Preparation, Characterization and Application of Optical Switch Probes

Abstract

Optical switches represent a new class of molecular probe with applications in high contrast imaging and optical manipulation of protein interactions. Small molecule, organic optical switches based on nitrospirobenzopyran (NitroBIPS) and their reactive derivatives and conjugates undergo efficient, rapid and reversible, orthogonal optically-driven transitions between a colorless spiro (SP) state and a colored merocyanine (MC) state. The excited MC-state also emits fluorescence, which serves as readout of the state of the switch. Defined optical perturbations of SP and MC generate a defined waveform of MC-fluorescence that can be isolated against unmodulated background signals by using a digital optical lock-in detection approach or to control specific dipolar interactions on proteins. The protocols describe general procedures for the synthesis and spectroscopic characterization of NitroBIPS and specifically labeled conjugates along with methods for the manipulation of dipolar interactions on proteins and imaging of the MC-state of NitroBIPS within living cells.

INTRODUCTION

Fluorescence microscopy is widely used to image changes in the distribution of specific proteins and their complexes within living cells. However, fluorescence imaging in living cells and tissue is challenging when imaging low levels of fluorescently tagged protein. In particular, the signal from endogenous fluorescent molecules is often so high that the fluorescent protein of interest must be present at a very so high level in fact that it leads to the complete inhibition or super-activation of the protein activity. Moreover, autofluorescence signals, which amount to 1~10,000 fluorescein equivalents (Aubin, 1979), rule out super-resolution based imaging of single probe molecules in living cells. Various approaches have been used to reduce autofluorescence but more often these methods involve chemical fixation or stressing cells and to date there is no effect method to reduce autofluorescence signals within living, healthy cells.

We recently introduced a new imaging modality that allows for the isolation of specific modulated fluorescence signals from a new class of fluorophore against un-modulated signals arising from endogenous fluorophores (Marriott et al, 2008). Key to this approach is a probe or optical switch whose quantum yield for fluorescence emission can be modulated between two different values. Optical switches are characterized by their ability to undergo rapid and reversible, high-fidelity, orthogonal, optically-driven transitions between two states that have distinct structural or environmental and spectroscopic properties (Figure 1; Sakata et al., 2005a,b). Synthetic optical switches based on nitrospirobenzopyran (NitroBIPS), a well known photochrome (Fischer and Hirschberg, 1952), exists either as a pink colored, highly polarized and fluorescent merocyanine (MC) state or else in a colorless and poorly polarized spiro (SP) state (Figure 1). The colorless SP-state NitroBIPS changes to a colored MC-state by illumination with UV light and this MC-state can be turned back to the colorless SP-state by exposure to visible light or thermally. The excitation of colored MC-state results in decay of its excited state by either photochemistry to the UV-absorbing state or with emission of red photon to the same ground state. This process can be repeated many times through orthogonal, optical control of transitions between the two states and occurs without the release photoproduct (Inouye, 1994; Sakata et al., 2005b). Recent studies from our group have shown that the exposure of cells to short (50–100 ms) pulses of 365 nm light does not lead to any obvious their effects on cell health (Marriott et al., 2008; Sakata et al., 2008).

Figure 1.

Photochromic properties of benzospiropyran. The merocyanine (MC) state was generated by irradiation the spiropyran (SP) state with UV light (365 nm). While the excitation of MC-state with visible light (546 nm) generated SP-state.

The quantum yields for optical switching of NitroBIPS depends on general and specific solvent effects, with higher yields in apolar solvents and lower efficiency transitions occurring in bulk water. We found that attaching NitroBIPS to proteins increases the quantum yield for switching compared to bulk water and moreover, high-fidelity transitions are most likely when a single NitroBIPS probe is attached to a unique site on the protein rather than random labeling of lysine residues (Medintz et al., 2004; Patolsky et al., 1998). Highly selective labeling of probes on proteins is achieved through coupling of the probe to the thiol group of cysteine. Fortunately, most proteins contain few if any free thiol groups or else they can be introduced using site-directed mutagenesis. An alternative approach for specific labeling of proteins with optical switches is through reactions with suicide substrates and in special cases through reaction of amino groups on the N-terminus or ε-amino group of lysine residues. Here then we describe protocols for the synthesis of reactive NitroBIPS probes and subsequent spectroscopic and photophysical characterization of the optical switching properties and fluorescence emission of the protein conjugates. Finally, we show how NitroBIPS-derived probes and protein conjugates are employed in conjunction with optical lock-in detection image microscopy to achieve high contrast imaging of specific proteins or cellular structures within living cells.

STRATEGIC PLANNING

Design of Optical Switch Probes

The NitroBIPS probes used in these protocols are synthesized as described in Protocols 1 and 2. The nitro group, introduced to position 6 in the BIPS molecule is used to shift the ground state equilibrium of the photochromic molecule toward the MC-state (Flennery, 1968; Hirshberg and Fischer, 1954; Song et al., 1995). NitroBIPS probes harboring an amino or thiol reactive functional group are synthesized with the aim of coupling to amino-groups on the surface of micron scale latex beads or to proteins (Sakata et al., 2005a). Specifically, the amino reactive optical switch, 1′,3′,3′-trimethyl-5′-(N-succinimidyloxycarbonylpropyl)-6-nitro-spiro(2H-benzopyran-2,2′,3H-indole) (5′-NHS ester of NitroBIPS, 5), is synthesized in several steps from commercially available 4-(4-aminophenyl)butanoic acid and 5-nitrosalicylaldehyde, as described in Protocol 1. On the other hand, the thiol reactive optical switch, 1′,3′,3′-trimethyl-6-nitro-8-iodomethylspiro(2H-benzopyran-2,2′,3H-indole) (8-Iodo-NitroBIPS, 7), is readily synthesized in 2 steps from commercially available 1,3,3-trimethyl-2-methyleneindoline and 3-chloromethyl-5-nitrosalicylaldehyde, as described in Protocol 2.

BASIC PROTOCOL 1

SYNTHESIS OF 1′,3′,3′-TRIMETHYL-5′-(N- SUCCINIMIDYLOXYCARBONYLPROPYL)-6-NITRO-SPIRO(2H-BENZOPYRAN- 2,2′,3H-INDOLE) (5′-NHS ESTER OF NITROBIPS)

1′,3′,3′-Trimethyl-5′-(N-succinimidyloxycarbonylpropyl)-6-nitro-spiro(2H-benzopyran-2,2′,3H-indole) (5) is synthesized according to the scheme presented in Figure 2. First, the 2,3,3-trimethyl-5-(ethyloxycarbonylpropyl)-3H-indole (1) is prepared by diazotization of 4-(4-aminophenyl)butanoic acid with sodium nitrite in the presence of hydrochloric acid, followed by reduction with stannous chloride dihydrate to yield the hydrazine derivative. This product is subsequently condensed with 3-methyl-2-butanone in the presence of sulfuric acid in ethanol. The hydroxylation of 1 with aqueous sodium hydroxide yields 2,3,3-trimethyl-5-(carboxypropyl)-3H-indole (2), which is subsequently subject to methylation with methyl iodide to yield 1,2,3,3-tetramethyl-5-(carboxypropyl)- 3H-indoleninium iodide (3). Condensation of 3 with 5-nitrosalicylaldehyde in ethanol in the presence of triethylamine yields 1′,3′,3′-trimethyl-5′-(carboxypropyl)-6-nitro-spiro(2H-benzopyran-2,2′,3H-indole) (4). Finally, treatment of 4 with N-hydroxysuccinimide in the presence of DCC affords the amino reactive N-hydroxysuccinimide ester 5.

Figure 2.

Synthesis of 1′,3′,3′-trimethyl-5′-(N- succinimidyloxycarbonylpropyl)-6-nitro- spiro(2H-benzopyran-2,2′,3H-indole).

Materials

• 4-(4-Aminophenyl)butanoic acid

• 25-ml Round-bottom flask

• Magnetic bar

• Stirring hotplate

• Ice bath

• Sodium nitrite

• Micro-centrifuge tube

• Ice bucket

• Fume hood

• Stannous chloride dehydrate

• 4-mL Glass vial

• Hydrochloric acid

• Water

• Filter paper

• Buchner funnel

• Suction flask

• 3-Methyl-2-butanone

• Sulfuric acid

• Anhydrous ethanol (EtOH)

• Nitrogen gas

• Oil bath Condenser

• 125-ml Separatory funnel

• Flask

• Saturated aqueous sodium carbonate

• pH paper

• Diethyl ether (Et₂O)

• Anhydrous magnesium sulfate

• Rotary evaporator

• Vacuum pump

• Methanol (MeOH)

• 1 M Aqueous sodium hydroxide

• 1 M Aqueous hydrochloric acid (HCl)

• Ethyl acetate

• 5-Nitrosalicylaldehyde

• Silica gel thin layer chromatography (250 μm, 60 Å with fluorescent indicator

• UV₂₅₄)

• 254/365-nm hand-held UV lamp

• 0.5-Inch diameter chromatography column with frit disc

• Silica gel 70–230 mesh

• Dichloromethane (DCM)

• Hexane

• Ethyl acetate (EtOAc)

• 10-ml Round bottom flask

• N,N′-Dicyclohexylcarbodiimide (DCC)

• N-Hydroxysuccinimide (NHS)

• Anhydrous tetrahydrofuran (THF)

Synthesis of 2,3,3-trimethyl-5-(carboxypropyl)-3H-indole (2)

• 1Suspend 4-(4-aminophenyl)butanoic acid (100 mg, 0.558 mmol) in a 1:1 mixture of concentrated hydrochloric acid (HCl) and water (2 ml) in a 25-ml round bottomed flask. Stir at 0 °C in an ice bath using a stirring plate.
• 2Weigh 42 mg of sodium nitrite (NaNO₂, 0.609 mmol) and add into a micro-centrifuge tube. Add 500 μl of water and pipette or sonify until a clear solution is obtained. Cool the solution on ice.
• 3Add, drop-wise, the sodium nitrite solution into the suspension obtained from step 1. Stir the mixture at 0 °C for 1 hour.

  The suspension will develop a clear yellow solution. It will generate gas bubbles.

• 4While waiting for the reaction in step 3, weigh 377 mg of stannous chloride dihydrate (SnCl₂•2H₂O, 1.670 mmol) and deposit into glass vial. Add 500 μl concentrated HCl and pipette or sonify until the solution clarifies.
• 5Add, drop-wise, the stannous chloride dihydrate solution into the reaction mixture from step 3. Stir at 0 °C for 1 hour and then filter to yield a white milk colored solid product.

  When adding the stannous chloride dihydrate solution, a white solid and gas bubbles are generated. The suspension should be cooled to precipitate the product. The suspension may be left in refrigerator for 2 hours before filtration.

• 6Dissolve the crude product from step 5 in 5 mL of ethanol in 25-ml round bottom flask. Add 729 μl of 3-methyl-2-butanone (1.125 mmol) and 56 μl of concentrated sulfuric acid (H₂SO₄, 0.560 mmol). Stir under reflux in an oil bath for 12 hours using a water-cooled condenser.
• 7Concentrate the reaction mixture by evaporation under reduced pressure. Add 10 ml of water, neutralize with aqueous sodium carbonate to pH 6–7, and extract with diethyl ether. Combine the organic extracts, dry over anhydrous magnesium sulfate and then evaporate under reduced pressure to dryness.

  During neutralization, the product will precipitate as evidenced through the yellow suspension.

• 8Dissolve the crude product in 1 ml of methanol in a 25-ml round bottom flask. Add 5 ml of 1 M aqueous sodium hydroxide and stir at room temperature for 1 hour.
• 9Add 1 M of aqueous HCl to the reaction mixture until a pH of ~1–2 is reached. Extract the product with ethyl acetate, combine the organic layers and dry over anhydrous magnesium sulfate followed by evaporation under reduced pressure yielding 98 mg of 2,3,3-trimethyl-5-(carboxypropyl)-3H-indole (2, 0.4 mmol).

Synthesis of 1,2,3,3-tetramethyl-5-(carboxypropyl)-3H-indoleninium iodide (3)

• 10Place the 98 mg of 2 (0.4 mmol) in a 25-ml round bottom flask and add 5 ml of DCM. Add 200 μl of methyl iodide (3.2 mmol) then stir and reflux (60 °C) in an oil bath for 12 hours using a water-cooled condenser apparatus.
• 11Cool the reaction mixture to room temperature and evaporate to dryness to yield a crude product 3, which is further dried using a vacuum pump.

Synthesis of 1′,3′,3′-trimethyl-5′-(carboxypropyl)-6-nitro-spiro(2H-benzopyran- 2,2′,3H-indole)(5′-carboxy-NitroBIPS, 4)

• 12Dissolve the crude product 3 in 8 ml of anhydrous EtOH in a 25-ml round bottom flask.
• 13Add 67 mg of 5-nitrosalicylaldehyde (0.4 mmol) and 56 μl of triethylamine (0.4 mmol) into the solution from step 12. Then stir and reflux in an oil bath under a water-cooled condensation apparatus for 2 hours.

  During refluxing the solution turns to a purplish red color.

• 14Cool the reaction down to room temperature and evaporate under reduced pressure to dryness.
• 15Dissolve the crude product in a small volume of DCM and load onto the top of a silica gel flash column equilibrated in hexane.
• 16Elute the column with a mixture of hexane and ethyl acetate (6/4). Combine the pure 5-carboxy-NitroBIPS-containing fractions using TLC as a guide and evaporate the product to dryness.

Synthesis of 1′,3′,3′-trimethyl-5′-(N-succinimidyloxycarbonylpropyl)-6-nitro- spiro(2H-benzopyran-2,2′,3H-indole) (5-NHS ester of NitroBIPS, 5)

• 17Place 122 mg of 4 (0.3 mmol), 93 mg of DCC (0.45 mmol), and 52 mg of NHS (0.45 mmol) in a 10-ml round bottom flask.

  Glassware and starting material should be dried for a more efficient reaction.

• 18Add 5 ml of anhydrous THF and stir the mixture overnight under N₂ gas at room temperature.
• 19Filter off the precipitate. Evaporate the filtrate to dryness to yield the 5′-NHS ester of NitroBIPS (5).

BASIC PROTOCOL 2

SYNTHESIS OF 1′,3′,3′-TRIMETHYL-6-NITRO-8-IODOMETHYLSPIRO(2H- BENZOPYRAN-2,2′,3H-INDOLE) (8-IODO-NITROBIPS, 7)

This compound is prepared by a halogenation exchange reaction of 1′,3′,3′-trimethyl-6-nitro-8-chloromethylspiro(2H-benzopyran-2,2′,3H-indole), which is synthesized by condensation of commercially available 1,3,3-trimethyl-2-methyleneindoline and 3-chloromethyl-5-nitrosalicylaldehyde. The synthetic scheme for 7 is presented in Figure 3.

Figure 3.

Synthesis of 1′,3′,3′-trimethyl-6-nitro-8-iodomethylspiro(2H-benzopyran- 2,2′,3H-indole).

Materials

• 1,3,3-Trimethyl-2-methyleneindoline

• 3-Chloromethyl-5-nitrosalicylaldehyde

• Anhydrous tetrahydrofuran (THF)

• Magnetic bar

• Stirring hotplate

• Nitrogen gas

• Fume hood

• Oil bath

• Condenser

• Rotary evaporator

• Vacuum pump

• Acetone

• Potassium iodide (KI)

• Filter paper

• Buchner funnel

• Suction flask

• Silica gel thin layer chromatography (250 μm, 60 Å with fluorescent indicator UV254)

• 254/365-nm hand-held UV lamp

• 0.5-Inch diameter chromatography column with frit disc

• Silica gel 70–230 mesh

• Dichloromethane (DCM)

• Hexane

• Ethyl acetate (EtOAc)

Synthesis of 1′,3′,3′-Trimethyl-6-nitro-8-chloromethylspiro(2H-benzopyran-2,2′,3H- indole) (8-Cloro-NitroBIPS, 6)

• 1Place 177 μl of 1,3,3-trimethyl-2-methyleneindoline (1 mmol) and 216 mg of 3-chloromethyl-5-nitrosalicylaldehyde (1 mmol) into a 25-ml round bottom flask.
• 2Add 10 ml of anhydrous THF and stir under reflux in an oil bath for 2 hours.
• 3Cool the reaction to room temperature and evaporate under reduced pressure to dryness.

Synthesis 1′,3′,3′-Trimethyl-6-nitro-8-iodomethylspiro(2H-benzopyran-2,2′,3H- indole) (8-Iodo-NitroBIPS, 7)

• 4Dissolve the crude product 6 in 10 ml of acetone in a 25-ml round bottom flask.
• 5Add 664 mg of anhydrous potassium iodide (4 mmol) to the solution and stir under N₂ gas at room temperature overnight.
• 6Filter off the precipitate. Evaporate the filtrate to dryness.
• 7Dissolve the crude product from step 6 into a small volume of DCM and load onto the top of a silica gel flash column equilibrated with hexane.
• 8Elute the column with a mixture of hexane and ethyl acetate (10/1). Combine the pure 8-Iodo-NitroBIPS-containing fractions using TLC as a guide and evaporate the product to dryness.

BASIC PROTOCOL 3

ABSORPTION CHARACTERIZATION OF NITROBIPS

The optical switch NitroBIPS undergoes rapid and reversible, optically-driven transitions between the SP- and MC-states. These two states have different UV-visible absorption spectra. Only the MC-state of NitroBIPS has an absorption band in visible region. The SP to MC transition is generated upon irradiation of the SP-state with near ultraviolet light (365 nm) while the MC- to SP-state is generated upon excitation of the MC-state with visible light (546 nm). The following protocol describes the method to measure the absorption spectra of NitroBIPS in the SP- and-MC states.

Materials

• 1′-,3′-Dihydro-1′,3′,3′-trimethyl-6-nitrospiro(2H-1-benzopyran-2,2′-(2H)-indole) (NitroBIPS) stock solution in methanol: 5 mM (Sigma-Aldrich, see recipe)

• Methanol (MeOH)

• 2- or 4-Window quartz cuvettes (Hellma)

• Spectrophotometer (Shimadzu PC1601)

• Hand-held UV lamp (365 nm)

• 530 nm output of an LED or 546 nm output of a 100 Watt mercury arc lamp

Prepare the NitroBIPS solution for the absorption measurement

• 1Prepare 1 ml of 50μM a NitroBIPS solution in MeOH by pipetting 10μl of a 5 mM NitroBIPS stock solution to 990μl of MeOH in a micro-centrifuge tube. Mix well by repeated pipetting.

  The 1 ml volume is suitable for a semimicro cuvette. In case of standard or macro cuvette (1 cm × 1 cm × 4 cm) one requires 3 ml of the NitroBIPS solution.

Absorption measurement

• 2Warm up deuterium and tungsten lamps about 15 minutes.
• 3Set the wavelength range from 250–750 nm in fast scan mode.
• 4Fill both cuvettes (sample and reference) with MeOH and record a reference spectrum over the same wavelength range (250–750 nm).
• 5Fill the sample cuvette with a freshly prepared NitroBIPS solution. The thermodynamically stable form of NitroBIPS in MeOH is the SP-state. Fill the reference cuvette with MeOH. Measures the absorption spectrum of the SP-state of NitroBIPS between 250–750 nm.

  If the absorption of interest peaks is higher than 2 absorbance units, dilute the sample and measure again. Ideally, the maximum absorption should be around 0.3.

• 6Irradiate the sample cuvette with 365 nm light for 1 minute using a hand-held UV-lamp. This will convert the SP-state of NitroBIPS to the MC-state.
• 7Immediately record the absorption spectrum of the MC-state of NitroBIPS.
• 8Irradiate the sample from step 7 with visible light (530 nm or 546 nm) to convert the MC-state of NitroBIPS to the SP-state.
• 9Record the absorption spectrum of the photo-generated SP-state of NitroBIPS.
• 10Repeat the operations from step 6 to step 9 to demonstrate high fidelity switch between the SP- and MC-states of NitroBIPS in MeOH.

  Figure 4 shows the absorption spectra of NitroBIPS in the SP- and MC-states in MeOH during a single cycle of optical switching.

Figure 4.

UV absorption spectra of NitroBIPS in SP- and MC-state in MeOH during a single cycle of optical switching.

BASIC PROTOCOL 4

FLUORESCENCE CHARACTERIZATION OF NITROBIPS

As described above NitroBIPS has two distinct and optically inter-convertible states, the colorless SP-state and the colored MC-state. Excitation of MC with 546 nm light generates the MC-excited state, which decays by one of two independent processes. The first process is the excited-state intramolecular reaction that reforms the SP-state and proceeds with a quantum yield as high as 0.8 in apolar solvents (Hobley et al., 2002), whereas the competing process involves the return of the excited MC to the same MC-ground state with emission of a red photon at ~618 nm. This reaction has a low quantum yield between 0.05 and 0.1 (Chibisov and Görner, 1997). The following protocol describes the method used to record the fluorescence emission spectrum of MC-NitroBIPS.

Materials

• 1′,3′-Dihydro-1′,3′,3′-trimethyl-6-nitrospiro(2H-1-benzopyran-2,2′-(2H)-indole) (NitroBIPS) stock solution in methanol: 5 mM (Sigma-Aldrich, see recipe)

• Methanol (MeOH)

• 4-Window quartz cuvettes (Hellma)

• Fluorescence spectrophotometer (SLM-AB2)

• Hand-held UV lamp (365 nm)

• 530 nm output of an LED or 546 nm output of a 100 Watt mercury arc lamp

Prepare the NitroBIPS solution for fluorescence spectrum measurement

• 1Prepare a sample solution as detailed in Basic Protocol 2.

  Only the MC-state of NitroBIPS has a red fluorescence emission.

• 2Warm up the xenon arc lamp in the fluorometer for about 15 minutes.

Emission spectrum

• 3Find the maximum visible absorption wavelength of NitroBIPS using Protocol 2- it should be close to 530 nm.
• 4Fix the excitation wavelength at maximum MC-absorption as indicated in step 3 (530 nm) and scan the fluorescence emission between 400–750 nm to get a better idea of the maximum emission wavelength.
• 5Measure the fluorescence spectrum of freshly prepared MC-state of NitroBIPS. To avoid the so-called inner filter effect it will be necessary to decrease the absorption of the MC-state at 530 nm to an absorption value that is less than 0.2

  Irradiation of the NitroBIPS solution with 365 nm light for about 1 minute will generate the MC-state of NitroBIPS - record the fluorescence intensity immediately. If the spectrum is off-scale, adjust the sensitivity (voltage) and measure the MC-fluorescence intensity again.

• 6Identify the emission wavelength that gives the maximum fluorescence intensity around 610~620 nm.

Excitation spectrum

• 7Fix the emission wavelength at 620 nm from previous experiment. Record the excitation spectrum of the MC-state of NitroBIPS between 400~600 nm.
• 8Identify the maximum excitation wavelength from the spectrum, around 530 nm. In some cases the excitation spectrum may need corrected to account for variation in the energy of the mercury arc lamp over the range of the excitation wavelengths.

Fluorescence emission spectrum

• 9Fix the excitation and emission wavelengths from the previous experiments (excitation at 535 nm and emission at 630 nm).
• 10Collect fluorescence spectrum of NitroBIPS between 550–750 nm.

  Figure 5 is the fluorescence emission spectrum of NitroBIPS in MeOH.

Figure 5.

Fluorescence spectra of NitroBIPS in MeOH in SP- and MC-state.

BASIC PROTOCOL 5

MICROSCOPIC CHARACTERIZATION OF NITROBIPS

The 5′-NHS ester of NitroBIPS (5) was designed to covalently link NitroBIPS to amino derivatized latex beads, and proteins through their lysine residues, for microscope-based imaging for the characterization of optical switching between the SP- and MC-states. The red MC-fluorescence is used to image the NitroBIPS labeled bead and to record the kinetics of transitions between the SP- and MC-states.

Materials

• Polybead^® amino microspheres: 3 μm (Polysciences, Inc.)

• Phosphate buffer saline (PBS): 1× (see recipe)

• 5′-NHS ester of NitroBIPS, stock solution in DMF: 5 mM (see recipe)

• Glass-bottom petri dish: 35 mm diameter incorporating a 14 mm glass coverslip, number 0 thickness (Mat Tek Corporation)

• Inverted fluorescence microscope (Zeiss) adapted for pulse probe microscopy (see Figure 6)

• Two of 100 Watt mercury arc lamp

• Computer controlled mechanical shutter (Melles Griot)

• Interference filter for excitation of Cy3 emission

• UV-pass filter (UG11, Schott glass)

• EMCCD camera (Hamamatsu)

Figure 6.

Schematic represent the inverted fluorescence microscope (Zeiss) adapted for pulse probe microscopy.

Preparation of NitroBIPS-labeled latex beads

• 1Stir a suspension of amino-derivatized latex beads and add 1 drop into a micro-centrifuge tube. Add 200 μl of PBS and suspend by pipetting.
• 2Add 10 μl of 5 mM of a 5′-NHS ester of NitroBIPS stock solution to suspension of beads. Mix well by pipetting or vortexing.
• 3Stir the sample at 4 °C for 2 hours.
• 4Centrifuge for 5 minutes at 10,000 ×g. Discard supernatant and resuspend pellet in 500 μl of PBS.

  This step is used to remove free, unbound 5′-NHS ester of NitroBIPS.

• 5Repeat step 4 for four times. Resuspend the final pellet with 200 μl of PBS.
• 6Place 2 μl of the NitroBIPS-labeled beads suspension on the cover glass/petri dish.

Microscope based optical switching of NitroBIPS labeled latex beads

• 7Turn on the microscope lamps and EMCCD camera 15 minutes before the measurement.
• 8With reference to Figure 6, use the in-plane 100W Hg-arc lamp with a closely associated 546 nm interference filter for continuous excitation of the sample with 546 nm light. Remove the Cy3 excitation filter from the filter cube set.
• 9Use a second Hg-arc lamp with integrated UG11 filter and shutter, orthogonally aligned to the 546 nm excitation light path, to deliver pulses of 365 nm light to the sample. Direct the 365 nm light into the excitation optical path using a dichroic mirror from a fluorescein filter set aligned 45° with respect the excitation light path. Set the shutter controls to deliver pulses of 365 nm light to the sample with 100 ms duration every 1 second.
• 10Use a 100× oil immersion objective and immersion oil to image the MC- fluorescence from beads.
• 11Adjust the coarse focusing knob to view beads bound to the surface the coverslip and use the fine focus knob to generate a sharper image of the immobilized beads.

  During focusing, both UV light and green light must be turned on to drive the SP to MC transition and to elicit emission of red fluorescence from MC-state.

• 12Record the MC-fluorescence intensity from individual beads on the EMCCD camera at video rate and store the images as a single movie file.
• 13Initiate recording of the MC-fluorescence movie in concert with activating the mechanical shutter over at least 5 cycles.

  Set the camera frame rate at 33 frames/s or as fast as possible.

Analyze data

• 14Select the area of interest from beads in the movie file recorded from step 13. Use the integrated software on the camera or a dedicated program to measure the average intensity of MC-fluorescence within selected areas for each frame in the movie.
• 15Export the measured data, plot and analyze the average MC-fluorescence intensity as a function of time using an excel spreadsheet or other graphic software.

  When using a 100 ms UV pulse, the first few frames collected at 33 f/s that follow the 365 nm pulse may be buried within the signal arising from the tail of the 365 nm pulse. These overlap frames can usually be deleted without affecting the analysis.

  Figure 7 shows the fluorescence intensity changes corresponding to optical switch cycles of NitroBIPS in individual beads over 8 cycle of optical switching.

Figure 7.

(A) Fluorescence intensity image of NitroBIPS-labeled beads. (B) The MC-fluorescence change corresponding to optical switch cycles of NitroBIPS in individual bead (red circle in A) over 8 cycle of optical switching.

PROTOCOL 6

ACTIN-LABELED 8-IODO-NITROBIPS

The thiol reactive optical switch, 8-Iodo-NitroBIPS (7), was designed for coupling NitroBIPS to proteins via their cysteine residues. In this protocol, 8-IodoNitroBIPS is conjugated to G-actin via alkylation of cysteine-374. The conjugate is purified from free probe using size-exclusion chromatography. The process for coupling and purification of the NitroBIPS conjugate of G-actin is described below.

Materials

• Purified lyophilized actin (Cytoskeleton)

• G-buffer (see recipe)

• G-buffer with DTT (see recipe)

• 8-Iodo-NitroBIPS stock solution in DMF: 5 mM (see recipe)

• Micro-centrifuge tube

• Pipette

• Pitette tips

• PD-10 column (GE Healthcare)

Preparation of G-actin conjugated 8-Iodo-NitroBIPS

• 1Resuspend 1 mg of actin powder with 500 μl of G-buffer and transfer to a 1.5-ml micro-centrifuge tube. Add 660 μl of G-buffer and mix well by gentle pipetting to generate a 20 μM solution of G-actin.
• 2Pipette 500 μl of the G-actin solution from step 1 to a micro-centrifuge tube, add 4 μL of 5 mM 8-Iodo-nitroBIPS stock solution and mix rapidly, and thoroughly, by vortexing for a few seconds. Incubate the reaction at room temperature in the dark for 1 hour.

  The reaction can also proceed overnight at 4 °C in the dark.

Purification of NitroBIPS-labeled G-actin

• 3Gently apply the solution (500 μl) from step 2 on top of a PD-10 column equilibrated in G-buffer with DTT.
• 4Elute the labeled conjugate with G-buffer with DTT. Collect fractions of 1 ml and use absorption at 350 nm to identify the protein conjugate. For a PD-10 column the protein will usually elute after 2 ml and is complete by 5 ml. Pool the fractions containing the conjugate.

  The pool of eluted conjugate is usually diluted about two-fold compared to the applied volume.

Absorption spectroscopy of the G-actin conjugate

• 5Follow the same procedure detailed in Protocol 3.

  Figure 8 shows the absorption spectrum of NitroBIPS-G-actin conjugate in the SP- and MC-state.

Figure 8.

UV absorption spectra of NitroBIPS-G-actin conjugate in the SP- and MC- state.

PROTOCOL 7

LIVE CELL IMAGING OF NITROBIPS

Measurements of MC-fluorescence intensity may be used to quantify the state of NitroBIPS optical switch, i.e., the percentage of SP- and MC-state, in the sample at any time while providing a sensitive signal to image the distribution of the MC-state of NitroBIPS in cells. This protocol details the method used to record and image the MC-fluorescence of NitroBIPS in living cells.

Materials

• HEK-293 cells

• Cell culture medium (see recipe)

• Glass-bottom petri dish: 35 mm diameter incorporating a 14 mm glass coverslip, number 0 thickness (Mat Tek Corporation)

• Water bath

• Incubator (37 °C) with 5 % carbon dioxide and moisture

• Tissue culture hood

• 8-Iodo-NitroBIPS stock solution in DMF: 5 mM (see recipe)

• Pipette

• Pipette tips

• Inverted fluorescence microscope (Zeiss) adapted for pulse probe microscopy (see Figure 6)

• Two of 100 Watt Mercury arc lamp or laser power source

• Computer controlled mechanical shutter (Melles Griot)

• Interference filter for excitation of Cy3 emission

• UV-pass filter (UG11, Schott glass)

• EMCCD camera (Hamamatsu)

Culture cell

• 1Grow HEK-293 cells in a glass-bottom petri dish. The final volume of the cell culture medium in the dish is 2 ml. Check the cell density every day until 60–80 % confluent.

Prepare BIPS sample

• 2Pipette 2 ml of cell culture medium into a sterile micro-centrifuge tube.
• 3Pipette 4μl of a 5 mM solution of 8-Iodo-NitroBIPS stock solution into the micro-centrifuge tube from step 2 (the final concentration is 10 μM). Mix well by pipetting.

Loading BIPS into living HEK 293 cell

• 4Remove the old cell culture medium by suction or pipetting.
• 5Rapidly add the new cell culture medium containing 8-Iodo-NitroBIPS from step 3.
• 6Incubate cells in a CO₂-incubator for 1 hour.
• 7Remove the old cell culture medium and add new 2 ml of fresh cell culture medium.
• 8Repeat step 7 for 3 times to remove any free 8-Iodo-NitroBIPS.

Imaging of living cell

• 9Follow the procedure detailed in Protocol 5: steps 7–13

Analyze data

• 10Follow the procedure detailed in Protocol 5: steps 14–15.

  Figure 9 shows the MC-fluorescence intensity change in living HEK-293 cell during several cycles of optical switching.

Figure 9.

(A) Fluorescence intensity image of NitroBIPS in live HEK-293 cell. (B) The MC-fluorescence change of NitroBIPS in a region of interest (red circle in A) in live HEK-293 cell during optical switch cycles.

REAGENTS AND SOLUTIONS

Use Milli-Q water in all recipes and protocol steps.

NitroBIPS 5 mM stock solution

• 1 mg NitroBIPS

• Dissolve in 620 μl MeOH

• Store at −20 °C

Phosphate buffered saline (1×)

• 8 g sodium chloride

• 0.2 g potassium chloride

• 1.44 g sodium phosphate dibasic (Na₂HPO₄)

• 0.24 g potassium phosphate monobasic (KH₂PO₄)

• Dissolve in 800 ml water.

• Adjust pH to 7.4 with HCl. Add water to 1000 ml

• Store at room temperature.

5′-NHS ester of NitroBIPS 5 mM stock solution

• 1 mg 5′-NHS ester of NitroBIPS

• Dissolve in 395 μl DMF

• Store at −20 °C

G-Buffer

• 5 mM Tris-HCl

• 0.2 mM CaCl₂

• 0.2 mM ATP

• Dissolve in 1000 ml water and adjust pH to 8.0

• Store at 4 °C

G-Buffer with DTT

• 1 mM DTT

• Dissolve in G-buffer

• Store at 4 °C

8-Iodo-NitroBIPS 5 mM stock solution

• 1 mg 8-Iodo-NitroBIPS

• Dissolve in 432 μl DMF

• Store at −20 °C

Cell culture medium

• 10% fetal bovine serum

• 100 units/ml penicillin G sodium

• 100 μg/ml streptomycin

• 4 mM L-glutamine

• Add to 500 ml Dulbecco’s Modified Eagle’s Medium (DMEM)

• Store at 4 °C and warm to 37 ° before use.

COMMENTARY

Background Information

A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cell processes and behavior. Since signaling pathways are often initiated by small number of receptor proteins and confined to specific loci in the cell, these studies will require microscope techniques and development of new probes that can generate high contrast and high-resolution images of changes in the distribution and interactions of specific proteins in a cell (Betzig et al., 2007; Yan and Marriott, 2003). We are improving these imaging techniques through the development of new optical switch reagents. Nitrospirobenzopyran (NitroBIPS) is an optical switch that undergoes, rapid and reversible, optically-driven deterministic transitions between an SP- and MC-state (Bertelson, 1999; Seto, 1990). The SP to MC transition is triggered by exciting the SP-state with near ultraviolet light from 320~420 nm and has a high 2-photon absorption cross-section, which peaks at 720 nm. The MC-state on the other hand has an absorption maximum that is sensitive to its molecular environment and ranges from 485 nm to 585 nm (Mao et al., 2008; Sakata et al., 2005a; Sakata et al., 2005b). Excitation of the MC-state of NitroBIPS with 546 nm light generates the MC-excited state that can decay via two independent processes each with a unique quantum yield. The first process is an excited state intramolecular reaction that reforms the SP-state and has a high quantum yield (Hobley et al., 2002) whereas the second process involves a return back to the ground state with the emission of a red photon and has a low quantum yield between 0.05 and 0.1 (Chibisov and Görner, 1997). However, even while weak, the MC-fluorescence of NitroBIPS is useful for imaging and analysis of the progress of optical switching in cells and tissue (Marriott et al., 2008; Mao et al., 2008; Sakata et al., 2008). Other optical switches are known having MC-state absorption spectra, such as spironaphthoxazine (NISO) that allow for orthogonal control of two switch probes (Sakata et al., 2005a). The modulated signal from the optical switch is truly unique and can be isolated from all other unmodulated signals such as autofluorescence using an optical lock-in detection (OLID) approach developed in Marriott et al. (2008). We are currently improving the fluorescence quantum yield of optical switch probes to increase the sensitivity of OLID microscopy to the detection level of single molecules.

Critical Parameters

The purification process using silica gel column chromatography should be done as quickly as possible in order to avoid complications from the acidity of silica gel, light, and the solvent. In particular, these effects can generate the MC-state of NitroBIPS, which is retained in the column and decreases the yield. To ensure a high yield of product, organic solvents should be anhydrous grade while performing the reaction under an atmosphere of nitrogen gas to reduce oxygen and moisture. Of course all glassware used for these reactions should be oven-dried.

For microscope based characterization of optical switching of NitroBIPS, it is best to use a sub-saturating level of illumination. A 100 ms UV pulse in this experiment is used to convert a sub-population of NitroBIPS from SP- to MC-state; however, complete conversion of molecules in the image field can be realized by using a longer irradiation time and higher power. The profile of the MC-fluorescence intensity decay depends on the energy of the 546 nm illumination energy. The higher the illumination energy, the faster the MC-fluorescence will decay.

Troubleshooting

When characterizing the optical switching efficiency of NitroBIPS using MC-fluorescence, some attention should be given to improving the fluorescence signal from MC through control of the illumination energy of the 365nm and 546 nm light. In general, one wants to generate as much MC using 365 nm illumination in the shortest period of time. On the other hand, it is necessary to record several data points to quantify the decay of MC-fluorescence using 546 nm light. If the energy of the 546 nm light is too high then the MC-state will rapidly convert to the SP-state, i.e., faster than the video capture rate. In this case it is possible to reduce the energy of the 546 nm light either by using the light level control switch on the lamp or by inserting a neutral density filter in the 546 nm light path.

Anticipated Results

For Protocol 1 in step 5, if the suspension is not cooled then one can expect a lower yield of product. If the purification of the product using silica gel is rapid one can expect yields for 5-carboxy-NitroBIPS (4) and 8-Iodo-NitroBIPS of about 80%. If the glassware, solvent, and starting material are dry, the yield of 5′-NHS ester of NitroBIPS (5) is usually close to 100%.

Time Considerations

The synthesis of 5′-NHS ester of NitroBIPS requires about 4 days, with one overnight reaction for preparation of indole, an overnight reaction for the methylation and an overnight reaction for the preparation of the NHS derivative. The synthesis of 8-Iodo-NitroBIPS requires 1 day with an overnight reaction for the halogenation exchange. The coupling of 5′-NHS ester of NitroBIPS to amino-latex beads requires 2 hours at room temperature, but this could be lengthened to increase the labeling ratio. Characterization of the optical switch in solution can be completed in one day. Characterization of the optical switch in the microscope including optimization of light levels can be realized within 1 day. Labeling of proteins with 8-Iodo-NitroBIPS and purification of the conjugate can be realized within 1 day. Growing HEK-293 cells to ~60–80% of confluent requires 1–3 days. The procedure to load and image 8-Iodo- NitroBIPS within cells can be carried out within 1 day.