Materials
All reagents were purchased from Sigma Aldrich and used as received except where specified. Methylene chloride was dried using a solvent purification system. Cellulose nanofibrils were obtained from the University of Maine and used as received (AR-CNFs) or autoclaved at 100 oC in base to remove lignin (AC-CNFs).
Fluorescence Lifetime Imaging Microscopy
FLIM was conducted on a custom build microscope at the National Institute of Standards and Technology (NIST). The excitation was performed using a Ti-Sapphire laser with a pulse width of 140 fs passed through SHG frequency doubling optics that emitted at 514 nm with an average pulse power of 0.5 μW. The sample excitation used an air objective with a numerical aperture of 0.75. The images were collected by raster scanning the laser focus using an X-Y piezo stage. The resulting fluorescence was collected through the same objective and sent to single photon counting modules (Becker and Hickl) for lifetime measurements through a notch filter to remove excitation light. The images were then analyzed using SPCImage NG software package and thresholded at 50 counts to remove background noise. The decay curves were fit using an algorithmically estimated IRF for each pixel. Phasors were generated using the time domain methodology with equation 1 and equation 2, where g(w) and s(w) are x and y coordinates of a cartesian plot. The phasors were then exported and replotted using Origin Pro 2019.
Where w is the angular repetition frequency of the excitation source; n is the harmonic frequency, I(t) the decay at each time in each pixel.
Synthesis of 4-(2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)phenol (PPB).
PPB was synthesized using a modified procedure common to the literature.39 4-hydroxy benzaldehyde (219.0 mg, 1.79 mmol) and freshly distilled 3-ethyl-2,4-dimethylpyrrole (379.8 mg, 3.42 mmol) were dissolved in 5 mL of methylene chloride under nitrogen atmosphere. One drop of trifluoroacetic acid (TFA) was added and the reaction was stirred for 1.5 h after TLC confirmation of aldehyde consumption. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 488.0 mg, 2.5 mmol) dissolved in 5 mL methylene chloride was added to the solution through a purged syringe. The reaction was stirred for 1 h. The reaction flask was lowered into an ice bath and brought to 0 oC. Diisopropylethylamine (2.55 g, 17.9 mmol) was added using a purged syringe dropwise and the reaction was stirred for 0.5h. While still in the ice bath, boron trifluoride etherate (2.32 g, 17.9 mmol) was added using a syringe, dropwise due to the presence of an exotherm. The reaction was stirred for 10 h. The reaction mixture was dispersed in 100 mL methylene chloride and washed three times with 100 mL saturated aqueous sodium bicarbonate, followed by three 100 mL washes with deionized water. The organic layer was dried over anhydrous sodium sulfate. PPB was isolated via column chromatography as a vibrant red solid with silica gel as the stationary phase and chloroform/ethyl acetate/hexanes (v:v, 1:1:3) solvent as mobile phase in a 25% yield. 1HNMR: (CDCl3): d (ppm) 7.1 (m, 2H, aromatic), 6.9 (m, 2H, aromatic), 2.5 (3H, S, CH3), 2.3 (4H, q, CH2), 2.2 (3H, S,CH3), 1.3 (9H, S, CH3), 1.0 (3H, t, CH3). Mass Spec, +M 396.4
Synthesis of 10-(4-(3,5-dichlorophenoxy)phenyl)-2,8-diethyl-5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine (mDTEB)
PPB (10 mg, 18.3 μmol) and potassium carbonate (10 mg, 72.0 mmol) were placed in dry THF (2 mL) in a two necked round bottom flask under an argon atmosphere. The flask was lowered into an ice bath and allowed to stir for 30 minutes. Cyanuric chloride dissolved in dry THF (1 mL) was added using a purged syringe dropwise to the reaction mixture. The reaction was stirred for 10 h. The mDTEB was isolated by column chromatography using acidic alumina as the stationary phase and hexanes/ethylacetate (v:v 1:1) as the mobile phase. The dye structure was confirmed using 1H NMR and used immediately to functionalize the cellulose. 1HNMR: (CDCl3): d (ppm) 7.2 (m, 2H, aromatic), 6.6 (m, 2H, aromatic), 2.5 (3H, S, CH3), 2.3 (4H, q, CH2), 2.2 (3H, S,CH3), 1.3 (9H, S, CH3), 1.0 (3H, t, CH3).
Cellulose Labeling.
Attachment of mDTEB to CNF
Scheme 2. mDTEB was dissolved in acetone (4mg/mL) and added to a stirring dispersion of CNFs in a 1 mmol solution of aqueous sodium carbonate. Cellulose repeat unit is shown as a tetrasaccharide of glucose to make the chemistry more readily observable.
Cellulose nanofibrils (CNFs with 97 mass% water) from the University of Maine (4.5 g of dry wt.) were added to Na2CO3 solution (150 mL 50 mM) and stirred for 30 min. An additional 100 mL of water was added to facilitate stirring. mDTEB (2 mg) was dissolved in 500 µL of acetone, then added to the alkaline CNF suspension and stirred for 72 h in the dark at room temperature. When the reaction was completed, the modified CNFs were isolated by centrifugation (500 rad/s (5000 rpm) for 20 min). After centrifugation, the excess of mDTEB was removed by washing labeled CNFs with 3 x 100 mL of an ethyl acetate – water mixture. Purification was carried out using a Speed Mixer (DAC 400 Mixer range, FlackTek INC, US) at 157rads/s 1500 rpm for 10 min followed by centrifugation at 4000 rpm for 10 min. The purification step was repeated until no fluorescent signal was detected in the washing liquor. The CNFs were resuspended in water and repeatedly centrifuged until the ethyl acetate solvent was completely removed. The labeled CNFs were analyzed using a NIST built, time correlated, single photon counting FLIM instrument utilizing a femtosecond Ti-sapphire laser. Samples were excited using 514 nm laser light. An air objective with a numerical aperture of 0.9 was used for imaging. The FLIM images were acquired by building up fluorescence decay curves at each pixel. The decay curve was subsequently fit using a single or multi exponential to determine the lifetime at that pixel. The images presented are 30 μm x 30 μm (256 pixels x256 pixels) with an integration time of 40 ms/pixel.