Fluorescence lifetime probes reveal organelle viscosity
was dissolved in DCM (60 ml) and DDQ (0.46 g, 2 mmol) was added and the reaction stirred
under N2, shielded from light for 45 minutes. Then triethylamine (0.87 ml, 5.88 mmol) was
added followed immediately by the addition of BF3. (OEt2)2 (0.62 ml, 5.02 mmol) and the reac-
tion was stirred at room temperature overnight. The reaction mixture was washed consecu-
tively with water (100 ml), NH4Cl (100 ml, 0.5 M), NaHCO3 (100 ml, 0.5M) and finally with
water (100 ml). The organic layer was dried over MgSO4, filtered and evaporated to give a dark
viscous oil which was purified by Biotage automated chromatography [DCM/EtOAc] to give
BODIPY 3 as an orange-green solid 0.13 g (15.5%) BODIPY 3 (0.1 g, 0.24 mmol) and triphe-
nyphosphine TPP (0.19 g, 0.72 mmol) were reacted as a solid melt at 90˚C, after 8 hrs TLC
[Silica gel: DCM/Hexane 8:2] still showed the presence of BODIPY 3 as well as an orange fluo-
rescent base-line spot which moved as a single spot on TLC [Silica gel: DCM/Acetone/MeOH
10:2:1]. A further portion of TPP (0.19 g, 0.72 mmol) was added and heating continued for a
further 8 hrs after which the reaction was complete by TLC. The reaction was cooled to room
temperature, resulting in a red-brown solid that was triturated with diethyl ether, filtered and
dried to give a red-brown powder. This was purified by flash chromatography [Silica gel:
DCM/Acetone/MeOH 10:2:1], the crude was loaded on top of the column as a concentrated
solution in DCM, eluted with 5 column volumes of DCM resulting in the separate elution of
both TPP and unreacted BODIPY 3. FMR-1 was then eluted with DCM/Acetone/MeOH,
coming off the column as a bright orange streaky band. Fractions were pooled and evaporated
to give pure FMR-1 as a bright orange solid 0.13 g (80%). 1H NMR (500 MHz, CDCl3) δH
7.89-7.84 (m, 8H), 7.8-7.76 (m, 3H), 7.7-7.66 (m, 6H), 7.5 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.8
Hz, 2H), 6.95 (d, J = 4.3 Hz, 2H), 6.53 (dd, J = 4.3, 1.9 Hz, 2H), 4.25 (t, J = 5.8 Hz, 2H), 4.06-
3.95 (m, 2H), 2.28 (t, J = 6.4Hz, 2H), 1.9 (q, J = 7.8Hz, 2H); 13C NMR (500 MHz, CDCl3) δC
161.34, 147.39, 143.29, 134.99, 134.75, 133.74, 132.45, 131.34, 130.49, 126.24, 118.66, 118.24,
117.98, 114.64; 19F NMR (500 MHz, CDCl3) δF -145.11; MS (ESI) m/z found 601.2384
[M-Br]+ calculated for C37H3BF2N2OP, 601.2392.
Calibration
Methanol/glycerol fractions ranging from 0 to 90% glycerol were used for the calibration, con-
taining 13 μM of FMR-1. Absorption spectra were collected on a Hitachi U4100 Emission
Spectrometer, and emission and excitation spectra were collected using a Horiba FluoroMax-4
Spectrofluorometer. For all spectral measurements, Thorlabs 3500 μL quartz cuvettes with
four clear sides containing around 3mL of sample solution was used.
For quantum yield measurements, both PM546 (* 0.4 μM, quantum yield 0.95) and Alexa
Fluor488 (* 0.4 μM, quantum yield 0.92) were used as reference dyes. Methanol was used as
baseline for PM546 and FMR-1 (refractive index 1.3292), water for Alexa Fluor488 (refractive
index 1.333). The quantum yield was calculated using
2
Absref
I
n
QY ¼ QYref
2
nref Abs Iref
Where n is the refractive index of the solvent, I is the integrated fluorescence intensity, and
Abs is the absorbance at the excitation wavelength (kept between 0.02-0.05 to ensure linear
response) [29].
Fluorescence lifetime measurements were conducted on an inverted TCS-SP2 microscope
(Leica Microsystems, Germany) and SPC-150 TCSPC boards (Becker & Hickl, Germany). 200
μL of sample solution was pipetted into a well in an uncoated 8-well plate from Ibidi (Inte-
grated BioDiagnostics). The instrument response function (IRF) was made from NaI-
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