The research leading to these results has received funding from the European Union&#39;s Horizon 2020 research and the innovation program under the Marie Sk&#322;odowska-Curie grant agreement No. 674990 (EXCILIGHT), and from EPSRC, EP/L02621X/1.

NOTE: These are the instructions to perform a single time-resolved, long-lived luminescence measurement in oxygen-free conditions at room temperature and including the procedure of sample degassing. The text describes the protocol for either solid or liquid samples and, because most of the steps are identical in both cases, the steps of the protocol that only apply to one of the two types are indicated as &#34;film&#34; or &#34;solution&#34;. The samples and films used in the protocol can be of any kind; therefore, the sample preparation and/or content are irrelevant and are not disclosed.
CAUTION: The handling of organic solvents presents a risk. Consult the Material Safety Data Sheet (MSDS) prior to using them. All operations with solvents must be performed under a working fume cupboard. Liquid nitrogen presents a risk, so it is important to use appropriate personal protective equipment (PPE) when handling it, which includes face and hand protection (mask, gloves). Upon evaporation, liquid nitrogen undergoes a 600-fold increase in its volume; therefore, never handle liquid nitrogen in a fully closed container. Instead, use appropriate Dewar flasks. Wear eye/face protection when working with glass equipment under vacuum, due to the risk of implosion. Most aromatic molecules, and especially those newly synthesized, present a known or unknown health risk. Use standard laboratory PPE and procedures to avoid contact with the material. A class 4 laser is used in the protocol. Working with lasers is dangerous and a suitable training is required. Protective equipment (i.e., goggles) covering the spectral region of the laser emission must be worn at all times.
1. Degassing the Samples
<ol>
	<li>Degassing a solution
	<ol>
		<li>Prepare 4 mL of a solution of approximately 10-5 M of a given luminescent compound (i.e., a phosphorescent metal complex or TADF emitter) in a chosen solvent (i.e., toluene, methylcyclohexane, ethanol, etc.). 
		NOTE: For the purpose of this protocol, we use 3,11-di(10H-phenothiazin-10-yl)dibenzo[a,j]phenazine as an emitter dissolved in methylcyclohexane as a solvent.</li>
		<li>Pour the solution (4 mL) into the degassing cuvette and close the valve.</li>
		<li>Record the photoluminescence spectrum of the air-saturated solution using a standard spectrofluorometer. Use the same excitation wavelength as in the time-resolved experiment. 
		NOTE: Here, 355 nm is used. 
		NOTE: Record the photoluminescence spectrum in the wavelength range from 365 nm to 800 nm. Make sure the fluorescence was previously recorded in a normal fluorescence cuvette.</li>
		<li>Connect the vacuum pump to the inlet neck of the degassing cuvette.</li>
		<li>Hold the inlet neck of the cuvette, and slowly put the cuvette into liquid nitrogen. Shake it occasionally, while the cuvette is in liquid nitrogen. To ensure the whole solution is frozen, shake the round flask to check the cuvette.</li>
		<li>Turn on the vacuum pump and open the inlet valve. With the solution frozen, keep the vacuum on for 10 min. Close the inlet valve and turn off the vacuum pump.</li>
		<li>Slowly place the cuvette into isopropanol. Shake the cuvette occasionally until the solvent is melted. If the degassing has been successful, air coming out of the solution should be observed upon the first cycle, in the form of bubbles. 
		NOTE: Solvent loss occurs mainly in the first freezing/unfreezing cycle, due to the inner walls of the cuvette wetted with solution. Any solution outside of the flask used for freezing shows a visible volatility due to it still being at room temperature. If degassing is performed to record a degassed-to-air-saturated factor, the best practice is to compare photoluminescence intensities of the solution after the degassing with an air-saturated solution obtained from that previously degassed. Opening the inlet valve and stirring the solution for a few minutes will give an air-saturated solution again.</li>
		<li>Repeat steps 1.1.5 - 1.1.7 altogether 3 - 5x, depending on the solvent used.</li>
		<li>Warm up the solution in the cuvette to room temperature using a water bath or wait for the temperature to equilibrate.</li>
		<li>Record the photoluminescence spectrum of the degassed solution as in step 1.1.3. 
		NOTE: This step is to ensure a successful degassing and to verify if any change to the photoluminescence is observed upon degassing. If a degassed-to-air-saturated factor is recorded, please refer to the note after step 1.1.7.</li>
	</ol>
	</li>
	<li>Degassing a solid film
	<ol>
		<li>Place the pre-prepared film on a size-fitting substrate in the sample holder and screw it tightly. A typical sample is a doped polymer film [i.e., poly(methyl methacrylate), cyclo olefin polymer) &#60; 0.5 mm thick, deposited on a quartz disc substrate of 12 x 1 mm. 
		NOTE: For the purpose of this protocol, a film of 3,11-di(10H-phenothiazin-10-yl)dibenzo[a,j]phenazine (1% w/w) doped in cyclo olefin polymer is used. To prepare such a sample on the disc substrate given, use 10 mg of a polymer solution in toluene (100 mg/mL) and mix them with 0.1 mg of a luminescent compound in toluene solution (1 mg/mL). Dry-film the sample for 30 min at 90 &#176;C or, for the same amount of time, place it under a rotary pump vacuum (10-3 - 10-2 mbar) at room temperature.</li>
		<li>Cover the holder with a vacuum shroud, ensuring its windows are facing the laser beam and the collimating lenses. Lock the shroud by closing the venting valve and turn on the roughing vacuum pump.</li>
		<li>Once the pressure in the sample space reaches 10-1 mbar, turn on the turbomolecular pump. Wait 30 min to 1 h in order to degas the sample thoroughly. 
		Note: The degassing time depends on the sample used. Thick polymer samples may take longer to degas. In order to find the conditions in which the sample is degassed, we suggest noting the time it takes to degas the sample when performing a steady state experiment (with the fluorometer). Monitoring the emission intensity over time from the start to the moment where no more changes in intensity are observed can be used as a measure of the time necessary to fully degas the sample.</li>
	</ol>
	</li>
</ol>
2. Turning on the Equipment and Setting Up the Experiment
<ol>
	<li>Turning on the laser
	<ol>
		<li>Turn on the laser system.</li>
		<li>Adjust the output pump power and wait about 30 min to warm up and stabilize the beam.</li>
		<li>Using a power meter, measure the laser fluence. The reading should be approximately 100 &#181;J per pulse (maximum pulse energy). Adjust the laser pulse energy if necessary and use a neutral density filter to adjust the excitation pulse energy to the specified level, if required. 
		NOTE: For the safety of the user and to avoid sample damage, laser pulse energy should not exceed 100 &#181;J per pulse in a typical experiment. If the photoluminescence of the sample is very bright, the laser power can be reduced so the detector is not saturated.</li>
	</ol>
	</li>
	<li>Setting up the equipment
	<ol>
		<li>Turn on the measuring system. 
		NOTE: The measuring system comprises a laser (described above), an iCCD camera, a spectrograph, and a computer, and these should be turned on at this step of the protocol.</li>
		<li>Turn on the 4 Spec software and spectrometer control program. Set up the measurement parameters (i.e., the spectrograph slit width, the wavelength range, and the number of collected scans).</li>
		<li>To access the camera&#8217;s control setup, choose Window | Camera. Make sure the camera is turned on by this time. The software connects with the camera now. Set the delay and the integration time for the zero-time parameters: 981 ns of delay and 10 ns of integration time. These parameters can then be used to verify if the measurement set-up is aligned. Set a trigger for &#8211;Trig. Then, send the parameters to the camera with the Send it button.
		<ol>
			<li>Set Scan Sequence | Scans per exp. to 100, which indicates 100 frames are recorded (100 pulses of the laser are used) to produce one spectrum at the delay and gate times given. Using Window | Camera control, set the MCP gain voltage to 850 V. With the given parameters, the wavelength range used is roughly 400 - 700 nm (depending on the current calibration).</li>
		</ol>
		</li>
		<li>Initialize the monochromator and set the slit and monochromator position/grating appropriate to the spectral range and intensity of the samples&#8217; emission.
		<ol>
			<li>Set the spectrometer position to 650, the turret to 1, and the axial entrance to 1. Press Enter and then click Run. Make sure the spectrometer is running, which indicates the command has successfully been sent.</li>
		</ol>
		</li>
		<li>Calibrate the system for the selected wavelength range. This is done using a pre-prepared calibration file. Click File | Load calibration curve and choose the appropriate calibration file on the 4 Spec program. Once the file is loaded, the calibration is done. 
		NOTE: Use the calibration file for spectrometer position 650.
		<ol>
			<li>Prepare the set of delay times and corresponding integration times that will be used to collect the spectra during the measurement.</li>
			<li>Remember the zero time, as all the delay times set in the software will be a sum of the zero time and the actual delay time. For delay times 0, 10, &#8230;, and 90 ns, use a 10 ns integration time. For delay times 100, 200, &#8230;, and 900 ns, use a 100 ns integration time. For delay times 1, 2, &#8230;, and 9 &#181;s, use a 1 &#181;s integration time, and finally, for delay times 10, 20, &#8230; 90 &#181;s, use a 10 &#181;s integration time. This can be extended up to the 100 ms time window dictated by the laser pulse, but this region will not be used in the protocol.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Placing the sample
	<ol>
		<li>Placing a solution
		<ol>
			<li>Fit a cuvette holder in the sample area or fit the cuvette in a cryostat if temperature control is required. 
			NOTE: The use of a degassing cuvette used for temperature-dependent studies is similar to the use of degassing cuvettes as explained in step 1.1, but with the dimensions adjusted to fit the cryostat.</li>
			<li>Place the degassing cuvette into the holder and secure it using a laboratory stand.</li>
			<li>Ensure, by careful observation of the photoluminescence, that the laser beam hits the cuvette.</li>
			<li>Cover the sample unit to avoid any room light being recorded by the detector and to reduce the risk of laser scattering.</li>
		</ol>
		</li>
		<li>Placing a film
		<ol>
			<li>Place a cryostat in the sample area. Continue as in step 1.2.</li>
			<li>Ensure, by careful observation of the photoluminescence, that the laser beam hits the sample. If necessary, adjust the beam path or the cryostat position.</li>
			<li>Cover the sample unit to avoid any room light being recorded by the detector and to reduce laser scattering.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Aligning the sample and the laser beam path
	<ol>
		<li>Cover the laser beam path with a shutter and run a single background (Ctrl+D).</li>
		<li>Uncover the beam path and record the spectrum (Ctrl+R).</li>
		<li>(film only) The emission should be in the middle of the vertical dimension of the camera image. If not, adjust the laser beam position/sample position in the vertical direction.</li>
		<li>(film only) Repeat steps 2.4.2 and 2.4.3 until the emission is adjusted.</li>
		<li>If the spectrum does not fit in the horizontal dimension of the image, adjust the monochromator position accordingly. Use an appropriate calibration curve for the new monochromator position. 
		NOTE: The calibration is done by the use of a standard Hg-Ar calibration lamp with a broad emission range (as sharp peaks) from UV to NIR. The spectrum of the lamp is recorded at a specified monochromator position and, using the camera software, the pixel position is translated into wavelength, as the calibration lamp peak positions are known.</li>
		<li>If the emission intensity at its maximum is lower than 106, increase the MCP gain voltage or broaden the monochromator slit. The saturation of the detector is observed as a deformation of the spectrum maxima or by the appearance of artifacts in the image. If this is observed, reduce the laser power, the MCP gain voltage, or the monochromator slit. 
		NOTE: Saturating the detector should be avoided, as this can damage the MCP. The signal intensity can be regulated also by adjusting the size of the laser beam spot. The laser beam intensity can be also reduced by using a neutral density filter.</li>
		<li>When optimized, the system is ready to use.</li>
	</ol>
	</li>
	<li>Setting up the experiment
	<ol>
		<li>Cover the laser path using a shutter.</li>
		<li>Measure the background emission using the Ctrl+D shortcut.</li>
		<li>Open the automatic measurement script and input the name of the experiment file into the textbox. Press Enter and input the starting line of the experiment file. Press Enter again and input the last line of the experiment file. Then, press Enter at the end to run the script. The automatic script allows the measurement of the emission at a set of different delay times given in the file.</li>
		<li>Once finished, select one spectrum and scale. Export the spectra to the file by clicking File | Export | Curve(s) as text and then choose a name and a directory. The results are now ready to be processed by the appropriate software.</li>
	</ol>
	</li>
</ol>
3. Finishing the Experiment
<ol>
	<li>When all planned experiments have been finished, turn off the equipment, proceeding in the opposite order as it was turned on in.</li>
	<li>(film only) Remove the sample from the cryostat.
	<ol>
		<li>Open the venting valve, release the clamp, and remove the vacuum shroud.</li>
		<li>Remove the sample from the sample holder.</li>
		<li>Place the vacuum shroud back to the cryostat.</li>
	</ol>
	</li>
	<li>(solution only) Remove the degassing cuvette from the holder and clean it. 
	CAUTION: Dispose of solvents in accordance with the institute&#39;s waste management rules. Nitric acid is corrosive. Take care when using it. Use PPE. Operate only under a working fume cupboard. 
	NOTE: The general cleaning procedure is specific to the cuvette type and the inlet valve used. Some valves should not be removed; thus, the cleaning must be performed without removing the valve.
	<ol>
		<li>Open the inlet valve and dispose of the solution.</li>
		<li>Rinse the cuvette with acetone, taking care to wash all inner walls. Repeat the rinse 3x.</li>
		<li>If in doubt of the cleanliness of the cuvette, wash it with water and then fill it with concentrated nitric acid (HNO3) and leave it overnight. Then wash it thoroughly with deionized water and dry it.</li>
	</ol>
	</li>
</ol>

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F., Penfold, T. J., Monkman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+spin-vibronic+coupling+mechanism+of+thermally+activated+delayed+fluorescence.">Revealing the spin-vibronic coupling mechanism of thermally activated delayed fluorescence.</a> Nature Communications. 7, 13680 (2016).</li><li>Pander, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+chalcogenophene-based+monomers+with+pyridine+acceptor+unit.">Synthesis and characterization of chalcogenophene-based monomers with pyridine acceptor unit.</a> Electrochimica Acta. 210, 773-782 (2016).</li><li>Data, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemically+Induced+Synthesis+of+Triphenylamine-based+Polyhydrazones.">Electrochemically Induced Synthesis of Triphenylamine-based Polyhydrazones.</a> Electrochimica Acta. 230, 10-21 (2017).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+efficient+thermally+activated+delayed+fluorescence+materials+for+pure+blue+organic+light+emitting+diodes.">Design of efficient thermally activated delayed fluorescence materials for pure blue organic light emitting diodes.</a> Journal of the American Chemical Society. 134, 14706-14709 (2012).</li><li>Mehes, G., Nomura, H., Zhang, Q., Nakagawa, T., Adachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+electroluminescence+efficiency+in+a+spiro-acridine+derivative+through+thermally+activated+delayed+fluorescence.">Enhanced electroluminescence efficiency in a spiro-acridine derivative through thermally activated delayed fluorescence.</a> Angewandte Chemie International Edition. 51 (45), 11311-11315 (2012).</li></ol>

The authors have nothing to disclose.

Degassing a solution is one of the most critical points in this method. Plastic inlet valves become worn easily and the system stops being hermetic. If in doubt, it is advised to check the cuvette with a known material with an established degassing factor. The cuvettes are also fragile; therefore, degassing should be performed with caution.
As the system typically requires a pulsed Nd:YAG laser, a proper maintenance of the laser unit must be performed regularly. The pumping flashlamp should be replaced regularly, and this should only be done by a qualified technician or another experienced person.
As the laser requires 30 min to warming up, it is a good practice to turn on the laser before degassing the sample. Once the sample is degassed, the laser should be ready for taking the measurements. However, the degassing time for a film is difficult to determine using this equipment. Therefore, it is worth to perform a steady-state experiment with a conventional fluorometer to estimate the degassing time (a stabilization of the photoluminescence intensity upon pumping down).
For short-lived emitters (i.e., those whose fluorescence decays within a few nanoseconds), there will be only a few spectra recorded, as the emission decay lasts for a short period of time. In this case, TCSPC or a streak camera would perform much better. On the other hand, long-lived emitters can be problematic if the emission lasts for more than 100 ms (i.e., phosphorescence). To expand the effective time window, a nitrogen laser is used in these cases. This allows reducing the repetition rate of the laser to 1 Hz and extending the time window to 1 s.
The protocol shown here is only exemplary and is dedicated to a new and inexperienced user. An experienced operator can modify the protocol in various different ways. There is a potential to further develop the system to extend the camera&#39;s sensitivity in red and (NIR) by replacing the photocathode, as mentioned in the Introduction.
The data analysis in the case of this experiment is a time-consuming job, as each experiment gives ca. 100 spectra. The spectra have to be divided by the integration time to reconstruct the luminescence decay, and often also normalized (divided by the maximum, standardized, or area-normalized) in order to facilitate an analysis of the spectra at different delay times. During the analysis, differences in the spectra (i.e., gradual red or blue shifts) are being looked for. If the measurement is performed in function of the temperature, then the spectra can show the presence of delayed fluorescence or phosphorescence, or both, depending on the temperature or time delay used. Transient decays are obtained by plotting the integrated luminescence spectra against the time delay, after dividing each spectrum by their respective integration time. The photoluminescence transient decay is obtained and can be fitted in order to calculate the radiative lifetime of the prompt and the delayed fluorescence or phosphorescence.

Time-resolved spectroscopy is an essential tool in the studies of novel materials for the application of organic light-emitting diodes (OLED)1,2,3. These techniques are especially important for the latest generations of OLED emitters [i.e., as thermally activated delayed fluorescence (TADF)4,5,6,7,8 or phosphorescent9,10,11 molecules], where photoluminescence processes can be observed in a broad timescale (up to seconds). Interestingly, such techniques can also be used to investigate electroluminescence in devices, over suitable time regimes12,13. The methods described above are, in general, focused on following time-dependent properties that involve photoluminescence signals such as the decay lifetime, the shape and energy of the emission spectra, and its dependence upon temperature or other factors.
Overall, the most popular method of time-resolved spectroscopy is time-correlated single photon counting (TCSPC) or its modifications, such as multichannel TCSPC. This method is especially suited to follow fast decays with a very high accuracy, usually on the nanosecond timescale. However, it has a major disadvantage, as it does not allow following the changes in the photoluminescence spectrum in an easy way. This is resolved using streak cameras14,15. However, both methods are not suited to follow long-lived luminescence decays. In this case, time-gated methods and multichannel scaling are the methods of election.
In this work, we discuss the time-gated acquisition of photoluminescence signals in a time range from less than a nanosecond up to 0.1 - 1 s in a single experiment16,17,18. Moreover, the quality of the spectra is excellent due to the high sensitivity of the detector that is used (an iCCD camera). This allows the observation of very fine changes in the emission spectrum and the investigation of the excited state dynamics in detail, identifying the emission of different excited species in one molecular system. The versatility of this equipment has been confirmed by several recent publications19,20,21,22,23,24,25,26. The excitation source is either an Nd:YAG laser with a 10 Hz repetition rate, providing a set of harmonics (266 nm, 355 nm, and 532 nm) or a nitrogen laser (337 nm) of a changeable repetition rate between 1 - 30 Hz.
The principle of the work of iCCD cameras is based on the image intensifier, which not only intensifies the incoming light but also works as a shutter (gate). The intensifier consists of a photocathode that is sensitive to a specific spectral range [i.e., ultraviolet (UV), visible, red, and near-infrared (NIR)], a micro-channel plate (MCP), and a phosphor. By changing the photocathode, it is possible to adapt the camera to a specific use. The photocathode converts incoming photons into photoelectrons that are multiplied in the MCP and then hit the phosphor screen generating photons. These photons, through a system of lenses, are focused onto a CCD chip and are converted into an electrical signal. For further details, please refer to the manufacturer&#39;s webpage27.
To collect emission spectra throughout the range from 1 ns to 100 ms with sufficient signal-to-noise ratio, the integration (exposure) time increases exponentially along with exponentially increasing the time delay. This is dictated by the properties of the photoluminescence decay, which follows exponential laws in most systems.
The method described here can be applied to several sample sizes and forms, including those with an uneven surface, powders, or small crystals19. The sample holder is easily adapted to support several different cuvettes, including standard and degassing cuvettes or flow cuvettes. All samples with photoluminescence in a range of 350 - 750 nm can be investigated by this equipment. The system is also equipped with a liquid nitrogen cryostat to perform temperature-dependent measurements of solid and liquid samples down to 77 K and a closed-cycle helium cryostat to perform measurements of solid samples down to 15 K. This allows studying such phenomena like TADF and phosphorescence. In summary, any compound or any kind of sample that emits photoluminescence in the specified region and time range and which absorbs the excitation laser light can be investigated in this equipment.
The removal of molecular oxygen is a particularly important issue in the investigation of the photophysics of molecules with a long-lived emission. Therefore, an experimental procedure of degassing samples (solutions and films) is also described in detail here. Quenching by oxygen affects long-lived luminescence and is a major problem in the investigation of delayed fluorescence and phosphorescence. However, this quenching effect also facilitates the investigation of the contribution of triplet excited states to the overall luminescence. This is accounted for measuring the photoluminescence intensity ratio of a degassed solution/film to air-saturated conditions17,23. As triplets are quenched by oxygen, the degassing-to-air emission ratio gives direct information about the contribution of the long-lived states that are responsible for the long-lived emissions (and so delayed fluorescence or phosphorescence). This can then be used to extract information about the yields of triplet formation in organic TADF emitters. Molecular oxygen exists in a triplet ground state as a biradical. Upon absorption of energy of ca. 1 eV, triplet oxygen undergoes a transition to a singlet excited state. Typically, excited state molecules have an energy of singlet and triplet higher than 1 eV. This energy can, therefore, be transferred to oxygen upon collision. As a result, the molecule returns to a ground state or undergoes intersystem crossing.
One of the most popular methods of degassing solutions is bubbling them with a neutral gas with no oxygen content, usually very pure nitrogen or argon. This technique is very helpful in different research areas (i.e., electrochemistry or photophysics)28,29,30,31. However, while this is a simple procedure and even effective for most purposes, simply purging a solution with a neutral gas is not always the most adequate way, as removing oxygen in trace amounts is almost impossible by this method. Moreover, severe solvent loss can occur due to its volatility, which may lead to changes in the concentration of the sample under study. However, this can be prevented by a saturation of the gas with the solvent used in the solution.
The technique described here is based on a different principle. It allows reducing solvent losses to a minimum and provides repeatable levels of oxygen removal. The technique requires special, usually home-made degassing cuvettes comprising a quartz cell for the acquisition of the luminescence signal - fluorescence or phosphorescence - and a small glass flask with a spherical shape for freezing/unfreezing, and a valve. Degassing is performed under repeating freezing/unfreezing cycles. Oxygen extraction is performed in a vacuum, with the sample in the flask compartment, and while the sample is frozen, followed by letting the sample equilibrate at room temperature, with the vacuum valve closed - during this period, solution melting occurs, and the oxygen dissolved in the liquid phase is released. This requires using the cuvette itself, a regular rotary vacuum pump, and a liquid nitrogen source for cooling. The method can be used with a variety of solvents, preferably those of a low melting point such as toluene, ethanol, methylcyclohexane, 2-methyltetrahydrofuran. Degassing solutions using this technique is fast, efficient, and reliable.
Figure 1 shows with a scheme how TADF and RTP luminescence in organic molecules is generated. Prompt fluorescence, delayed fluorescence, and phosphorescence can all be recorded with the same measurement setup. With this technique, not only luminescence decays but also time-resolved emission spectra can be recorded. This enables the characterization of the molecular system and the facile identification of RTP and TADF emitters. As Figure 3 shows, a TADF emitter will normally show the same emission spectrum over the whole decay, while an RTP emitter shows a short-lived fluorescence and a long-lived phosphorescence that differ in the emission spectra.

<table><tbody><tr><td>Degassing cuvette</td><td></td><td></td><td>Not commercial product</td></tr><tr><td>Nd:YAG laser</td><td>EKSPLA</td><td>EKSPLA NL204-0.5K-TH</td><td></td></tr><tr><td>Gated iCCD camera</td><td>Stanford Computer Optics</td><td>4Quick Edig</td><td></td></tr><tr><td>Spectrograph</td><td>Horiba Instruments inc.</td><td>TRIAX180</td><td></td></tr><tr><td>Liquid nitrogen cryostat</td><td>Janis Research</td><td></td><td></td></tr><tr><td>Helium closed cycle cryostat</td><td>Cryomech</td><td></td><td></td></tr><tr><td>Fluorolog fluorometer</td><td>Jobin Yvon</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td>Technical</td></tr><tr><td>Cyclo olefin polymer</td><td>Zeon</td><td>Zeonex 480</td><td></td></tr><tr><td>Toluene</td><td>ROMIL</td><td>H771</td><td>Toluene SpS</td></tr></tbody></table>

time-resolved photophysical characterization of triplet-harvesting organic compounds at an oxygen-free environment using an iccd camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Photoluminescence spectra of a platinum-based phosphor solution in toluene were recorded before and after degassing (Figure 2). The air-saturated solution is almost non-emissive, while the degassed solution shows a bright photoluminescence. Figure 3 shows a decay profile of a TADF emitter in toluene solution (Figure 3a) and the time-resolved spectra recorded in the same experiment (Figure 3b) with a phosphorescence spectrum recorded at 80 K, as well as a decay profile of a room temperature phosphorescent molecule in a solid polymer host (Figure 3c) and the time-resolved spectra recorded in that same experiment (Figure 3d) with a phosphorescence spectrum recorded at 80 K.
Figure 3 presents two sets of data recorded in different sample form (solution and solid film) of two different molecules. In Figure 3a, two time regimes can be distinguished: below ~100 ns, the prompt fluorescence decay is observed, while at later times, it is the delayed fluorescence decay that is observed. As seen in Figure 3b, the spectra associated with prompt and delayed fluorescence nearly overlap with each other, as is expected, because this emission is originated from the same electronic state. Phosphorescence that was recorded at low temperature is shown for comparison. TADF emitters typically have a small singlet-triplet energy gap; therefore, the phosphorescence spectrum may be very close to the fluorescence. Figure 3c shows the decay of a room temperature phosphorescent organic molecule. The decays may appear similar, but a comparison of the spectra (Figure 3d) confirms that the delayed emission is not fluorescence, but phosphorescence. The lack of points between the short and the long time regimes is typical if the long-lived emission has got an especially long lifetime (i.e., &#62; 10 ms). The reason is that, in this time regime, the prompt fluorescence is already too weak to be observed as it has already decayed, but the long-lived emission, when integrated using a considerably shorter time than its radiative lifetime integration times, is still not strong enough to be detected. Phosphorescence spectra recorded at room and low temperature differ significantly as the molecule shows rigidochromism.
It is worth to note that the experiment enables the recording of emission spectra and intensity with not only up to 9 decades of time, but also 8 - 9 decades of intensity. The spectra are smooth and of good quality.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56614/56614fig1.jpg" /> 
Figure 1: Scheme showing the differences between triplet-harvesting molecules: delayed fluorescent (TADF) and phosphorescent (RTP). The protocol presented here (if extended by temperature-dependent measurements) can be used to investigate these molecules and record their key properties. Note: in some RTP molecules, the prompt fluorescence may not be observed. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56614/56614fig2.jpg" /> 
Figure 2: Photoluminescence spectra showing an increase of photoluminescence intensity after degassing a solution. The figure shows the effect of degassing a toluene solution of a platinum-based phosphorescent metalocomplex using the protocol presented in this work. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56614/56614fig3.jpg" /> 
Figure 3: Representative results. (a) This panel shows the luminescence decay transient of a TADF emitter in toluene. (b) This panel shows the representative spectra recorded in the same experiment as is shown in panel a, with the phosphorescence spectrum recorded at 80 K. (c) This panel shows the luminescence decay transient of an RTP molecule in cyclo olefin polymer. (d) This panel shows spectra recorded in the same experiment as is shown in panel c, along with a low-temperature phosphorescence spectrum. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56614/56614fig4.jpg" /> 
Figure 4: Schematic of the measurement system. The Nd:YAG laser produced third harmonics at 355 nm. The laser light hit the sample, which absorbed part of the light and emitted photoluminescence shortly after. The photoluminescence was then collimated and focused onto a spectrograph where it was refracted. The light was then recorded by the iCCD camera, which enabled recording the spectra in the time domain. Note the configuration of the solid and liquid samples. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56614/56614fig5.jpg" /> 
Figure 5:&#160;Photograph of a degassing cuvette used in room-temperature measurements. The cuvette consists of quartz fluorescence cell, a glass freezing flask, and a valve. All elements are connected with glass tubes. Note that the cuvette is not a commercially available item. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56614/56614fig6.jpg" /> 
Figure 6:&#160;Comparison of a regular degassing cuvette and a cuvette used for low-temperature experiments. The cuvette for low-temperature measurements is very similar to the regular one. However, it is fitted with a long glass tube to fit the dimensions of the liquid nitrogen cryostat, and the quartz fluorescence cell is made of one piece of quartz; therefore, it is resistant to temperature changes in a wide range of temperatures. <a href="https://www.jove.com/files/ftp_upload/56614/56614fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera at JoVE.com

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

time-resolved-photophysical-characterization-triplet-harvesting

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Introduction to Molecular Spectroscopy

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8.8K Views.  University of Durham. Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Video: Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioengineering

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

Engineering

Integrating a Triplet-triplet Annihilation Up-conversion System to Enhance Dye-sensitized Solar Cell Response to Sub-bandgap Light

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents and hence higher efficiencies. Photon up-conversion by way of triplet-triplet annihilation (TTA-UC) is an attractive technique for using these otherwise wasted low energy photons to produce photocurrent, while not interfering with the photoanodic performance in a deleterious manner. Further to this, TTA-UC has a number of features, distinct from other reported photon up-conversion technologies, which renders it particularly suitable for coupling with DSC technology. In this work, a proven high performance TTA-UC system, comprising a palladium porphyrin sensitizer and rubrene emitter, is combined with a high performance DSC (utilizing the organic dye D149) in an integrated device. The device shows an enhanced response to sub-bandgap light over the absorption range of the TTA-UC sub-unit resulting in the highest figure of merit for up-conversion assisted DSC performance to date.

An integrated device, incorporating a dye-sensitized solar cell and triplet-triplet annihilation up-conversion unit was produced, affording enhanced light harvesting, from a wider section of the solar spectrum. Under modest irradiation levels a significantly enhanced response to low energy photons was demonstrated, yielding a record figure of merit for dye-sensitized solar cells.

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs O2 sensitive luminescent sensor foils (planar optodes) in combination with a specialized camera system for imaging luminescence lifetime in the frequency-domain. Planar optodes are prepared by dissolving the O2-sensitive indicator dye in a polymer and spreading the mixture on a solid support in a defined thickness via knife coating. After evaporation of the solvent, the planar optode is placed in close contact with the sample of interest - here demonstrated with the roots of the aquatic plant Littorella uniflora. The O2 concentration-dependent change in the luminescence lifetime of the indicator dye within the planar optode is imaged via the backside of the transparent carrier foil and aquarium wall using a special camera. This camera measures the luminescence lifetime (&#181;s) via a shift in phase angle between a modulated excitation signal and emission signal. This method is superior to luminescence intensity imaging methods, as the signal is independent of the dye concentration or intensity of the excitation source, and solely relies on the luminescence decay time, which is an intrinsically referenced parameter. Consequently, an additional reference dye or other means of referencing are not needed. We demonstrate the use of the system for macroscopic O2 imaging of plant rhizospheres, but the camera system can also easily be coupled to a microscope.

Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System

We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants.

We describe a method to image dissolved oxygen (O2), in 2D at high spatial (&#60; 50-100 &#181;m) and temporal (&#60; 10 s) resolution. The method employs ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Biology

Time-Resolved Photoluminescence Spectroscopy of Semiconductor Nanocrystals and Other Fluorophores

Time-resolved photoluminescence (TRPL) is a key technique for understanding the photophysics of semiconductor nanocrystals and light-emitting materials in general. This work is a primer for setting up and conducting TRPL on nanocrystals and related materials using single-photon-counting (SPC) systems. Basic sources of error in the measurement can be avoided by consideration of the experimental setup and calibration. The detector properties, count rate, the spectral response, reflections in optical setups, and the specific instrumentation settings for single photon counting will be discussed. Attention to these details helps ensure reproducibility and is necessary for obtaining the best possible data from an SPC system. The main aim of the protocol is to help a student of TRPL understand the experimental setup and the key hardware parameters one must generally comprehend in order to gain useful TRPL data in many common single-photon-counting setups. The secondary purpose is to serve as a condensed primer for the student of experimental time-resolved luminescence spectroscopy.

This paper presents an experimental how-to on time-resolved photoluminescence. The hardware used in many single photon-counting setups will be described and a basic how-to will be presented. This is intended to help students and experimenters understand the key system parameters and how to correctly set them in time-resolved photoluminescence setups.

Time-resolved photoluminescence (TRPL) is a key technique for understanding the photophysics of semiconductor nanocrystals and light-emitting materials in ...

Decoding Transient Absorption - Processing, Fitting, and Interpreting Data

An Introduction to Processing, Fitting, and Interpreting Transient Absorption Data

Transient absorption (TA) spectroscopy is a powerful time-resolved spectroscopic method used to track the evolution of excited-state processes through changes in the system&#39;s absorption spectrum. Early implementations of TA were confined to specialized laboratories, but the evolution of commercial turn-key systems has made the technique increasingly available to research groups across the world. Modern TA systems are capable of producing large datasets with high energetic and temporal resolution that are rich in photophysical information. However, processing, fitting, and interpreting TA spectra can be challenging due to the large number of excited-state features and instrumental artifacts. Many factors must be carefully considered when collecting, processing, and fitting TA data in order to reduce uncertainty over which model or set of fitting parameters best describes the data. The goal of data preparation and fitting is to reduce as many of these extraneous factors while preserving the data for analysis. In this method, beginners are provided with a protocol for processing and preparing TA data as well as a brief introduction to selected fitting procedures and models, specifically single wavelength fitting and global lifetime analysis. Commentary on a number of commonly encountered data preparation challenges and methods of addressing them is provided, followed by a discussion of the challenges and limitations of these simple fitting methods.

Author Spotlight: Exploring Light-Driven Chemical Reactions and Energy-Harnessing Devices in Photochemical Research

This protocol is a beginner's entryway into processing, fitting, and interpreting transient absorption spectra. The focus of this protocol is the preparation of datasets, and fitting using both single wavelength kinetics and global lifetime analysis. Challenges associated with transient absorption data and its fitting are discussed.

Transient absorption (TA) spectroscopy is a powerful time-resolved spectroscopic method used to track the evolution of excited-state processes through ...

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the local flexibility and dynamics of the deoxyribonucleic acid (DNA)-binding forkhead (FKH) domain of the FoxP1 transcription factor. FoxP1 dimerizes through a three-dimensional domain-swapping (3D-DS) mechanism, forming a disordered intermediate with or without DNA. Since 3D-DS involves an intrinsically disordered region, understanding its behavior is crucial for elucidating the structural and functional properties of FoxP1. Using a single-cysteine-labeled FoxP1, we conducted single-molecule fluorescence anisotropy (smFA) experiments, applying dynamic anisotropy Photon Distribution Analysis (daPDA) and time-resolved anisotropy Burst Variance Analysis (traBVA) approaches to probe local flexibility and dynamics. This protocol provides a detailed, step-by-step guide for smFA measurements, emphasizing time-resolved analyses, variance, and probability distribution techniques to capture structural dynamics across different timescales. This approach enabled us to relate dynamics and heterogeneity to FoxP1 dimerization and DNA binding, highlighting the complex action mechanism that characterizes this transcription factor.

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the ...