Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems

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Rational Design of Bioavailable Photosensitizers for

Manipulation and Imaging of Biological Systems

Graphical Abstract

Authors

Thomas C. Binns, Anthony X. Ayala,

Jonathan B. Grimm, ...,

S₁

N

O

N⁺

Mark H. Ellisman, Minoru Koyama,

Luke D. Lavis

−

CO₂

S₀

fluorophore

Correspondence

add halogens

O → S

lavisl@janelia.hhmi.org

S₁

I

N

O

N⁺

N

S

N⁺

T₁

In Brief

³O₂

−

Binns et al. describe photosensitizers

based on rhodamine dyes. Incorporation

of halogen or sulfur atoms into the

rhodamine structure substantially

CO₂

S₀

¹O₂

photosensitizer

chromophore-assisted

light-inactivation

targeted

DAB

cell ablation

photopolymerization

increases singlet oxygen quantum yield.

Combined with the HaloTag-labeling

system, these photosensitizers allow

light-mediated destruction of proteins,

ablation of entire cells, or deposition of

electron microscopy contrast agents.

×

Highlights

d

Incorporation of iodine or sulfur into rhodamine dyes

increases ¹O₂quantum yield

Rhodamine photosensitizers are compatible with live-cell-

labeling strategies

Generating ¹O₂allows photoablation of entire cells in culture

or in vivo

Photopolymerization of 3,3⁰-diaminobenzidine gives contrast

in electron microscopy

Binns et al., 2020, Cell Chemical Biology 27, 1–10

September 17, 2020 ª 2020 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2020.07.001

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Rational Design of Bioavailable Photosensitizers

for Manipulation and Imaging of Biological Systems

Thomas C. Binns,^1,2,3Anthony X. Ayala,¹Jonathan B. Grimm,¹Ariana N. Tkachuk,¹Guillaume A. Castillon,⁴

Sebastien Phan,⁴Lixia Zhang,¹Timothy A. Brown,¹Zhe Liu,¹Stephen R. Adams,⁵Mark H. Ellisman,⁴Minoru Koyama,¹

and Luke D. Lavis^1,6,

*

¹Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA

²Graduate School, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

³College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

⁴Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, USA

⁵Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, USA

⁶Lead Contact

*Correspondence: lavisl@janelia.hhmi.org

https://doi.org/10.1016/j.chembiol.2020.07.001

SUMMARY

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological sys-

tems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be

utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited.

Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely

used rhodamine scaffold and demonstrate their efﬁcacy for chromophore-assisted light inactivation, cell

ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These

chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to

high-resolution imaging.

INTRODUCTION

phyrins and phthalocyanines, transition-metal complexes, and

ﬂuorescent dyes appended with heavy atoms (Xiong et al.,

The ability to measure and manipulate biological systems using 2019). Extant compounds possess excellent photophysical

light-driven processes has revolutionized biology. The excited properties but often exhibit limited bioavailability and can be difﬁ-

state that occurs after a molecule absorbs light can be har- cult to target to speciﬁc cells or subcellular regions. Recent work

nessed in different ways. Fluorescent dyes absorb and then on genetically encoded photosensitizers based on ﬂavoproteins

emit light of different wavelengths, allowing sensitive visualiza- has produced useful reagents, such as miniSOG, that can be ex-

tion of biological processes inside cells. Photoactivatable pressed in speciﬁc cells or subcellular regions (Bulina et al., 2006;

(‘‘caged’’) compounds use the excited state energy to break Riani et al., 2018; Ruiz-Gonzalez et al., 2013; Shu et al., 2011), but

chemical bonds and release bioactive molecules with high these protein-based photosensitizers have relatively short exci-

spatiotemporal precision. Optogenetic reagents use light to tation wavelengths (Torra et al., 2019). KillerRed and SuperNova

drive conformational changes in proteins to affect cellular func- are protein-based photosensitizers with longer excitation wave-

tion. Finally, photosensitizing molecules absorb light energy lengths and well-documented utility (de Rosny and Carpentier,

and then transfer it to adjacent molecules, facilitating specialized 2012; Takemoto et al., 2013), but their potency is lower than

chemistries inside cells and tissue.

A key application of photosensitizers is the generation of reac-

small-molecule photosensitizers (Onukwufor et al., 2020).

We set out to develop a hybrid small molecule:protein photo-

tive oxygen species (ROS) through the interaction of the triplet sensitizer system that combines the ﬂexibility and performance

excited state of the photosensitizer with molecular oxygen (³O₂) of small-molecule dyes with the subcellular speciﬁcity of genet-

(Baptista et al., 2017; Xiong et al., 2019). The resulting singlet ox- ically encoded reagents. To complement the recent develop-

ygen (¹O₂) and other ROS can be leveraged to manipulate or ment of impressive noncovalent hybrid photosensitizer systems

interrogate biological systems in a variety of ways: chromo- (He et al., 2016; Marek and Davis, 2002; Tour et al., 2003) based

phore-assisted light inactivation (CALI) of proteins, targeted abla- on labeling strategies that require a constant supply of small-

tion of cells, or photooxidation and subsequent polymerization of molecule ligand (Ayele et al., 2019), we aimed to use the covalent

diaminobenzidine (DAB) to provide contrast in light and electron HaloTag-labeling system developed by Promega (Los et al.,

microscopy (EM). This broad utility has led to the development of 2008). This ‘‘self-labeling’’ enzyme-derived tag system has

different classes of photosensitizers useful in living systems. been used in many contexts ranging from single cells

Small-molecule photosensitizers fall into three main classes: por- (Chong et al., 2018) to animals (Abdelfattah et al., 2019) and

Cell Chemical Biology 27, 1–10, September 17, 2020 ª 2020 Elsevier Ltd.

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I

A

B

X

N

O

N⁺

N

O

N⁺

HO

O

HO

X

O

Y

O

X

NIS

halogenation

CH₃CN

87%

−

CO₂

−

CO₂

Y

CO₂

Y

Janelia Fluor 549 (JF₅₄₉, 6)

Janelia Fluor 567 (JF₅₆₇, 7)

Fluorescein (1)

Eosin (2): X = Br, Y = H

Rose Bengal (3): X = I, Y = Cl

N

S

N

S

N⁺

i. t-BuLi

Br Br

N⁺

N

S

N⁺

THF, −78 °C

N

O

O→S

8

+

O

−

ii. 9, −20 °C to rt

CO₂

O

N

46%

O

Oxazine 1 (4)

Methylene Blue (5)

Janelia Fluor 570 (JF₅₇₀, 10)

9

JF₅₄₉(6)^b

C

D

E

Eosin (2)^a

JF₅₆₇(7)

567

JF₅₇₀(10)

570

7 ex

10 ex

1.0

0.5

0.0

λ_max(nm)

_em(nm)

ε (M⁻¹cm⁻¹

526

544

112,000

–

549

571

7 em

10 em

λ

590

593

0.5

0.0

101,000

134,000

3.47

12,600

101,000

0.79

83,600

117,000

2.24

)

ε_max(M⁻¹cm⁻¹

)

c

d

–

K_L–Z

Φ_f

0.20

0.57

0.88

0.04

0.63

400

500

600

700

400

500

600

700

Wavelength (nm)

Φ_so

0.002

0.44

0.088

Figure 1. Photosensitizer Structures, Synthesis, and Photophysical Characterization

(A) Relationship of classic photosensitizer chemical structures to parent ﬂuorophores. Eosin (2) and Rose Bengal (3) are halogenated derivatives of ﬂuorescein (1)

and methylene blue (5) is a sulfur-containing analog of oxazine 1 (4).

(B) Iodination of JF₅₄₉(6) to yield JF₅₆₇(7) and organolithium-mediated reaction of 8 and 9 to yield JF₅₇₀(10).

(C and D) Normalized ﬂuorescence excitation (ex) and emission (em) spectra of (C) 7 and (D) 10.

(E) Table of photophysical properties for eosin (2), JF₅₄₉(6), JF₅₆₇(7), and JF₅₇₀(10).

Notes: ^adata for 2 taken from (Fleming et al., 1977; Gandin et al., 1983; Seybold et al., 1969); ^bdata for 6 taken from (Grimm et al., 2015, 2017b); ^ce_maxmeasured in

triﬂuoroethanol + 0.1% (v/v) triﬂuoroacetic acid; and ^dK_L–Zmeasured in 1:1 (v/v) dioxane:water.

provides rapid, speciﬁc labeling of protein fusions with synthetic with a relatively high atomic number enhances the rate of inter-

ﬂuorophores. Previous examples of HaloTag-compatible photo- system crossing from the singlet excited state to the triplet

sensitizers are typically ﬂuorescein derivatives, such as eosin excited state. This triplet state then interacts with molecular ox-

(Figure 1A) and related compounds. Such ligands are excited ygen (³O₂), generating singlet oxygen (¹O₂) with concomitant

by relatively short wavelengths (Lee et al., 2008; Li et al., 2018; relaxation of the dye to the ground state; the efﬁciency of singlet

Takemoto et al., 2011), which are phototoxic on their own oxygen generation can be quantiﬁed as the singlet oxygen quan-

(Douthwright and Sluder, 2017; Magidson and Khodjakov, tum yield (F_SO).This principle is illustrated by the halogenation of

2013), and require ester-masking groups to render the molecules ﬂuorescein (1, F_SO= 0.03) to yield classic photosensitizers eosin

cell permeable (Takemoto et al., 2011). We now report HaloTag- (i.e., eosin Y; 2, F_SO= 0.57) and Rose Bengal (3, F_SO= 0.76, Fig-

compatible photosensitizer ligands based on our cell- and tis- ure 1A), where the halogen substituents also shift the absorption

sue-permeable azetidine-containing ‘‘Janelia Fluor’’ rhodamine wavelengths to the red (Fleming et al., 1977; Gandin et al., 1983;

scaffold (Grimm et al., 2015, 2017b). These dyes are red shifted Seybold et al., 1969). Likewise, replacement of the oxygen in the

compared with eosin and do not need ester-masking groups for phenoxazine dyes, such as laser dye oxazine 1 (4), with sulfur

membrane permeability. We demonstrate the utility of these yields well-known photosensitizer methylene blue (5, Figure 1A)

probes for CALI, targeted cell ablation in culture and in vivo, (Becker et al., 1990; Francisco et al., 2017). This substitution also

and photoinduced DAB deposition for EM. This work expands elicits a bathochromic shift in wavelength with the increase

the repertoire of the HaloTag-labeling system to ablation of cells in F_SO

.

in intact animals and generating contrast for EM.

We sought to apply this heavy atom effect to rhodamine dyes,

which are widely used ﬂuorescent labels but have received

limited attention as photosensitizer scaffolds (Pal et al., 1996).

A key advantage of rhodamine dyes over ﬂuorescein-based

compounds, such as eosin (2) and Rose Bengal (3), is based

on charge. Fluorescein derivatives are anionic at physiological

RESULTS

Rational Design and Synthesis of Janelia Fluor

Photosensitizers

Many classic small-molecule photosensitizers exploit the ‘‘heavy pH and, as highlighted above, require ester-masking groups to

atom effect’’ (Gold, 1987), where the incorporation of an atom allow efﬁcient entry into cells. These ester bonds are notoriously

2

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unstable in aqueous solution, particularly for halogenated deriv- the lactone-zwitterion equilibrium and not signiﬁcant differences

atives such as 2 and 3 (Lavis et al., 2006, 2011); their use in vivo in the inherent absorptivity of the two chromophores.

typically requires injection of DMSO solutions directly into tissue

We then measured the ﬂuorescence quantum yield (F_f) and

(Sawinski et al., 2009). In contrast, simple rhodamine dyes are singlet oxygen quantum yield (F_SO) of both potential photo-

net neutral and exist in equilibrium between a lipophilic lactone sensitizer dyes using Rose Bengal (3; F_SO= 0.76) (Ezquerra

and polar zwitterion. This ‘‘dynamic amphipathicity’’ allows rho- Riega et al., 2017) as a standard. JF₅₆₇(7) showed a relatively

damines to rapidly traverse biological membranes without the low F_f= 0.04 and a high F_SO= 0.44. In contrast, JF₅₇₀(10)

need for auxiliary groups, allowing facile loading into living cells showed a higher F_f= 0.63 balanced by a more modest

and even animals (Abdelfattah et al., 2019; Grimm et al., 2017b). F_SO= 0.088. These F_SOvalues were substantially higher

We recently developed an improved class of rhodamines, the Ja- than the parent ﬂuorophore JF₅₄₉(6), which gave a low

nelia Fluor dyes, which incorporate four-membered azetidine F_SO= 0.002. For comparison, eosin (2) has a reported

rings in place of the common dimethylamino groups found in F_SO= 0.57 (Gandin et al., 1983) and the F_SOfor methylene

many classic rhodamines. This simple substitution results in sub- blue (5) has been measured at 0.07 in aqueous solution (Fran-

stantial improvements in photophysical properties (Grimm et al., cisco et al., 2017). These data show that both halogenation

2015) and facilitates rational ﬁne-tuning of the spectral and and sulfur substitution substantially increase F_SOcompared

chemical features of these molecules (Grimm et al., 2017b). with the parent dye and that these F_SOvalues are comparable

For example, the prototypical Janelia Fluor 549 (JF₅₄₉, 6, Fig- with those of established photosensitizers, such as eosin and

ure 1B) exhibits a 2-fold improvement in both brightness and methylene blue.

photostability in cellular-imaging experiments compared with

the parent tetramethylrhodamine compound (Grimm et al., Synthesis of HaloTag Ligands and CALI

2015). Since increased excited state lifetime and photostability To enable attachment of these dyes to speciﬁc proteins in living

are important for photosensitizer performance, we sought to systems, we prepared derivatives of both 7 and 10 containing

extend this azetidine-incorporation strategy to photosensitizer the chloroalkane HaloTag ligand moiety at the optimal 6-position

`

dyes. We envisioned either halogenation (a la 3) or sulfur substi- on the pendant phenyl ring (7_HTLand 10_HTL; Figure 2A) (Los et al.,

`

tution (a la 5) applied to rhodamine compounds, such as 6. For 2008). These were synthesized in analogous fashion to the free

the halogenation strategy, we found that straightforward treat- dyes. The JF₅₆₇-HaloTag ligand (7_HTL; Figure S1A) involved

ment of JF₅₄₉(6) with N-iodosuccinimide afforded compound 7 direct iodination of 6-carboxy-JF₅₄₉(11) to give the diiodo 6-car-

with incorporation of two iodine atoms at the 4- and 5-positions boxy-JF₅₆₇(12), which could be amidated with the HaloTag

of the xanthene moiety (Figure 1B). To accomplish the sulfur sub- ligand amine (13) to yield 7_HTL. The synthesis of 10_HTLwas

stitution, we adapted our recent synthetic approach to rhoda- more involved (Figure S1B), starting with the coupling of 3-bro-

mine dyes (Grimm et al., 2017a) through lithium-bromide ex- mothiophenol (14) and 3-bromoiodobenzene (15) to give diary-

change of brominated diarylthioether 8 followed by addition to lthioether 16. Cross-coupling with azetidine gave 17, which

phthalic anhydride (9) to yield compound 10 (Figure 1B).

was regioselectively brominated with NBS to give 8. Metalation

We then examined the spectral and chemical properties of of 8 with t-BuLi and addition to diorthoester methyl benzoate

these two dyes compared with eosin (2) and JF₅₄₉(6; Figures 18 followed by deprotection gave 6-carboxy-JF₅₇₀(19). Amida-

1C–1E). As in other halogenated xanthene dyes (e.g., 2 and 3), tion with the HaloTag ligand amine (13) yielded 10_HTL

the iodo substituents in rhodamine 7 elicited a bathochromic

We evaluated the properties of 7_HTLand 10_HTL(Figure 2A) as

.

shift with an absorption maximum (l_max) of 567 nm and an emis- conjugates with puriﬁed HaloTag protein (HT) in vitro. The equi-

sion maximum (l_em) of 590 nm in aqueous buffer; we named this librium between the open, colored zwitterionic form and the

dye ‘‘Janelia Fluor 567’’ (JF₅₆₇) based on the l_max. Likewise, the closed, colorless lactone of rhodamine dyes can be leveraged

replacement of the bridging oxygen with a sulfur in 10 caused a to create chromogenic and ﬂuorogenic ligands—molecules

red shift in its spectra with l_max/l_em= 570 nm/593 nm; we gave that show large increases in absorption and ﬂuorescence upon

this dye the moniker ‘‘Janelia Fluor 570’’ (JF₅₇₀). We then inves- binding their cognate biomolecular target. Since JF₅₆₇(7) ex-

tigated the absorptivity of these dyes in aqueous solution. hibited a relatively low extinction coefﬁcient in aqueous solution

Although the sulfur-containing JF₅₇₀(10) showed high absorptiv- and a relatively low K_L–Z(Figure 1E), we predicted that the JF₅₆₇

-

ity with an extinction coefﬁcient (e) of 83,600 M^À1cm^À1, the halo- HaloTag ligand (7_HTL) would show increased absorption upon

genated derivative JF₅₆₇(7) gave a substantially lower e = 12,600 binding the HaloTag protein. Indeed, incubation of this photo-

M

^À1cm^À1. As mentioned above, rhodamines exist in equilibrium sensitizer dye with HaloTag protein elicited an 8.6-fold increase

between a colorless, nonﬂuorescent lactone form, and a in absorption due to binding of the ligand to the HaloTag protein

colored, ﬂuorescent zwitterionic form. We suspected that the (Figure 2B), similar in magnitude to the prototypical ﬂuorogenic

electron-withdrawing iodo substituents in compound 7 shifted rhodamine dye tetramethyl-Si-rhodamine (Lukinavicius et al.,

this equilibrium toward the colorless lactone form. We therefore 2013). In contrast, the JF₅₇₀-HaloTag ligand did not show a sub-

measured the equilibrium constant (K_L–Z) and maximum extinc- stantial increase in absorption (Figure 2C), as expected from the

tion coefﬁcient (e_max) using triﬂuoroethanol containing 0.1% properties of the free dye (Figure 1E). We further investigated the

(v/v) triﬂuoracetic acid, a solvent system that promotes formation properties of the HaloTag conjugates and compared them with

of the open form (Grimm et al., 2017b; Zheng et al., 2019) (Fig- eosin-HaloTag ligand (2_HTL); this was prepared by treatment of

ure 1E). Under these conditions we found that both dyes exhibit the commercially available diacetate compound (2_HTLAc₂)

high absorptivity (e_max> 100,000 M^À1cm^À1), demonstrating that (Takemoto et al., 2011) with porcine liver esterase (Figures 2D

the difference in observed e in aqueous solution is due to a shift in and S2A). JF₅₆₇-HaloTag ligand (7_HTL) and JF₅₇₀-HaloTag ligand

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A

B

C

D

X

Br

1.0

0.8

0.6

0.4

0.2

0

1.0

0.8

0.6

0.4

0.2

0

N⁺

7_HTL+HT

10_HTL+HT

N

Y

RO

Br

O

OR

Br

7_HTL

10_HTL

O

−

CO₂

O

HN

O

Cl

2_HTLAc₂: R = Ac

2_HTL: R = H

7

_HTL: X = I, Y = O

400 450 500 550 600 650 700

Wavelength (nm)

400 450 500 550 600 650 700

Wavelength (nm)

PLE

10_HTL: X = H, Y = S

7

_HTL–HT

G

10_HTL–HT

E

2_HTL–HT

(Eosin)

F

N

O

N⁺

561 nm

(JF₅₆₇

)

(JF₅₇₀

)

*

6

_HTL+ 561 nm

*

532

577

λ_abs(nm)

λ_em(nm)

577

600

7_HTL+ 561 nm

10_HTL+ 561 nm

508 nm

−

CO₂

553

602

O

*

ε (M⁻¹cm⁻¹

)

70,000

83,000

0.65

60,000

0.05

HN

2

_HTL+ 508 nm

O

0.26

Φ_f

Cl

6_HTL

0

−20

−40

% Change in EGFP emission

I

H

J

t = 0 min

10_HTL

brightfield

6_HTL

t = 0 min brightfield

t = 0 min

blank

555 nm

*

6

_HTL+ 555 nm

*

7_HTL+ 555 nm

10_HTL+ 555 nm

508 nm

*

ns

10_HTL

t = 30 min brightfield

t = 30 min

6_HTL

t = 30 min brightfield

t = 30 min

2_HTL+ 508 nm

0

20

40

60

80

% Cells ablated

Figure 2. Properties of HaloTag Ligands and Their Utility in CALI and Cell Ablation

(A) Chemical structures of JF₅₆₇-HaloTag ligand (7_HTL) and JF₅₇₀-HaloTag ligand (10_HTL).

(B and C) Absorbance spectra of (B) 7_HTLand (C) 10_HTLHaloTag protein (HT).

(D) Chemical structure of eosin-Ac₂-HaloTag ligand (2_HTLAc₂) and conversion to 2_HTLby incubation with porcine liver esterase (PLE).

(E) Table of photophysical properties for 7_HTL, 10_HTL, and 2_HTLattached to HaloTag protein.

(F) Chemical structure of JF₅₄₉-HaloTag ligand (6_HTL).

(G) Comparison of EGFP CALI using 561 nm excitation light (laser; 10 W/cm²) alone (n = 19), 561 nm excitation light (laser; 10 W/cm²) with 6_HTL(n = 15), 7_HTL

(n = 23), and 10_HTL(n = 14), 508-nm centered excitation light (LED; 10 W/cm²) alone (n = 35), or 508-nm centered excitation light (LED; 10 W/cm²) with 2_HTL

(n = 30); center line indicates median; box limits indicate upper and lower quartiles; whiskers indicate min-max; data pooled from two experiments and analyzed

via Kruskal-Wallis test with Steel-Dwass-Critchlow-Fligner post-hoc test.

(H and I) Representative confocal ﬂuorescence (561 nm laser excitation) and bright-ﬁeld microscopy images of U2OS cells expressing HaloTag-TOMM20 fusion

protein incubated with JF₅₇₀-HaloTag ligand (10_HTL) (H) or JF₅₄₉-HaloTag ligand (6_HTL) (I) before (t = 0 min) and after (t = 30 min) wide-ﬁeld irradiation with 560-nm

centered light. Scale bars, 13 mm.

(J) Comparison of cell ablation efﬁcacy using 6_HTL, 7_HTL, and 10_HTLwith 555 nm excitation light or 2_HTLusing 508-nm centered excitation light in U2OS cells stably

expressing HaloTag-TOMM20 fusion protein exposed to 55 mW/cm²excitation light for 3 min; center line indicates median; box limits indicate upper and lower

quartiles; whiskers indicate min-max; data analyzed via Welch’s ANOVA and Games-Howell post-hoc test (n = 5, 10.89 mm²microplate wells); n = 5 except for

blank measurement where n = 10.

Statistical signiﬁcance in (G and J) reported as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figures S1–S3

(10_HTL) showed similar F_fvalues to the free dyes (7 and 10) with a and therefore constitute the most red-shifted HaloTag-compat-

slight bathochromic shift of 8–12 nm due to attachment of the ible photosensitizers described.

HaloTag ligand moiety and conjugation to the HaloTag protein

For an initial application, we tested these photosensitizers in

(Figures 1C–1E , 2E, S2B, and S2C). Both dyes exhibited a sub- CALI of proteins in live cells. CALI allows the targeted inactiva-

stantial bathochromic shift (ꢀ45 nm) compared with the conju- tion of proteins via excitation of a conjugated photosensitizer,

gate of the eosin-HaloTag ligand (2_HTL, Figures 2E and S2D) which produces damaging oxidants, typically singlet oxygen

4

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(¹O₂) or other radical products such as superoxide (Baptista pathogenic microbes (Hu et al., 2018; Wang et al., 2017), or for

et al., 2017; Jay, 1988; Liao et al., 1994; Ochsner, 1997). These basic science experiments to study the effect of the ablation of

reactive species act with an effective radius of 4–6 nm (Beck speciﬁc cell lineages on animal development or behavior.

et al., 2002; Linden et al., 1992) via methionine oxidation and pro-

We ﬁrst tested the ablative utility of the JF dyes in cell culture,

tein crosslinking (Yan et al., 2006). Unlike conventional genetic targeting the HaloTag ligands to the nucleus or mitochondria us-

knockout methods, the CALI technique gives temporal control ing HaloTag fusions to either histone H2B (‘‘HaloTag-H2B’’) or

of protein ablation, which may allow for functional knockouts the mitochondrial outer membrane import protein TOMM20

of vital proteins (Vitriol et al., 2007; Wojtovich et al., 2016) with (‘‘HaloTag-TOMM20’’). We found that both JF₅₆₇-HaloTag ligand

faster time resolution compared with RNAi (Li et al., 2018). (7_HTL) and JF₅₇₀-HaloTag ligand (10_HTL) were effective ablative

CALI has been found useful in studying mitochondrial electron agents of U2OS cells stably expressing HaloTag-H2B when

transport chain physiology (Wojtovich et al., 2016) and memory excited with 555-nm centered light (3 min, 55 mW/cm²; Fig-

maintenance (Takemoto et al., 2017), making bioavailable and ure S3B), ablating 35% and 19% of cells, respectively, when

targetable photosensitizers of particular interest in various areas measured 24 h after illumination. This was better or equivalent

of biology.

to the eosin-Ac₂-HaloTag ligand (2_HTLAc₂; 16% ablation;

For a proof-of-concept CALI experiment, we expressed 3 min, 55 mW/cm², 508-nm centered light) and substantially

EGFP-HaloTag fusion proteins in U2OS cells and then labeled larger than the JF₅₄₉-HaloTag ligand (9) or DMSO controls, which

with photosensitizer ligands JF₅₆₇-HaloTag ligand (7_HTL), JF₅₇₀

-

both showed ꢀ1% ablation under the same illumination condi-

HaloTag ligand (10_HTL), and eosin-Ac₂-HaloTag ligand tions (Figure S3C). Similar trends were observed with HaloTag-

(2_HTLAc₂), with the JF₅₄₉-HaloTag ligand (6_HTL; Figure 2F) and TOMM20-expressing cells but at higher overall efﬁcacy with

vehicle-only (DMSO) as controls (Figure 2G). CALI efﬁcacy was JF₅₆₇-HaloTag (7_HTL), JF₅₇₀-HaloTag ligand (10_HTL), and eosin-

assessed by measuring the decrease in EGFP ﬂuorescence Ac₂-HaloTag ligand (2_HTLAc₂), showing ablation of 65%, 48%,

upon illumination of the cells with photosensitizer excitation light; and 35% of cells, respectively, compared with ꢀ2% in the

we chose 561 nm for the JF ligands and 508 nm for the eosin JF₅₄₉-HaloTag ligand (6_HTL

)

and DMSO-only controls

ligand to give comparable absorption cross-sections (Fig- (Figures 2H–2J and S3D). Increasing illumination intensity

ure S3A). JF₅₆₇-HaloTag ligand (7_HTL

and JF₅₇₀-HaloTag (147 mW/cm²) resulted in >80% ablation for both the experi-

)

ligand (10_HTL) both showed effective CALI, showing a decrease ments using the Janelia Fluor photosensitizer HaloTag ligands

in EGFP ﬂuorescence after brief photosensitizer excitation (Figures S3E and S3F). These compounds also showed negli-

(10 s, 561 nm, 10 W/cm²). Interestingly, cells labeled with gible ‘‘dark’’ toxicity, demonstrating that the cellular ablation is

JF₅₄₉-HaloTag ligand (6_HTL) showed a small but signiﬁcant in- light mediated (Figures S3G–S3H). Ablation was also negligible

crease in average ﬂuorescence intensity under the same condi- in the absence of HaloTag fusion proteins, demonstrating that

tions, possibly due to the diminutive amount of generated ROS damage due to nonspeciﬁcally bound dyes is nominal

promoting further maturation of EGFP chromophores. CALI ex- (Figure S3I).

periments with the eosin ligand (2_HTLAc₂) were more compli-

cated due to the overlap of the EGFP and eosin absorbance Targeted Cell Ablation In Vivo

spectra. Cells labeled with 2_HTLAc₂and excited with 508-nm We then tested the utility of this cell ablation strategy in vivo using

centered light elicited a signiﬁcant decrease in EGFP ﬂuores- the zebraﬁsh model system. Transgenic casper zebraﬁsh

cence, but the dye-free control experiment also showed signiﬁ- (Mitfa^w2/w2roy^a9/a9) embryos expressing a neuron-speciﬁc elavl3

cant bleaching of EGFP by the 508-nm centered light dose. GAL4-VP16 driver were injected with a construct resulting in the

These data demonstrate that both iodination and sulfur substitu- co-expression of mCerulean3 ﬂuorescent protein and the mito-

tion yield photosensitizer JF dyes that perform similarly to an es- chondria-targeted HaloTag-TOMM20 fusion (Figure 3A). These

tablished compound in CALI applications. This experiment also neurons were labeled with JF₅₇₀-HaloTag ligand (10_HTL) or the

showcases the longer absorbance maxima of 7_HTLand 10_HTL

,

previously described bioavailable JF₅₈₅-HaloTag ligand (20_HTL;

which could allow concurrent use of these compounds with Figure S4A) (Grimm et al., 2017b) by incubation of these trans-

GFP-based reporters or blue light-excited optogenetic reagents. genic larval ﬁsh with these ligands in the system water (33 mM).

Attempts to use 7_HTLin vivo resulted in poor labeling (data not

Targeted Cell Ablation in Cell Culture

shown), likely due to the lipophilic iodo substituents combined

Photosensitizers can also be utilized to ablate entire cells (Miller with the shift in K_L–Ztoward the lactone form. Both 10_HTLand

and Selverston, 1979), presumably via CALI of vital biomolecules 20_HTLdyes labeled neurons throughout the zebraﬁsh brain. We

or oxidative damage to organelles. Reported targets for effective then illuminated the approximate forebrain region (zone 1, Fig-

cell ablation include the nucleus and mitochondria (He et al., ure 3B) of ﬁsh labeled with 10_HTLwith 3 W/cm²and ﬁsh incu-

2016; Shirmanova et al., 2015). For nuclear-targeted photosen- bated with 20_HTLwith 12 W/cm²; the higher illumination power

sitization, the hypothesized mechanisms of ablation include used for the JF₅₈₅accounts for the differences in absorption co-

DNA damage leading to death of irradiated cells. For mitochon- efﬁcients at 560-nm centered light. We measured cell viability by

drial-targeted photosensitizers, cell death is presumably caused counting cells using the mCerulean3 ﬂuorescence signal, ﬁnding

by mitochondrial membrane damage and subsequent cyto- that, in zone 1 (ꢀforebrain) of JF₅₇₀-labeled ﬁsh, only ꢀ3% of

chrome c leakage leading to apoptosis or decreased energy cells were viable 24 h post-illumination, whereas use of the

output potential leading to necrosis. Photoablation of cells can JF₅₈₅-HaloTag ligand preserved 93% of the neurons in ﬁsh (Fig-

be utilized for clinical applications such as photodynamic ther- ures 3C and 3D). Neuronal viability was also quantiﬁed in zones

apy of malignancies (Zhang et al., 2018), photoinactivation of roughly corresponding to the midbrain and hindbrain regions

Cell Chemical Biology 27, 1–10, September 17, 2020

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Figure 3. Photoablation of Neurons in Larval

A

B

D

Zone 1:

~forebrain

Zebraﬁsh

10×UAS:TOMM20-HaloTag-2a-mCer3

HaloTag

(A) Experimental scheme of larval zebraﬁsh

neuronal ablation experiment.

Zone 2:

~midbrain

(B) Cartoon of larval zebraﬁsh brain ‘‘zones’’ used

in this experiment; yellow circle indicates approx-

imate area of irradiation.

Tg[elavl3:Gal4]

ligand

post-irradiation

imaging

loading

irradiation

5

Zone 3:

~hindbrain &

spinal cord

0

3

4

6

(C) Representative ﬂuorescence microscopy im-

ages of zebraﬁsh pre-irradiation (t = 0 min), and one

day post-irradiation (t = 24 h) expressing HaloTag

fusions to the mitochondrial protein TOMM20

(magenta) co-expressing mCerulean (cyan) and

Time (days)

C

t = 0

t = 24 h

*

Zone 1

Zone 2

Zone 3

incubated with either JF₅₇₀-HaloTag ligand (10_HTL

,

top; 3 W/cm²excitation) or JF₅₈₅-HaloTag ligand

(20_HTL, bottom; 12 W/cm²). Scale bars, 100 mm.

(D) Quantiﬁcation of cell ablation efﬁcacy using

JF₅₇₀-HaloTag ligand (10_HTL) with JF₅₈₅-HaloTag

ligand (20_HTL) as control; n = 6 for 10_HTLexperi-

ments and n = 5 for 20_HTLexperiments; center line

indicates median; box limits indicate upper and

lower quartiles; whiskers indicate min-max; data

analyzed via independent t tests.

ns

t = 0

t = 24 h

10_HTL

20_HTL

0

50

100

Viability 24 h post-irradiation (%)

Statistical signiﬁcance reported as follows:

*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

See also Figure S4.

(zones 2 and 3, Figures 3B, S4B, and S4C) to assess off-target chromatin-nucleosome interface scale (5–24 nm; Figures 4E–

ablation due to light scattering (Dobrucki et al., 2007) or damage 4H) (Ou et al., 2017).

to neuronal processes projecting into the area of illumination.

This off-target ablation was modest in zone 2, with 10_HTLzebra-

ﬁsh retaining 80% of labeled cells, and negligible in zone 3, which

DISCUSSION

showed no signiﬁcant ablation (Figure 3D), indicating that cell

death is largely contained to the area of illumination.

Light-driven chemical reactions allow the manipulation of biolog-

ical systems with high spatiotemporal control. Photosensitizers

generate ROS, which can be harnessed in a variety of applica-

tions ranging from inactivation of individual proteins to the syn-

DAB Photopolymerization for EM

Finally, we tested the JF photosensitizers as agents for DAB thesis of imaging contrast agents in situ. We transformed the

polymerization, which results in the generation of a visibly opa- cell-permeable Janelia Fluor scaffold into a photosensitizer sys-

que, osmophilic material. This polymer is an effective contrast tem through rational design. The resulting dyes JF₅₆₇(7) and

agent in both light microscopy and EM. Photosensitizing ﬂuo- JF₅₇₀(10) show comparable F_SOvalues with classic photosensi-

rescent dyes have a rich history in both EM and correlative light tizers (Figure 1). Their HaloTag ligand derivatives 7_HTLand 10_HTL

microscopy and EM (Deerinck et al., 1994; Gaietta et al., 2002; are as effective as the established eosin ligand (2_HTL) in CALI and

Grabenbauer et al., 2005; Hoffmann et al., 2010; Liss et al., cell culture ablation experiments but are red shifted and can be

2015; Shu et al., 2011), and this photopolymerization comple- introduced into biological systems without the use of esterase-

ments peroxide-mediated DAB deposition using horseradish sensitive masking groups (Figure 2). This bioavailability of 10 al-

peroxidase (Hopkins et al., 2000) and APEX (Lam et al., 2015; lows extension of this compound’s utility to in vivo experiments

Martell et al., 2012). The hybrid system comprised by the (Figure 3). Both dyes can also be utilized to photopolymerize

photosensitizer Janelia Fluor ligands and the HaloTag com- DAB and show utility in high-resolution EM (Figure 4). Although

bines the modularity and performance of small-molecule pho- JF₅₆₇(7) exhibits a substantially higher F_SOthan JF₅₇₀(10, Fig-

tosensitizers, such as eosin (Deerinck et al., 1994), with the ure 1E) in vitro, the performance of 7_HTLin CALI (Figure 2G)

genetic speciﬁcity of protein-based reagents, such as miniSOG and cell ablation in culture (Figure 2J) was only modestly better

(Shu et al., 2011), but with longer excitation wavelengths. We than 10_HTL; both dyes gave comparable DAB photodeposition

expressed HaloTag-H2B fusions in human osteosarcoma (Figures 4A–4D). These results, combined with the utility in vivo

cells, labeled with either JF₅₆₇-HaloTag ligand (7_HTL) or JF₅₇₀

-

(Figure 3), recommend JF₅₇₀(10) as a general-purpose photo-

HaloTag ligand (10_HTL), ﬁxed the cells, imaged them by light sensitizer, with JF₅₆₇(7) reserved for applications that require

microscopy, performed DAB photopolymerization and osmium high amounts of ¹O₂. Having established these photosensitizing

tetroxide treatment, and then imaged the sample using trans- modiﬁcations to the core Janelia Fluor rhodamine scaffold, we

mission electron microscopy (TEM). Photooxidation of DAB can combine them into a single compound, apply them to other

was speciﬁc to cells expressing HaloTag-H2B fusion proteins, rhodamine variants, and then further ﬁne-tune these dyes to

and this labeling strategy enabled high-resolution EM in the nu- improve bioavailability and modulate their spectral properties

cleus (Figures 4A–4D). Histones could be visualized as elec- (Grimm et al., 2015, 2017b). Such chemical tools will enable so-

tron-dense puncta in TEM images and the use of tomographic phisticated cell ablation experiments in vivo and advanced imag-

techniques enabled 3D EM with resolution approaching the ing experiments such as correlated light microscopy and EM.

6

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Figure 4. Photooxidation of DAB and EM

(A–D) Representative images of human osteosarcoma (HOS) cells expressing HaloTag-H2B fusion protein labeled with JF₅₆₇-HaloTag ligand (7_HTL; top) or JF₅₇₀

-

HaloTag ligand (10_HTL, bottom). (A) Pre-photooxidation ﬂuorescence microscopy images. (B) Pre-photooxidation differential interference contrast (DIC) light

microscopy images. (C) Post-photooxidation DIC light microscopy images. (D) TEM images post-photooxidation and OsO₄treatment of the entire ﬁeld of view

and regions of interest (ROIs) 1–4. ROIs 1 and 3 are from cells with low HaloTag-H2B expression and show low staining from DAB photooxidation and OsO₄

treatment. ROIs 2 and 4 show increased electron density of the chromatin resulting from speciﬁc labeling of 7_HTLor 10_HTLand subsequent photooxidation of DAB

and osmiﬁcation; arrows highlight representative HaloTag-H2B-containing nucleosomes. Scale bars, 10 mm (A–D) and 200 nm (ROIs 1–4).

(E) Representative electron tomogram slices in the nucleus of an HOS cell expressing HaloTag-H2B fusion proteins labeled with 10_HTL, followed by photo-

oxidization of DAB and osmiﬁcation (cf. ROI 4). Arrows denote a cluster of nucleosomes in the volume. Scale bar, 200 nm.

(F) 3D volumetric rendering extracted from the electron tomogram in (E).

(G) Magniﬁed view at the segmented chromatin denoted in (F).

(H) Manual matching of stained nucleosomes in (G) with a simpliﬁed nucleosome structure consisting of a disc of 11 nm diameter and 5.5 nm height. These

imaging experiments were duplicated with similar results.

SIGNIFICANCE

these compounds exhibit a bathochromic shift in absorption

spectra, allowing illumination with less phototoxic wave-

We report the rational design of photosensitizers derived lengths of light or multiplexing with ﬂuorescent proteins or

from the azetidine-containing Janelia Fluor rhodamine scaf- optogenetic reagents. These compounds also diffuse in

fold. For use in biological systems, we synthesize their Hal- and out of cells and tissue without the need for chemical

oTag ligand variants and demonstrate the utility of these modiﬁcation, making use of these chemical tools straight-

compounds in disparate applications. Compared with forward. These molecules can be used in a variety of biolog-

commercially available photosensitizer HaloTag ligands, ical experiments, such as destroying individual proteins with

Cell Chemical Biology 27, 1–10, September 17, 2020

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Resource

tions (with inventors J.B.G. and L.D.L.) are assigned to HHMI. L.D.L. serves

high spatiotemporal control, ablating genetically deﬁned

on the editorial board of Cell Chemical Biology. The authors declare no other

cells in a developing or learning animal, or providing multi-

competing interests.

modal contrast in light and electron microscopy.

Received: April 1, 2020

STAR+METHODS

Revised: June 4, 2020

Accepted: June 29, 2020

Published: July 21, 2020

Detailed methods are provided in the online version of this paper

and include the following:

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d RESOURCE AVAILABILITY

B Lead Contact

B Materials Availability

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SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

chembiol.2020.07.001.

Chong, S., Dugast-Darzacq, C., Liu, Z., Dong, P., Dailey, G.M., Cattoglio, C.,

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Thanks to Paul Tillberg, Claire Deo, Fadi Jradi, Maria Ioannou, and Charles Kim

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Melissa Ramirez, and Kevin McGowan of the Janelia Research Campus Mo-

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Kevin McGowan of the Janelia Research Campus Cell and Tissue Culture

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AUTHOR CONTRIBUTIONS

ꢀ ꢁ

Ebner, M., Lucic, I., Leonard, T.A., and Yudushkin, I. (2017). PI(3,4,5)P(3)

Conceptualization, T.C.B., J.B.G., Z.L., S.R.A., and L.D.L.; Methodology,

T.C.B., A.X.A., J.B.G., A.N.T., G.A.C., S.P., T.A.B., Z.L., S.R.A., M.K., and

L.D.L.; Investigation, T.C.B., A.X.A., J.B.G., A.N.T., G.A.C., S.P., L.Z., T.A.B.,

S.R.A., and M.K.; Formal Analysis, T.C.B., J.B.G., A.N.T., G.A.C., S.R.A.,

and M.K.; Writing – Original Draft, T.C.B. and L.D.L.; Writing – Review & Edit-

ing, T.C.B. and L.D.L.; Visualization, T.C.B., J.B.G., G.A.C., M.K., and L.D.L.;

Supervision, T.A.B., Z.L., S.R.A., M.H.E., M.K., and L.D.L.; Project Administra-

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10 Cell Chemical Biology 27, 1–10, September 17, 2020

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

Invitrogen

IDENTIFIER

Chemicals, Peptides, and Recombinant Proteins

Singlet Oxygen Sensor Green

Thermo Fisher Scientiﬁc

Catalog #S36002

JF₅₆₇(7)

Janelia Research Campus; this paper

Promega

N/A

JF₅₆₇–HaloTag ligand (7_HTL

)

N/A

JF₅₇₀(10)

N/A

JF₅₇₀–HaloTag ligand (10_HTL

)

N/A

Eosin-Ac₂–HaloTag ligand (2_HTLAc₂)

N/A

Eosin Y

Sigma Aldrich

Sigma Aldrich Catalog #E4009

JF₅₄₉(6)

Janelia Research Campus; (Grimm et al., 2015)

Janelia Research Campus; (Grimm et al., 2017b)

Sigma Aldrich

N/A

JF₅₈₅–HaloTag ligand (20_HTL

)

N/A

Rose Bengal

Sigma Aldrich Catalog #330000

N/A

JF₅₄₉–HaloTag ligand (6_HTL

)

Janelia Research Campus; (Grimm et al., 2015)

Sigma Aldrich

Porcine liver esterase (PLE)

Sigma Aldrich Catalog #E3019

NucBlueꢀ Live ReadyProbesꢀ Reagent

NucRedꢀ Dead 647 ReadyProbesꢀ Reagent

Gateway LR Clonase II Enzyme Mix

Invitrogen

Thermo Fisher Scientiﬁc

Catalog #R37605

Invitrogen

Thermo Fisher Scientiﬁc

Catalog #R37113

Invitrogen

Thermo Fisher Scientiﬁc

Catalog #11791020

Durcupanꢀ ACM

Sigma Aldrich

Sigma Aldrich Catalog #44610

Experimental Models: Cell Lines

Human: U2OS cell line

ATCC

RRID: CVCL_0042

Human: U2OS cell line expressing

HaloTag-H2B

Janelia Research Campus; ATCC; HT-H2B

sequence; (Chen et al., 2014)

Modiﬁed cells of RRID: CVCL_0042

Human: U2OS cell line expressing

HaloTag-TOMM20

Janelia Research Campus; ATCC;

TOMM20 sequence from Michael

Davidson, FSU

Modiﬁed cells of RRID: CVCL_0042

Modiﬁed cells of RRID:CVCL_0312

Human: HOS cell line: HaloTag-

Histone H2B (HT-H2B)

UCSD; ATCC

Experimental Models: Organisms/Strains

Mitfa^w2/w2roy^a9/a9(Casper) zebraﬁsh

Janelia Research Campus; (Kimura et al., 2008)

N/A

(Danio rerio), Tg(elavl3:Gal4-VP16)

Recombinant DNA

pHaloTag-EGFP

Thomas Leonard & Ivan Yudushkin;

(Ebner et al., 2017).

http://n2t.net/addgene:86629;

RRID:Addgene_86629

pRSET.HaloTag

Janelia Research Campus; pRSET:

Invitrogen; HaloTag: Promega

pRSET - V35120; HaloTag - N/A

10xUAS:TOMM20-HaloTagv7-p2a-

mCerulean3, myl7:EGFP

Janelia Research Campus; Tol2 destination

vector:(Kwan et al., 2007); Kozak sequence:

(Grzegorski et al., 2014); Native TOMM20:

Accession NM_001002698; NP_001002698);

HaloTagv7: Promega; P2A sequence –

(Lo et al., 2015); mCerulean3 sequence:

(Markwardt et al., 2011).

N/A

(Continued on next page)

Cell Chemical Biology 27, 1–10.e1–e7, September 17, 2020 e1

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Continued

REAGENT or RESOURCE

Software and Algorithms

SOURCE

IDENTIFIER

XLSTAT v2020.1.1 add-in to Microsoft

Excel 2019 v1908 build 11929.20838

Addinsoft

http://www.xlstat.com/en/

GraphPad Prism 7.04

GraphPad Software

https://www.graphpad.com/scientiﬁc-

software/prism/

Fiji ImageJ Distribution 2.0.0-rc-69/1.52i

Amira 6.3

(Schindelin et al., 2012)

Thermo Fisher Scientiﬁc

https://ﬁji.sc/

N/A

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact, Luke D.

Lavis: lavisl@janelia.hhmi.org.

Materials Availability

Aliquots of JF₅₆₇–HaloTag ligand (7_HTL) and JF₅₇₀–HaloTag ligand (10_HTL) are available by request from the Lead Contact.

Data and Code Availability

This study did not generate new datasets or code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cells utilized in CALI and ablation experiments were U2OS cells (human female, osteosarcoma), some with genetic alterations for the

expression of various HaloTag fusion proteins: transiently transfected EGFP; stably expressed HaloTag–histone H2B fusions; stably

expressed HaloTag–TOMM20 fusions. U2OS cells for CALI experiments were maintained in Dulbecco’s Modiﬁed Eagle Medium

(DMEM; 4.5 g/L glucose and sodium pyruvate, À^L-glutamine, Àphenol red; Corning) supplemented with 10% (v/v) fetal bovine serum

(FBS; Gibco), 2 mM ^L-glutamine (Gibco), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco), and 5% CO₂at 37^ꢁC. U2OS cells

for ablation experiments were maintained in DMEM (high glucose, Àglutamine, Àphenol red; Gibco) containing 13 GlutaMax (Gibco),

10% v/v FBS (Gibco) and 5% CO₂at 37^ꢁC. 13 Antibiotic–Antimycotic (Gibco) was used for maintenance only (excluded from imag-

ing/treatment formulations) and cells were passaged at approximately 80% conﬂuence with 0.25% Trypsin–EDTA with phenol red

(Gibco). Human osteosarcoma (HOS) cells (female) expressing HaloTag–histone H2B fusions utilized for electron microscopy exper-

iments were cultured on 35 mm MatTek dishes (MatTek Corp) in DMEM (Gibco) containing 10% v/v FBS (Gibco) without antibiotic.

Embryonic Casper (Mitfa^w2/w2roy^a9/a9) zebraﬁsh (Danio rerio) were utilized from the 1–2 cell stage through day 6 post-fertilization as

detailed below. All zebraﬁsh experiments presented in this study were conducted in accordance with the animal research guidelines

from the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee and Institutional

Biosafety Committee of Janelia Research Campus.

HaloTag protein expressed by and subsequently puriﬁed from BL21 chemically competent E. coli.

METHOD DETAILS

General Chemical Synthesis Details

Commercial reagents were obtained from reputable suppliers and used as received. All solvents were purchased in septum-sealed

bottles stored under an inert atmosphere. All reactions were sealed with septa through which a nitrogen atmosphere was introduced

unless otherwise noted. Reactions were conducted in round-bottomed ﬂasks or septum-capped crimp-top vials containing Teﬂon-

coated magnetic stir bars. Heating of reactions was accomplished with a silicon oil bath or an aluminum reaction block on top of a

stirring hotplate equipped with an electronic contact thermometer to maintain the indicated temperatures.

Reactions were monitored by thin layer chromatography (TLC) on precoated TLC glass plates (silica gel 60 F₂₅₄, 250 mm thickness)

or by LC/MS (Phenomenex Kinetex 2.1 mm 3 30 mm 2.6 mm C18 column; 5 mL injection; 5–98% MeCN/H₂O, linear gradient, with

constant 0.1% v/v HCO₂H additive; 6 min run; 0.5 mL/min ﬂow; ESI; positive ion mode). TLC chromatograms were visualized by

UV illumination or developed with p-anisaldehyde, ceric ammonium molybdate, or KMnO₄stain. Reaction products were puriﬁed

by ﬂash chromatography on an automated puriﬁcation system using pre-packed silica gel columns or by preparative HPLC (Phenom-

enex Gemini–NX 30 3 150 mm 5 mm C18 column). Analytical HPLC analysis was performed with an Agilent Eclipse XDB 4.6 3

150 mm 5 mm C18 column under the indicated conditions. High-resolution mass spectrometry was performed by the High Resolution

Mass Spectrometry Facility at the University of Iowa.

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NMR spectra were recorded on a 400 MHz spectrometer. ¹H and ¹³C chemical shifts were referenced to TMS or residual

solvent peaks. Data for ¹H NMR spectra are reported as follows: chemical shift (d ppm), multiplicity (s = singlet, d = doublet, t = triplet,

q = quartet, dd = doublet of doublets, m = multiplet), coupling constant (Hz), integration. Data for ¹³C NMR spectra are reported by

chemical shift (d ppm) with hydrogen multiplicity (C, CH, CH₂, CH₃) information obtained from DEPT spectra.

JF₅₆₇(7)

JF₅₄₉(6; 500 mg, 1.22 mmol) was taken up in CH₃CN (40 mL), and N-iodosuccinimide (1.10 g, 4.87 mmol, 4 eq) was added portion-

wise over 10 min. After stirring the reaction at room temperature for 3 h, it was concentrated to half volume, diluted with water, and

extracted with CH₂Cl₂(23). The combined organic extracts were washed with brine, dried over anhydrous MgSO₄, ﬁltered, and

evaporated. Flash chromatography on silica gel (0–30% EtOAc/toluene) afforded 699 mg (87%) of JF₅₆₇(7) as a purple solid. ¹H

NMR (CDCl₃, 400 MHz) d 8.00 (dt, J = 7.3, 1.1 Hz, 1H), 7.65 (td, J = 7.4, 1.3 Hz, 1H), 7.60 (td, J = 7.4, 1.2 Hz, 1H), 7.19 – 7.11

(m, 1H), 6.58 (d, J = 8.7 Hz, 2H), 6.21 (d, J = 8.7 Hz, 2H), 4.24 (q, J = 7.4 Hz, 4H), 4.17 (q, J = 7.4 Hz, 4H), 2.25 (p, J = 7.4 Hz, 4H);

¹³C NMR (CDCl₃, 101 MHz) d 169.4 (C), 155.2 (C), 153.0 (C), 152.4 (C), 135.0 (CH), 129.8 (CH), 128.2 (CH), 127.2 (C), 125.1 (CH),

124.1 (CH), 110.32 (CH), 110.27 (C), 84.3 (C), 69.1 (C), 54.8 (CH₂), 16.3 (CH₂); Analytical HPLC: t_R= 12.5 min, >99% purity (10–

95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min ﬂow; ESI; positive ion mode; detection

at 575 nm); HRMS (ESI) calcd for C₂₆H₂₁I₂N₂O₃[M+H]⁺662.9636, found 662.9654.

JF₅₆₇–HaloTag Ligand (7_HTL

)

6-Carboxy-JF₅₄₉(11; TFA salt; 100 mg, 1.22 mmol) was taken up in CH₃CN (8 mL), and N-iodosuccinimide (317 mg, 1.41 mmol, 8 eq)

was added. After stirring the reaction at room temperature for 18 h, 1 M NaOH (5 mL) was added. Following 5 min of vigorous stirring,

the mixture was acidiﬁed with 1 M HCl (5.5 mL), diluted with water, and extracted with 15% i-PrOH/CHCl₃(33). The combined

organic extracts were dried over anhydrous MgSO₄, ﬁltered, and evaporated. The crude material was puriﬁed by reverse phase

HPLC (10–75% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive). The pooled HPLC product fractions were partially

concentrated to remove MeCN and extracted with 10% MeOH/CH₂Cl₂(33). The organic extracts were dried over anhydrous MgSO₄,

ﬁltered, and evaporated to afford 6-carboxy-JF₅₆₇(12) as a dark purple solid (30 mg, 21%, TFA salt).

6-Carboxy-JF₅₆₇(12; TFA salt; 30 mg, 36.6 mmol) was combined with DSC (22.5 mg, 87.8 mmol, 2.4 eq) in DMF (2 mL). After adding

Et₃N (30.6 mL, 219 mmol, 6 eq) and DMAP (0.4 mg, 3.7 mmol, 0.1 eq), the reaction was stirred at room temperature for 1 h. HaloTag(O2)

amine (HTL-NH₂, 13; TFA salt; 37.1 mg, 110 mmol, 3 eq) in DMF (250 mL) was added, and the reaction was stirred an additional 2 h at

room temperature. It was subsequently diluted with saturated NaHCO₃and extracted with EtOAc (23). The combined organic ex-

tracts were washed with brine, dried over anhydrous MgSO₄, ﬁltered, and concentrated in vacuo. The residue was puriﬁed by reverse

phase HPLC (20–70% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive); pooled HPLC product fractions were

partially concentrated to remove MeCN, diluted with saturated NaHCO₃, and extracted with CH₂Cl₂(23). The organics were dried

over anhydrous MgSO₄, ﬁltered, and evaporated to afford 18.5 mg (55%) of JF₅₆₇–HaloTag ligand (7_HTL) as a purple solid. ¹H NMR

(CDCl₃, 400 MHz) d 8.07 – 7.97 (m, 2H), 7.53 – 7.48 (m, 1H), 6.75 (bs, 1H), 6.57 (d, J = 8.7 Hz, 2H), 6.21 (d, J = 8.7 Hz, 2H), 4.25 (q, J =

7.4 Hz, 4H), 4.19 (q, J = 7.4 Hz, 4H), 3.66 – 3.57 (m, 6H), 3.55 – 3.48 (m, 4H), 3.37 (t, J = 6.6 Hz, 2H), 2.25 (p, J = 7.4 Hz, 4H), 1.78 – 1.69

(m, 2H), 1.53 – 1.45 (m, 2H), 1.45 – 1.36 (m, 2H), 1.35 – 1.27 (m, 2H); Analytical HPLC: t_R= 13.1 min, >99% purity (10–95% MeCN/H₂O,

linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min ﬂow; ESI; positive ion mode; detection at 575 nm); HRMS

(ESI) calcd for C₃₇H₄₁ClI₂N₃O₆[M+H]⁺912.0768, found 912.0782.

Bis(3-bromophenyl)sulfane (16)

An oven-dried round-bottom ﬂask was charged with CuI (604 mg, 3.17 mmol, 0.1 eq) and K₂CO₃(8.77 g, 63.5 mmol, 2 eq). The ﬂask

was sealed and evacuated/backﬁlled with nitrogen (33). Isopropanol (125 mL) was added, followed by ethylene glycol (3.54 mL,

63.5 mmol, 2 eq), 3-bromothiophenol (14; 3.28 mL, 31.7 mmol), and 3-bromoiodobenzene (15; 4.45 mL, 34.9 mmol, 1.1 eq). The re-

action mixture was stirred at 80^ꢁC for 18 h. It was then diluted with saturated NH₄Cl (200 mL) and EtOAc (200 mL), vigorously stirred

for 30 min, and ﬁltered through Celite. The ﬁltrate was separated, and the aqueous layer was extracted again with EtOAc. The com-

bined organics were washed with brine, dried over anhydrous MgSO₄, ﬁltered, and evaporated. Flash chromatography (100% hex-

anes, linear gradient) afforded 8.93 g (82%) of dibromide 16 as a colorless oil. ¹H NMR (CDCl₃, 400 MHz) d 7.48 (t, J = 1.8 Hz, 2H), 7.40

(ddd, J = 7.8, 1.9, 1.2 Hz, 2H), 7.28 – 7.23 (m, 2H), 7.18 (t, J = 7.8 Hz, 2H); ¹³C NMR (CDCl₃, 101 MHz) d 137.3 (C), 133.8 (CH), 130.79

(CH), 130.75 (CH), 129.8 (CH), 123.3 (C); HRMS (EI) calcd for C₁₂H₈Br₂S [M]^+$341.8708, found 341.8732.

Bis(3-(azetidin-1-yl)phenyl)sulfane (17)

An oven-dried round-bottom ﬂask was charged with CuI (985 mg, 5.17 mmol, 0.2 eq), L-proline (1.19 g, 10.4 mmol, 0.4 eq), and

K₂CO₃(14.30 g, 103.5 mmol, 4 eq). The ﬂask was sealed and evacuated/backﬁlled with nitrogen (33). A solution of dibromide 16

(8.90 g, 25.9 mmol) in DMSO (100 mL) was added, and the reaction was ﬂushed again with nitrogen (33). Following the addition

of azetidine (10.46 mL, 155.2 mmol, 6 eq), the reaction was stirred at 100^ꢁC for 18 h. It was then cooled to room temperature, diluted

with saturated NH₄Cl, and extracted with EtOAc (23). The combined organic extracts were washed with water and brine, dried over

anhydrous MgSO₄, ﬁltered, and concentrated in vacuo. Puriﬁcation by ﬂash chromatography on silica gel (0–30% EtOAc/hexanes,

linear gradient) afforded 17 (6.08 g, 79%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) d 7.11 (t, J = 7.9 Hz, 2H), 6.68 (ddd, J = 7.7, 1.7,

1.0 Hz, 2H), 6.45 (t, J = 2.0 Hz, 2H), 6.30 (ddd, J = 8.1, 2.3, 0.9 Hz, 2H), 3.83 (t, J = 7.2 Hz, 8H), 2.33 (p, J = 7.3 Hz, 4H); ¹³C NMR (CDCl₃,

101 MHz) d 152.8 (C), 136.3 (C), 129.5 (CH), 119.9 (CH), 113.6 (CH), 110.1 (CH), 52.5 (CH₂), 17.1 (CH₂); HRMS (ESI) calcd for

C₁₈H₂₁N₂S [M+H]⁺297.1420, found 297.1428.

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Bis(5-(azetidin-1-yl)-2-bromophenyl)sulfane (8)

Sulﬁde 17 (6.00 g, 20.2 mmol) was taken up in DMF (100 mL). N-Bromosuccinimide (7.20 g, 40.5 mmol, 2 eq) was added portion-wise

over 5 min, and the reaction was then stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo; the re-

sulting residue was diluted with water and extracted with EtOAc (23). The combined organic extracts were washed with water and

brine, dried over anhydrous MgSO₄, ﬁltered, and concentrated in vacuo. The crude product was triturated with Et₂O, sonicated, and

ﬁltered. The ﬁlter cake was washed with Et₂O and dried to provide the title compound as a white solid. The ﬁltrate was concentrated,

chromatographed on silica gel (0–50% Et₂O/hexanes, linear gradient), and triturated as before to yield additional dibromide product.

The two crops of white powder were combined, affording 6.51 g (71%) of dibromide 8. ¹H NMR (CDCl₃, 400 MHz) d 7.40 – 7.34

(m, 2H), 6.23 – 6.18 (m, 4H), 3.76 (t, J = 7.3 Hz, 8H), 2.31 (p, J = 7.2 Hz, 4H); ¹³C NMR (CDCl₃, 101 MHz) d 151.9 (C), 135.6 (C),

133.3 (CH), 115.0 (CH), 112.3 (C), 112.1 (CH), 52.4 (CH₂), 16.9 (CH₂); HRMS (ESI) calcd for C₁₈H₁₉Br₂N₂S [M+H]⁺452.9630, found

452.9632.

JF₅₇₀(10)

A solution of dibromide 8 (200 mg, 0.440 mmol) in THF (10 mL) was cooled to À78^ꢁC under nitrogen. tert-Butyllithium (1.7 M in

pentane, 1.14 mL, 1.94 mmol, 4.4 eq) was added, and the reaction was stirred at À78^ꢁC for 30 min. It was then warmed to

À20^ꢁC, and a solution of phthalic anhydride (9; 143 mg, 0.969 mmol, 2.2 eq) in THF (10 mL) was added dropwise over 30 min via

addition funnel. The reaction was allowed to warm to room temperature overnight (18 h). Following the addition of AcOH (100 mL),

the mixture was diluted with MeOH, deposited onto Celite, and concentrated to dryness. Silica gel chromatography (0–10%

1

MeOH (2 M NH₃)/CH₂Cl₂, linear gradient; dry load with Celite) afforded 87 mg (46%) of JF₅₇₀(10) as a dark purple solid. H NMR

(CD₃OD, 400 MHz) d 8.14 – 8.08 (m, 1H), 7.68 – 7.59 (m, 2H), 7.25 (d, J = 9.3 Hz, 2H), 7.21 – 7.17 (m, 1H), 6.79 (d, J = 2.3 Hz, 2H),

6.55 (dd, J = 9.3, 2.3 Hz, 2H), 4.22 (t, J = 7.6 Hz, 8H), 2.51 (p, J = 7.6 Hz, 4H); ¹³C NMR (CD₃OD, 101 MHz) d 172.6 (C), 158.9 (C),

154.0 (C), 144.0 (C), 139.14 (C), 139.10 (C), 136.7 (CH), 131.3 (CH), 130.8 (CH), 130.4 (CH), 130.3 (CH), 120.7 (C), 113.9 (CH),

104.6 (CH), 52.6 (CH₂), 16.9 (CH₂); Analytical HPLC: t_R= 11.8 min, >99% purity (10–95% MeCN/H₂O, linear gradient, with constant

0.1% v/v TFA additive; 20 min run; 1 mL/min ﬂow; ESI; positive ion mode; detection at 550 nm); HRMS (ESI) calcd for C₂₆H₂₃N₂O₂S

[M+H]⁺427.1475, found 427.1489.

6-Carboxy-JF₅₇₀(19)

A solution of dibromide 8 (600 mg, 1.32 mmol, 1.5 eq) in THF (30 mL) was cooled to -78^ꢁC under nitrogen. tert-Butyllithium (1.7 M in

pentane, 3.11 mL, 5.28 mmol, 6 eq) was added, and the reaction was stirred at -78^ꢁC for 30 min. It was then warmed to -20^ꢁC, and a

solution of ester 18 (346 mg, 0.880 mmol, 1 eq) in THF (15 mL) was added dropwise over 30 min via addition funnel. The reaction was

allowed to warm to room temperature overnight (18 h). It was subsequently diluted with saturated NH₄Cl and water and extracted

with EtOAc (23). The combined organic extracts were dried over anhydrous MgSO₄, ﬁltered, and concentrated in vacuo. The result-

ing residue was redissolved in MeOH (10 mL), and 1 M HCl (1 mL) was added. After stirring the solution at room temperature for

30 min, it was diluted with toluene (10 mL), deposited onto Celite, concentrated to dryness, and puriﬁed by ﬂash chromatography

(0–20% MeOH/CH₂Cl₂, linear gradient, with constant 1% v/v AcOH additive; dry load with Celite) to provide the bis(2,2-bis(hydrox-

ymethyl)propyl) diester intermediate (608 mg, 94%, acetate salt).

The diester (608 mg, 0.827 mmol) was taken up in 2,2,2-triﬂuoroethanol (15 mL), and 25% w/w NaOH (5 mL) was added. The re-

action was stirred at room temperature for 7 days. It was then acidiﬁed with AcOH (5 mL), diluted with water, and extracted with 15%

i-PrOH/CHCl₃(33). The combined organic extracts were dried over anhydrous MgSO₄, ﬁltered, and evaporated. Flash chromatog-

raphy on silica gel (0–20% MeOH/CH₂Cl₂, linear gradient, with constant 1% v/v AcOH additive) afforded 342 mg (78%, TFA salt) of 6-

carboxy-JF₅₇₀(19) as a dark purple solid. ¹H NMR (CD₃OD, 400 MHz) d 8.41 (d, J = 8.2 Hz, 1H), 8.36 (dd, J = 8.2, 1.6 Hz, 1H), 7.87 (d,

J = 1.6 Hz, 1H), 7.15 (d, J = 9.4 Hz, 2H), 6.88 (d, J = 2.3 Hz, 2H), 6.62 (dd, J = 9.4, 2.3 Hz, 2H), 4.27 (t, J = 7.7 Hz, 8H), 2.53 (p, J = 7.6 Hz,

4H); ¹³C NMR (CD₃OD, 101 MHz) d 167.8 (C), 167.3 (C), 161.8 (C), 154.3 (C), 144.9 (C), 139.1 (C), 136.5 (CH), 136.0 (C), 135.9 (C), 132.9

(CH), 132.5 (CH), 131.8 (CH), 120.1 (C), 114.8 (CH), 104.6 (CH), 52.7 (CH₂), 16.9 (CH₂); Analytical HPLC: t_R= 9.8 min, >99% purity (10–

95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min ﬂow; ESI; positive ion mode; detection at

575 nm); HRMS (ESI) calcd for C₂₇H₂₃N₂O₄S [M+H]⁺471.1373, found 471.1376.

JF₅₇₀–HaloTag ligand (10_HTL

)

6-Carboxy-JF₅₇₀(19; acetate salt; 30 mg, 56.5 mmol) was combined with DSC (34.8 mg, 136 mmol, 2.4 eq) in DMF (2 mL). After adding

Et₃N (47.3 mL, 339 mmol, 6 eq) and DMAP (0.7 mg, 5.7 mmol, 0.1 eq), the reaction was stirred at room temperature for 1 h. HaloTag(O2)

amine (HTL-NH₂, 13; TFA salt; 57.3 mg, 170 mmol, 3 eq) in DMF (250 mL) was added, and the reaction was stirred an additional 1 h at

room temperature. Puriﬁcation of the crude reaction mixture by reverse phase HPLC (10–95% MeCN/H₂O, linear gradient, with con-

stant 0.1% v/v TFA additive) provided 32.2 mg (72%, TFA salt) of JF₅₇₀–HaloTag ligand (10_HTL) as a dark purple solid. ¹H NMR

(CD₃OD, 400 MHz) d 8.73 (t, J = 5.4 Hz, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.20 (dd, J = 8.3, 1.8 Hz, 1H), 7.75 (d, J = 1.7 Hz, 1H), 7.16

(d, J = 9.4 Hz, 2H), 6.90 (d, J = 2.3 Hz, 2H), 6.62 (dd, J = 9.4, 2.3 Hz, 2H), 4.28 (t, J = 7.6 Hz, 8H), 3.68 – 3.55 (m, 8H), 3.53 (t, J =

6.6 Hz, 2H), 3.42 (t, J = 6.5 Hz, 2H), 2.54 (p, J = 7.6 Hz, 4H), 1.76 – 1.67 (m, 2H), 1.54 – 1.45 (m, 2H), 1.45 – 1.29 (m, 4H); Analytical

HPLC: t_R= 12.5 min, >99% purity (10–95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive; 20 min run; 1 mL/min

ﬂow; ESI; positive ion mode; detection at 575 nm); HRMS (ESI) calcd for C₃₇H₄₃ClN₃O₅S [M+H]⁺676.2606, found 676.2622.

Spectroscopy

Spectroscopy of various dyes, photosensitizers and associated HaloTag Ligand variants was performed using 1 cm path length,

3.5 mL or 1 mL or 300 mL quartz cuvettes (Starna Cells). Absorption spectra were recorded on a Cary Model 100 spectrometer (Agi-

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lent). Fluorescence emission spectra were recorded on a Varian Cary Eclipse (Agilent). Absorbance and ﬂuorescence spectroscopy

were also carried out using a Flexstation 3 microplate reader (Molecular Devices) when noted. Fluorescence quantum yields were

recorded on a Quantaurus-QY (Hamamatsu). Spectroscopy measurements of free dyes were performed 10 mM HEPES, pH 7.3

at ambient temperature (22 2^ꢁC); the reported values of maximum absorption wavelength (l_abs), extinction coefﬁcient at l_abs(e),

maximum ﬂuorescence emission wavelength (l_em), and ﬂuorescence quantum yield (F_f) are averages (n = 3). Spectroscopy of

HaloTag conjugates were performed in Dulbecco’s phosphate-buffered saline (DPBS, Gibco).

Solutions prepared for spectroscopy contained 3–5 mM dye and 10 mM HaloTag protein when applicable. Spectra of compounds

bound to HaloTag protein were allowed a 30 min binding incubation period in the dark at ambient temperature prior to measurement.

Masked compound Eosin-Ac₂–HaloTag ligand (2_HTLAc₂) was incubated with 23.75 units/mL porcine liver esterase in the dark at

ambient temperature for 6 h prior to incubation with HaloTag protein and subsequent spectroscopy. The reported values of maximum

absorption wavelength (l_abs), extinction coefﬁcient at l_abs(e), maximum ﬂuorescence emission wavelength (l_em), and ﬂuorescence

quantum yield (F_f) are averages (n = 2)

K_L–Zwas calculated using the following formula: K_L–Z= (e_dw/e_max)/(1 – e_dw/e_max), where e_dwis the extinction coefﬁcient in a 1:1 v/v

dioxane:water solvent mixture, and e_maxis the extinction coefﬁcient in 0.1% v/v triﬂuoroacetic acid (TFA) in 2,2,2-triﬂuoroethanol

(TFE). The reported values are averages (n = 2).

HaloTag Protein Puriﬁcation

Puriﬁed HaloTag protein was obtained via pRSET vector expression in BL21 E. coli and subsequent puriﬁcation via immobilized metal

¨

afﬁnity chromatography (IMAC) using an AKTA Avant (GE Healthcare Life Sciences). Protein was dialyzed in DPBS for 48 h prior to

quantiﬁcation and use.

Singlet Oxygen Quantum Yield

Singlet oxygen quantum yields (F_SO) of free dyes were measured using the commercially available Singlet Oxygen Sensor Green

(SOSG; Thermo Fisher). Solutions of 5 mM dye + 30 mM SOSG in DPBS + 0.1% DMSO dye cosolvent + 0.6% MeOH SOSG cosolvent

at a volume of 30 mL were excited in 384-well microplate wells with light doses of increasing exposure times and constant power on a

Nikon Ti-Eclipse microscope outﬁtted with a Lumencor LED excitation system. SOSG emission was measured using a FlexStation 3

microplate reader and corrected for baseline emission and DMSO negative control contributions to emission. Values were ﬁrst

normalized to the estimated photons absorbed by each dye to correct for differential excitation efﬁciency based upon light source

output spectra and dye molar absorptivity spectra; absorption cross-section measurements of dyes for F_SOdetermination were

measured in DPBS. Well averages were then calculated (n = 3), and data were normalized to the standard dye Rose Bengal (3), which

has a F_SO= 0.76 (Ezquerra Riega et al., 2017).

Chromophore Assisted Light Inactivation (CALI) of EGFP in Live Cells

U2OS cells were transfected with pHaloTag-EGFP (Ebner et al., 2017) via the Amaxa Nucleofector II system from Lonza to transiently

express a HaloTag-EGFP fusion protein. These cells were labeled with 1 mM ligand in media + 0.1% DMSO cosolvent for 1 h followed

by 2 media washes. EGFP ﬂuorescence was imaged via 488 nm laser or 440 nm-centered excitation preceding and immediately

following a 10 s exposure to 10 W/cm²561 nm monochromatic (laser) or 508 nm-centered LED-based light; the 440 nm/508 nm com-

bination was utilized for the Eosin-Ac₂–HaloTag ligand (2_HTLAc₂). Change in mean EGFP signal due to CALI was calculated per manu-

ally segmented cell using the FIJI 2.0.0-rc-69/1.52i distribution of ImageJ (Schindelin et al., 2012).

Photoablation of Cells in Culture

U2OS cells expressing HaloTag–H2B (Chen et al., 2014), HaloTag–TOMM20, or no exogenous proteins were seeded in a 384-well,

tissue-culture treated microplate (Perkin Elmer) at a density of 2K cells/well and allowed to grow for 24 h. Cells were then labeled with

1 mM ligand in media + 0.1% DMSO cosolvent for 1 h followed by 2 media washes and exposed to various light of various power and

wavelength for 3 min, one well at a time, through the 43 objective of a Nikon Ti-Eclipse microscope at 5% CO₂, 37^ꢁC. After an addi-

tional incubation for 24 hr, cells were costained with NucBlue Live ReadyProbes Reagent and NucRed Dead 647 ReadyProbes Re-

agent (Thermo Fisher) and imaged. The images were analyzed using the FIJI 2.0.0-rc-69/1.52i distribution of ImageJ (Schindelin et al.,

2012) as follows. Image segmentation was carried out by autothresholding each image via the Triangle algorithm 10 manual value

adjustments until image noise was optimally suppressed followed by the binary watershed algorithm. Segmented cells were counted

via the Analyze Particles function (5-inf pixel size, 0.15–1.00 roundness). The percentage of ablated cells was determined by

measuring the number of dead cells stained in the NucRed Dead 647 ReadyProbe image and the total number of cells stained in

the NucBlue Live ReadyProbe image. For the representative confocal and brightﬁeld images (Figures 2H and 2I) U2OS cells express-

ing HaloTag–TOMM20 were labeled with 500 nM ligand in media, washed, and imaged using a Zeiss 880 inverted confocal micro-

scope with an EC Plan-Neoﬂuar 403/1.30 oil immersion objective and 561 nm laser excitation. Cells were imaged before and 30 min

after a 3 min irradiation dose delivered with an X-Cite 120PC Series illuminator using Zeiss ﬁlter set 45 (excitation: 560 nm with a

40 nm bandpass; beamsplitter: 585 nm; emission: 630 nm with a 75 nm bandpass).

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Cloning of Zebraﬁsh Ablation Construct

The zebraﬁsh construct 103UAS:TOMM20-HaloTag-p2a-mCerulean3, myl7:EGFP was assembled using the Tol2kit system with

Gateway LR Clonase II Enzyme Mix (Invitrogen). It was composed of the following components: p5E 103UAS, pME TOMM20-Hal-

oTagv7-p2a-mCerulean3, p3E PolyA (Tol2#302) and the destination vector, pDestTol2pCG2 (Tol2#395) (Grzegorski et al., 2014;

Kwan et al., 2007; Lo et al., 2015; Los et al., 2008; Markwardt et al., 2011).

In Vivo Ablation of Zebraﬁsh Neurons

Mitfa^w2/w2roy^a9/a9(Casper) zebraﬁsh (Kimura et al., 2008) were maintained under standard conditions at 28.5^ꢁC and a 14:10 h

light:dark cycle. Embryos (1–2 cell stage) of Tg(elavl3:Gal4-VP16) (Kimura et al., 2008) were injected with 25 ng/mL DNA plasmid en-

coding the co-expression cassette of mitochondria-localized HaloTag and mCerulean3 under the control of the 103UAS

promoter (103UAS:TOMM20-HaloTag-p2a-mCerulean3, myl7:EGFP), and 25 ng/mL Tol2 transposase mRNA diluted in E3 medium.

Subsequently, the injected embryos with broad expression of mCerulean3 were incubated in system water containing HaloTag dye

ligands: JF₅₇₀–HaloTag ligand (10_HTL) or JF₅₈₅–HaloTag ligand (20_HTL) at 30 mM at 4-day post-fertilization (dpf) for 12 h and then

washed in dye-free system water. Larvae at 5 dpf were then anesthetized in tricaine methanesulfonate (MilliporeSigma, E10521,

MO) dissolved in system water at 160 mg/L and then embedded in 1.6% low melting point agar (MilliporeSigma, 2070-OP, MO). Volu-

metric images of the entire brain were ﬁrst acquired with a confocal microscope (Zeiss, LSM880) with a 203 1.0 NA objective lens

(Zeiss, W Plan-Apochromat 203/1.0) to conﬁrm the distribution of the cells labeled with mCerulean3 and HaloTag ligand prior to abla-

tion. Then yellow, 560 nm-centered ablation light was shone on the forebrain region (Figures 3A and 3B; Zone 1) for 5 minutes through

the wideﬁeld illumination path equipped with a ﬂuorescent ﬁlter (Zeiss, Filter Set 45) and a mercury lamp (Leistungselektronik JENA

GmbH, HXP 120 V) with the ﬁeld diaphragm adjusted just to illuminate the target region. The power densities used for 10_HTLand

20_HTL3 W/cm²and 12 W/cm²respectively. Post-ablation confocal stacks were acquired right after the ablation and 1 day after

the ablation (6 dpf) with the imaging parameters identical to those used prior to ablation. Images were analyzed using the FIJI

2.0.0-rc-69/1.52i distribution of ImageJ (Schindelin et al., 2012) as follows. Zone ROIs were manually segmented using brightﬁeld

maximum intensity z-projections and kept consistent across image stack channels using FIJI’s ROI manager and crop function. FIJI’s

3D Object Counter module was utilized to count cells in the mCer ﬂuorescence channel image stacks obtained pre-irradiation and

24 h post-irradiation using an 8-bit minimum threshold pixel value of 11 and default size settings. The percentage of remaining cells

was determined by measuring the number of cells in pre-irradiation samples and the number of cells in 24 h post-irradiation samples.

DAB Photopolymerization for Electron Microscopy

HOS cells were incubated for 30 min with 5 mM dye in culture medium at 37^ꢁC. Cells were ﬁxed with 4% paraformaldehyde (Electron

Microscopy Sciences) in 5 mM CaCl₂, 0.1 M sodium cacodylate buffer, pH 7.4, incubating at room temperature for 5 min and 0^ꢁC (ice)

for 2 h. Cells were washed 53, 2 min each wash, in 0.1 M sodium cacodylate buffer and then blocked for 15 min in blocking buffer

(10 mM glycine, 10 mM potassium cyanide in 0.1 M sodium cacodylate buffer). After 33 washes with 0.1 M sodium cacodylate buffer,

cells were bathed in 2.5 mM DAB$(HCl)₄(Sigma) in 0.1 M sodium cacodylate buffer. Cells were placed on a cold-stage Leica SPE-II

confocal microscope and photooxidized by continuous epi-ﬂuorescence illumination (150 W Xenon Lamp) through an mCherry ﬁlter

cube (excitation: 542–582 nm) for 7 min. Cells were washed 53, 2 min each wash, with 0.1 M sodium cacodylate buffer and ﬁxed for

30 min with 2% glutaraldehyde at 4^ꢁC. After 33 additional washes with 0.1 M sodium cacodylate buffer, the sample was stained for

30 min with 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M sodium cacodylate buffer. Cells were washed with

doubly distilled water 53, 2 min each wash.

EM Sample Preparation and Imaging

After DAB polymerization and osmiﬁcation, HOS cells were dehydrated in ethanol and embedded in Durcupan epoxy resin as

described previously (Ou et al., 2015). Brieﬂy, the samples were incubated for 2 min in water containing increasing concentrations

of ethanol (20% v/v, 50% v/v, 70% v/v, and 90% v/v) followed by three successive 2 min incubations in 100% ethanol. The samples

were then inﬁltrated with durcupan resin (Sigma, 44610) with three successive 2 h treatments. Finally, the cells were incubated with

freshly prepared durcupan resin and cured for 2 d at 60^ꢁC. Epoxy embedded cells were cut into either 70–80 nm sections and

mounted onto 200 hexagonal mesh copper grid for TEM or 250 nm sections mounted onto slot grid covered with a luxel ﬁlm for elec-

tron tomography. For conventional transmission electron microscopy (TEM), ultrathin sections were imaged at 80 kV using an FEI

Tecnai microscope (FEI Company). For EM tomography, a mixture of 5 nm and 10 nm colloidal gold particles serving as ﬁducial

markers were deposited on both sides of the semi-thin (ꢀ250 nm) specimen sections after the sample grids were glow discharged.

The tomography data was collected on an FEI Titan Halo electron microscope operating at 300 kV with a DE64 direct electron de-

tector. The grids were loaded into a rotation holder speciﬁcally designed for multi-tilt tomography manufactured by Fischione Instru-

ments (Model 2040). During a tilt series, images were acquired tilting the goniometer holding the sample holder from À60^ꢁto +60^ꢁ

with 0.5^ꢁincrements. SerialEM was used to control the FEI Titan for the data acquisition (Mastronarde, 1997). The sample was rotated

along the z-axis by 45^ꢁfor the second tilt series, 90^ꢁ(relative to origin) for the third tilt series, and 135^ꢁ(relative to origin) for the ﬁnal tilt

series. Adjustments were made to the stage/sample height to ensure an identical eucentric height and magniﬁcation for each tilt se-

ries. The sequence of tilt series followed the multilevel access scheme previously described (Phan et al., 2017), which minimizes er-

rors associated with sample shrinkage evenly across all tilt series. EM tomography reconstruction: The entire set of EMT images were

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aligned and reconstructed using Transform-based Tracking, Bundle adjustment, and Reconstruction (TxBR) package (Lawrence

et al., 2006; Phan et al., 2012, 2017). 3D volume rendering was made with Amira 6.3.

QUANTIFICATION AND STATISTICAL ANALYSES

For spectroscopy measurements, n values are given in the Method Details section and represent the number of measurements of

different samples prepared from the same dye DMSO stock solution or HaloTag conjugate stock solution. For F_SOdetermination,

n values are given in the Method Details section and represent the number of separate photochemical experiments using the

same dye DMSO stock solutions of dye and SOSG sensor. For CALI experiments, n values are given in the legend of Figure 2

and represent the number of cells. For ablation experiments in culture, n values are given in the legends of Figure 2 and Figure S3

and represent the number of separate experiments counting all and dead cells using NucBlue Live and NucRed Dead. For ablation

experiments in vivo, n values are given in the legend of Figure 3 and indicate the number of ﬁsh. For electron microscopy experiments,

these were duplicated with similar results as indicated in the legend of Figure 4. For all whisker plots, the center line indicates median;

box limits indicate upper and lower quartiles; whiskers indicate min–max. Graphs were rendered using GraphPad Prism 7.04. Stat-

ictical tests were perfomed using the Addinsoft XLSTAT v2020.1.1 add-in to Microsoft Excel 2019 v1908 build 11929.20838. All data

analyzed statistically were assessed for normality and scedasticity assumptions when appropriate using the Shapiro-Wilk test or Lev-

ene’s test. Parametric or non-parametric tests are indicated in the ﬁgure legends and include the Student’s T-test, Mann–Whitney

U test, ANOVA with Tukey’s post-hoc, Welch’s ANOVA with Games–Howell post-hoc, and Kruskal–Wallis H test with Steel–

Dwass–Critchlow–Fligner procedure.

Cell Chemical Biology 27, 1–10.e1–e7, September 17, 2020 e7

Article Doi

Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems

DOI: 10.1016/j.chembiol.2020.07.001

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Article abstract of DOI:10.1016/j.chembiol.2020.07.001

Full text of DOI:10.1016/j.chembiol.2020.07.001

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