilibrium between a nonfluorescent lactone and a fluorescent zwitterion; tuning this equilibrium can make them fluorogenic. In this issue, Lavis and co-workers describe a new approach for rationally designing fluorogenic rhodamines.[1] They initiated this work by analyzing a set of rhodamines and determining that fluorogenicity is inversely related to the lactone-zwitterion equilibrium constant (KL-Z). Their data support a mechanism in which the rhodamine adopts a colorless lactone form in solution but favors the fluorescent zwitterionic form upon binding a macromolecular target. Their findings suggest that fluorogenicity is not a result of rhodamine aggregation, as previously thought.[2b]

Armed with this insight, Lavis and co-workers rationally designed new probes by systematically modifying rhodamine. First, they synthesized a new fluorinated rhodamine probe called Janelia Fluor 552 (JF 552). JF 552 has favorable spectral properties (λabs/λemit = 552 nm/575 nm), high quantum yield (Φ = 0.83), and good cell permeability. However, JF 552 was only modestly fluorogenic due to its KL-Z = 0.70. To fine-tune the KL-Z to favor the lactone, they combined features of JF 552 with JF 5254b (KL-Z = 0.068) to generate a new fluorogenic, green-emitting probe that they named Janelia Fluor 526 (JF 526). This rhodamine favors the nonfluorescent lactone (KL-Z = 0.0050), while retaining a high quantum yield (0.87) and extinction coefficient (ε = 118 000 M–1 cm–1) in the zwitterionic form (Figure ).

Figure 1

Lactone-zwitterion equilibrium of Janelia Fluor 526 (JF 526). Lavis and co-workers report that the equilibrium constant (KL-Z) is sufficient to predict the fluorogenicity of rhodamines, such as the JF 526. Upon binding to a biomolecular target, JF 526 preferentially adopts the fluorescent zwitterionic form.

The fluorophores in the current Lavis work have an absorbance maxima between 526 nm (JF 526) and 646 nm (JF 646). The spectral diversity of the new rhodamines is an important part of their appeal.

Combining spectrally distinct fluorophores enables researchers to acquire informative micrographs and make new discoveries. For example, the human protein atlas was created from thousands of four-color micrographs (www.proteinatlas.org).[5] Green was reserved for immunolabeling each protein one-at-a-time, while three distinct colors were dedicated to highlighting cell landmarks: the nucleus (blue), microtubules (orange), and endoplasmic reticulum (near-infrared). Using this approach, Lundberg and colleagues mapped 12 000 human proteins to 30 subcellular structures.[5] Imagine if the micrographs had included five or six colors each! This has become feasible as bright, spectrally distinct probes get added to the fluorophore palette.

Fluorogenic probes are useful for live cell imaging because they have a high signal-to-noise ratio. Lavis and co-workers demonstrated this feature using three fluorogenic rhodamines (Figure ). For three-color imaging, JF 526-pepstatin A highlighted lysosomes, SiR-Taxol labeled microtubules, and a JF 585 HaloTag ligand was used to label histone H2B in the nucleus. This is a high impact result—it is unprecedented for three fluorogenic rhodamines to be used at once for no-wash, live cell imaging.

Figure 2

Fluorogenic rhodamines enable no-wash, live cell imaging. (A) Structures of rhodamine ligands. (B) Fluorescence micrograph of a cell treated with three fluorogenic rhodamines, shown in panel A, to highlight the lysosomes (green), nucleus (orange), and microtubules (magenta). Cells were stably transfected to express a histone-H2B-HaloTag fusion protein, which was labeled with JF 585-HaloTag ligand (JF 585-HTL; 100 nM). JF 526-Pepstatin A (2 μM) labeled lysosomes and SiR-Taxol (1 μM) labeled tubulin. Cells were treated simultaneously with all three probes for 1 h and then imaged without washing. Micrograph is adapted from ref (1). Copyright 2019 American Chemical Society.

Super-resolution microscopy (SRM) is a set of imaging methods that enables features of cells to be observed with nanoscale resolution. While there are many fluorophores compatible with SRM, few were specifically designed for it. Chemists are actively contributing to the development of bright fluorophores for SRM. This topic was recently reviewed by Li and Vaughan.[6] Now Lavis and co-workers demonstrate that JF 526’s brightness and fluorogenicity make it a good probe for structured illumination microscopy (SIM) and stimulated emission depletion (STED) microscopy. However, JF 526 required a small modification to encourage photoswitching or “blinking”. Their new hydroxymethyl JF 526 spontaneously blinks, enabling single-molecule localization microscopy, a type of SRM, without the need for special buffers or photoactivation.[1]

In summary, Lavis and co-workers have demonstrated the value of expanding the fluorophore palette for coloring in the features of living cells. I will finish by describing three areas where I see opportunities for creating better, brighter fluorophores for multicolor microscopy. First, we need more fluorophores that absorb and emit in the near-infrared (NIR), a spectral region with low cellular absorbance, light scattering, and autofluorescence. The new Nebraska Red probes are a great start.[3] Second, it would be useful to have more fluorophores with a large difference between the absorption and emission maxima (i.e., Stokes shift). Such probes facilitate multicolor imaging of subcellular structures, as beautifully demonstrated by Gibbs and co-workers. They generated a five-color micrograph using a single laser line to excite five novel bodipy dyes with varying Stokes shifts.[7] Third, there is a need for more target-specific, small-molecule stains for organelles, which could replace postfixation immunolabeling. Human cells have over 30 subcellular structures[5]—only a few of these have a corresponding small-molecule stain. Of course, new fluorophores should also be biocompatible, photostable, and reasonably bright (e.g., QY > 0.3). It is time to move past the monotony of red–green–blue to a luminous future full of spectrally diverse fluorophores.