ADDITIONAL METHODS
All protein fusions
To construct the mEos4a fusions to the C-terminus of the fluorescent protein
(number of linker amino acids in parenthesis), the subsequent digests were
performed: human lamin A (18), NheI and BglII (cDNA source: David Gilbert, FSU;
NM_170707.2); human β-actin (18), NheI and BglII (cDNA source: Clontech,
Mountain View, CA; NM_001101.3); human light chain clathrin (15), NheI and BglII
(cDNA source: George Patterson, NIH; NM_001834.2); human α-tubulin (18), NheI
and BglII (cDNA source: Clontech; NM_006082); human H2B (10), BglII and NheI
(histones, cDNA source: George Patterson, NIH; NM_021058.3). To prepare the Nterminal fusions to mEos4a, the following digests were performed: human keratin
(18), EcoRI and NotI (cDNA source: Open Biosystems, Huntsville, AL;
NM_199187.1); human calnexin (14), AgeI and NotI (cDNA source: Origene,
Rockville, MD; NM_001746.3); human zyxin (6), BamHI and NotI (cDNA source:
Origene; NM_003461); human caveolin 1 (10), NheI and BglII (cDNA source:
Origene; NM_001753); human H2B (6), BamHI and NotI (histones, cDNA source:
George Patterson, NIH; NM_021058.3).
To construct the mEos4b fusions to the C-terminus of the fluorescent protein
(number of linker amino acids in parenthesis), the following digests were
performed: human H2B (10), BglII and NheI (histones, cDNA source: George
Patterson, NIH; NM_021058.3); human β-actin (18), NheI and BglII (cDNA source:
Clontech; NM_001101.3); human myotilin (14), AgeI and BspEI (cDNA source:
Origene; NM_006790.1); mouse CAF1 (10), AgeI and BspEI (chromatin assembly
factor, cDNA source: Akash Gunjan, Florida State University; NM_013733.3); human
α-tubulin (18), NheI and BglII (cDNA source: Clontech; NM_006082); human
lysosomal membrane glycoprotein 1 (20), BamHI and NotI (LAMP1, George
Patterson, NIH; NM_012857.1); human lamin A (18), NheI and BglII (cDNA source;
David Gilbert, FSU; NM_170707.2); mouse myosin IIA (14), NheI and BglII (cDNA
source: Origene; NM_022410.2); human light chain clathrin (15), NheI and BglII
(cDNA source: George Patterson, NIH; NM_001834.2); human zyxin (6), BamHI and
NotI (cDNA source: Origene; NM_003461); human TOMM20 (10), BamHI and NotI
(translocase outer mitochondria membrane 20, cDNA source: Origene;
NM_014765.2); human caveolin 1 (10), NheI and BglII (cDNA source: Origene;
NM_001753); human CENP-B (22), BamHI and NotI (cDNA source: Alexey
Khodjakov, Wadsworth Center, NY; NM_001810.5); rat connexin-43 (7), BamHI and
NotI (cDNA source: Matthias Falk, Lehigh U; NM_001004099.1); chicken c-src (7),
BamHI and EcoRI (cDNA source: Marilyn Resh, Sloan-Kettering Institute, New York;
XM_001232484.1); CytERM (17), AgeI and NotI (rabbit cytochrome p450, Erik
Snapp, Albert Einstein College of Medicine; XM_002718526.1); human H2B (6),
BamHI and NotI (histones, cDNA source: George Patterson, NIH; NM_021058.3);
mouse mannosidase 2 (10), NheI and BamHI (cDNA source: Jennifer LippincottSchwartz, NIH; NM_008549.2); human pyruvate dehydrogenase (10), AgeI and NotI
(PDHA1, cDNA source: Origene; NM_000284); yeast Lifeact (7), BamHI and NotI
(cDNA source: IDT, Coralville, IA); human vimentin (7), BamHI and NotI (cDNA
source: Robert Goldman, Northwestern University Medical School, Chicago, IL;
NM_003380.3); human vascular epithelial cadherin (10), AgeI and NotI (cDNA
source: Origene; NM_001795.3). Following digestion with the appropriate
restriction enzymes and gel purification, the plasmids were ligated together with
the similarly digested and gel-purified mEos4a or mEos4b cloning vector and
amplified using a Plasmid Maxiprep kit (Qiagen, Valencia, CA).
Time-lapse imaging
The utility of photoconversion for time-lapse imaging and tracking of distinct
protein populations was tested for several mEos4b fusions in transiently
transfected epithelial cells (HeLa CCL2). Both the green and red species were
sufficiently bright and photostable to allow for long-term time-lapse imaging.
Photoconversion of mEos4b fused to H2B was used to dynamically track cell
division. The exchange of photoconverted and native mEos4b labeling lamin A in the
nuclear lamina was visualized over the course of several hours. Similar experiments
were performed imaging actin dynamics and gap junction plaque growth behavior.
All results are shown in Additional Figures 1-5.
Confocal microscopy
Laser-scanning confocal microscopy was conducted using an Olympus FV1000,
equipped with argon-ion (457 and 488 nm) and helium–neon (543 nm) lasers and
proprietary filter sets. Confocal imaging was performed in Delta-T culture chambers
(Bioptechs; Butler, PA) under a humidified atmosphere of 5% CO2 in air.
Optical Properties
Spectroscopic characterization
Absorption spectra of purified proteins in 50 mM tricine buffer (pH 8.2) were
measured with an absorbance spectrometer (Lambda 35, Perkin-Elmer). mEos2
was additionally measured in 50 mM MOPS (pH 7.2) for comparison to literature
values. All protein solutions were first centrifuged (10,000 RPM for 5 minutes) to
remove scattering particles, with the supernatant used for measurements. Protein
concentration was determined by UV absorbance at 280 nm, using a predicted
extinction coefficient (in M-1cm-1) 280 = NTrp*5500 + NTyr*1490 + NCys*125
1,2.
The
extinction coefficient of the green form 505 was determined by the measured
absorbance at 505 nm divided by the protein concentration. We also determined
chromophore concentration for the green form using the alkali denaturation
method
3-6,
assuming a GFP-like extinction coefficient of 44,000 M-1cm-1 at 447 nm
at pH 13.0. This method gave values for 505 that were consistently 27% higher than
those found by protein concentration based on UV absorbance.
Fluorescence
correlation spectroscopy was additionally performed on several variants to
determine chromophore concentration, using eGFP at pH 9.5 as a concentration
reference, by the methods and instruments described in 7. The extinction coefficient
of the red form 570 was found by measuring the initial change in optical density
(OD) of the red and green forms of protein following irradiation with 385 nm light,
assuming in an incremental time period the loss in concentration of green-form
proteins by photoconversion equals the gain in concentration of red-form proteins.
We accounted for the fact that the decrease in OD at 505 nm rides on top of the tail
of the red-form absorbance peaked near 570 nm. Fluorescence excitation and
emission spectra were recorded on either a microplate reader (Safire2, Tecan) or a
luminescence spectrometer (LS55, Perkin-Elmer).
Photoconversion on purified
protein solutions was performed in cuvettes using a LED flood array (Loctite)
operating at 385 nm (14 nm full-width at half-maximum) that delivered an intensity
of 0.42 W/cm2 at the sample face. Quantum yields were measured in an integratingsphere spectrometer (Quantaurus, Hamamatsu) for both green-form and
photoconverted red-form proteins (each solution 3 ml of 0.1 OD at peak
absorbance).
Photoswitching/Bleaching
Time-course measurements were carried out on Tricine-buffered aqueous
droplets of protein isolated by oil
8
on a widefield epi-illumination microscope
(AxioImager.Z2, Zeiss) with a 20x 0.8 NA objective. Collinear laser excitation was
provided to the microscope at 405 nm (Sharp laser diode, Thorlabs), 488 nm
(Sapphire 488, Coherent) and 561 nm (Sapphire 561, Coherent) using beamcombining dichroics. In order to measure photoswitching, photoconversion, and
bleaching, sequences of light pulses were delivered to the sample, cycling between
405/488 for the green form or between 405/561 for the red form, generated by
computer-controlled shutters. Laser power was measured at the output of the
objective, and the laser beam area and uniformity at the sample was imaged and
measured using a CCD camera. We calculated the laser intensity from the measured
laser power and beam area. Fluorescence collected by the objective was passed
through appropriate dichroic/bandpass filter cubes (dichroic FF495-Di03 and
bandpass 525/50 for green form, dichroic Di01-R405/488/561/635 and bandpass
625/90 for red form; all Semrock), and detected by a fiber-coupled avalanche
photodiode APD (SPQM-AQRH14, Pacer), which sampled a sub-region of the
illuminated protein droplet. The APD signal was summed every 40-msec time
interval and stored on computer with its corresponding time point.
Two-photon action spectra
Two-photon spectra were obtained for 1 M protein solutions (as
determined by alkali denaturation) in 50 mM tricine buffer, pH 8.2 using a
Ti:sapphire laser (Chameleon Ultra II, Coherent) as excitation source on an inverted
microscope (IX81, Olympus) with a 60x 1.2 NA water-immersion objective. The
system, described in detail in 7, was set up to deliver a constant power (1 mW) at the
sample across the spectrum of the laser (710 – 1080 nm). Fluorescence was
collected by the objective, reflected off a dichroic, and passed through a bandpass
filter (550/88 for green form, 625/90 for red form, Semrock) and detected by a
fiber-coupled APD (SPQM-AQ14, Pacer).
Reference spectra were obtained for
fluorescein in 1 mM NaOH, pH 11.0 for the green form and tdTomato in 30 mM
MOPS, 100 mM KCl, pH 7.2 for the red form. Reference values of action spectra or
two-photon cross sections were taken from
9
for fluorescein and from
tdTomato.
PALM imaging of mEos4 variants fused to a variety of cellular proteins
Sample Preparation for PALM imaging
10
for
For PALM imaging, cells were seeded directly onto 35 mm, #0 thickness
coverglass MatTek dishes (MatTek Corporation; Ashland, MA) approximately 24
hours prior to transfection. The culture medium was a 1:1 mixture of DMEM and
Ham’s F12 supplemented with 12.5% cosmic calf serum, as described above.
Transfection was performed using the Effectene transfection reagent (Qiagen)
following the manufacturer’s instructions. Transfected cells were allowed to sit for
approximately 24-48 hours prior to fixation, to ensure proper localization of the
fusion construct. For keratin fusions, cells were fixed using -20oC methanol for 10
minutes and rehydrated in PBS. For α-tubulin fusions, cells were fixed in a 3%
(w/v) PFA, 0.05% (v/v) glut, 0.3% (v/v) Triton X-100 solution in HEPES buffer,
washed for approximately 5 minutes in a 0.1% solution of NaBH4 in PBS on ice, and
rinsed several times in PBS. The remaining fusions were all fixed in 2% (w/v) PFA
in PBS and washed twice with PBS containing 50 mM glycine.
PALM Imaging
The potential of mEos4a, mEos4b and mEos2 for PALM was compared by
imaging fusions of each FP to a variety of cellular components (Additional Figs. 15). Hardware settings, including frame rate, gain, binning, etc., were identical for all
acquisitions to facilitate comparison of images. Overall all three mEos variants
performed similarly well in PALM imaging experiments, with large photon counts
per molecule and many labeled molecules identified per run (Additional Fig. 4).
PALM imaging of mEos4a, mEos4b and mEos2 fusion constructs was performed on
a commercial Nikon (Nikon Instruments; Melville, NY) N-STORM super-resolution
system, featuring a Nikon Eclipse Ti inverted research microscope with a motorized
total internal reflection fluorescence (TIRF) illuminator. The instrument was
operated in an oblique pseudo-TIRF mode, where signal is maximized by utilizing
both TIR and reflected fluorescence. All imaging was performed using a Nikon
100X 1.49 NA Apo TIRF oil immersion objective and collected over a 40.96 μm ×
40.96 μm area onto an Andor iXon 897 EMCCD camera (Andor Technology, UK),
with a frame size of 256 × 256 pixels and a corresponding pixel size of 160 nm. All
imaging was performed using a Chroma Quad Band TIRF filter set (Chroma). Data
collection was performed until exhaustion of the fluorophore population for all
experiments, with molecule data shown for the noted frame ranges, corresponding
to the length of the shortest run for each fusion. All data were collected at a frame
rate of 6.7 Hz (150 ms/frame) with a camera EM gain multiplier value of 300,
conversion gain value of 1, and without binning. Readout was performed using a
561 nm diode laser from an Agilent (Agilent Technologies, Santa Clara, California)
MLC400B monolithic laser combiner. Activation was performed using a 405 nm
diode laser from the MLC400B, and applied as needed to ensure enough emitters
were consistently being identified for the software drift correction algorithms to
function properly (approximately 100 emitters identified per frame is considered
ideal). The imaging buffer was a 10 mM cysteamine (MEA), 1% (w/v) polyvinyl
alcohol solution in PBS.
Data analysis
Data collection and analysis was performed using Nikon’s NIS-Elements 4.11
software. For analysis, only single emitters with an intensity of at least 300 gray
levels above the local background were identified. Molecule data was further
constrained to show statistics for molecules emitting between 50 and 5000 photons
within the given frame range. Data was exported from the NIS Elements software
into Microsoft Access (Microsoft Corporation, Redmond, WA) for database
compilation and statistical analysis, followed by compilation of all statistical data
into a single spreadsheet in Microsoft Excel for ease of reference.
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