The authors would like to acknowledge Allan Bracker and Daniel Gammon at the Naval Research Laboratory for providing the samples being studied. This work was supported (in part) by the Defense Threat Reduction Agency, Basic Research Award # HDTRA1-15-1-0011, to University of California-Merced.

Note: The methodology described is specific to a particular software, although other software packages may be used instead.
1. Sample Preparation and Cool Down
<ol>
	<li>Fabricate the sample.
	<ol>
		<li>Grow the sample, using the Stranski-Krastanov growth method via molecular beam epitaxy creating two vertically-stacked self-assembled InAs/GaAs QDs that are separated by a 4 nm tunnel barrier as previously described.16 Embed the QDs in an electric field effect structure (i.e., Schottky diode) allowing for an electric field to be applied to the QDMs.17 
		Note: The use of QDMs is not a requirement for the method. Also, the semiconductors InAs/GaAs are not necessary, the technique will work for QDMs or QDs made from any semiconductor combination.</li>
		<li>Fabricate the samples so that individual QDs can be optically addressed. Do this by either adding an aperture mask to the top of the sample or making a low density sample with 108 QDs/cm2 or less depending on the focal spot size.18</li>
	</ol>
	</li>
	<li>Mount the sample into the chip header.
	<ol>
		<li>Apply an alloy consisting of 50% bismuth, 26.7% lead, 13.3% tin, and 10% cadmium on the ceramic chip header. Heat the chip using a hot plate until the alloy liquefies. Place the bottom of the sample onto the liquefied alloy attaching it to the chip header. 
		Note: The bottom of the sample is one of the electrodes of the Schottky diode and the solder point to the chip header is connected to a pin. Another alternate for attaching the sample is conductive silver epoxy.</li>
	</ol>
	</li>
	<li>Adhere 40 G&#160;gold wire from a point (top corner) of the sample to a pin on the chip.
	<ol>
		<li>Place a droplet of silver epoxy on the top corner of the sample and one droplet on one of the pin pads on the chip.</li>
		<li>Carefully lay the gold wire in the two droplets. 
		Note: The top of the sample is the other electrode of the Schottky diode that allows for application of an electric field.</li>
	</ol>
	</li>
	<li>Mount the chip and sample into the cryostat and ensure the sample has good thermal contact with the copper sample holder.
	<ol>
		<li>Apply indium foil between the chip and the cryostat&#39;s cold finger.</li>
		<li>Pressure mount the chip to the cold finger. Use two screws with washers and tighten firmly to ensure good thermal contact with the cryostat&#39;s copper cold finger.</li>
	</ol>
	</li>
	<li>Attach wires from the chip pins that are connected to both the top and bottom electrodes on the sample. Run these wires through the cryostat to the source meter. 
	Note: The source meter applies a bias to the sample&#39;s electrodes, exposing the QDs to an external electric field.</li>
	<li>Evacuate and bring the cryostat and sample chamber to vacuum. Start the turbo pump, evacuating to about 10-6 Torr in preparation for the cool down of the sample. 
	Note: Cooling and temperature control for the experiment is accomplished with the cryostat which is composed of a closed cycle refrigerator and an attached microscopy sample chamber.</li>
	<li>Start the cryostat&#39;s compressor. Allow the cryostat to cool the system until reaching the desired temperature. 
	Note: For the presented results the temperature was approximately 18 K. Once the sample is cooled, the setup is ready for the installation of the optics that will allow for the optical measurement to be taken.</li>
</ol>
2. Optics Setup
Note: For all set up procedures, run the laser, source meter, spectrometer and CCD by either using the software provided by the manufacturer or other custom program.
<ol>
	<li>For PL collection, place a long working distance 50X microscope objective and collimation lens in line with the lens that focuses the PL signal onto the spectrometer. Collect the spectrum through a 0.75 m monochromator where the signal is dispersed by an 1,100 mm-1 grating and detected using a liquid nitrogen cooled 1,340 x 100 pixel CCD camera.</li>
	<li>Using a white light source, illuminate the sample.
	<ol>
		<li>Focus the image of the sample through an external camera and the spectrometer by properly aligning the all optics on the detection side (i.e., collimation and focusing lens) and at zero wavelength get a clean focused image of the sample on the spectrometer CCD. 
		Note: It is helpful to have another external imaging camera to help with alignment and getting a crisp image of the sample.</li>
	</ol>
	</li>
	<li>After the detection side is set up, focus the laser onto the sample. Focus the beam spot to the smallest size possible on the sample by using a lens. Use a tunable diode laser with an excitation range that contains the ground state transition energies. Set the laser incident to the sample at an oblique angle. An advantage of oblique incidence is that it helps get rid of a large portion of the scattered laser light.</li>
	<li>Excite the sample at a higher non-resonant energy. Optimally, do this by exciting at an energy below the wetting layer. For the InAs/GaAs QDMs in this study this corresponds to 75 meV or more above the ground state transitions.</li>
	<li>Run the spectrum acquisition software in focus mode. Scan the sample across the laser spot using an X-Y translation stage which is attached to the cryostat microscopy sample housing. Do this until the spectrometer CCD captures the discrete lines of the ground-state transitions. Center the detection on one of the QDMs. 
	Note: Once a QDM is found, the optical setup is completed. For the samples used the ground state energies are around 1,300 meV.</li>
	<li>Generate a bias map.
	<ol>
		<li>Apply a potential via the source meter (step 1.5) connected to the electrodes on the sample; this applies a bias across the electrodes in turn generating an electric field to the QDMs. 
		Note: The bias range applied to the sample is 0-2 V across the Schottky diode structure. This is when the device is in reverse bias, and the electric field limits the amount of charges in the QDM allowing for the individual charge states to be visible.</li>
		<li>Take individual spectra at different incremented voltage values, depending on desired resolution this normally ranges from hundredths to thousandths of a volt. Combine these individual spectra together by using a custom program (e.g., LabView). 
		Note: The program can be easily coded using a wide range of different programs to combine the vector columns of the individual spectrums into a matrix, in the presented experiment this has been added in to stitch together the data in real-time.
		<ol>
			<li>Click the run button to take a bias map. This takes spectra at a set bias and make it a column vector, then adds each incremented bias spectra as another column. 
			Note: This generates a data matrix where the intensity values correspond to the PL intensity, rows represents energy/wavelength, and columns correspond to voltage. The bias map should be viewable as it is being run, thus allowing live feedback on the quality of the data. 
			Note: A bias map helps to identify different charge configurations and gives the proper information to complete the setup of both the excitation and detection paths.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Identify the transition that will be excited. Note the energy of the transition and the bias range of interest.
	<ol>
		<li>At this point decide how the laser excitation will be tuned through the transition. There are three different options to get the laser to excite into the ground state transition:
		<ol>
			<li>Tune the transition energy by temperature.18</li>
			<li>Use the Stark shift of the transition to achieve resonance with the laser energy.19 
			Note: A nice feature of the two methods mentioned above is that a tunable laser source is not needed, since the QDM transitions are tuned through a fixed laser energy.</li>
			<li>Alternatively, use a tunable laser source, stepping the laser energies through the transition. A detection signal will be present when the laser is resonant with the transition, this gives the measurement its laser limited spectral resolution. This will be the focus for the rest of the protocol.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>With the transition identified and the experimental parameters set, select both excitation and detection energies for the measurement.
	<ol>
		<li>Choose the excitation energy as that of the transition. Choose the detection as the excited transition&#39;s energy minus the energy of the longitudinal optical (LO) phonon associated with the semiconductor alloy. Using these values, choose the proper edge pass filters for the measurement; they must have cutoffs in between the excitation and detection energies. 
		Note: For the experimental results presented, the driven transition was the neutral ground state exciton shown in Figure 3, observed at 1,301.7 meV and the -1 LO phonon emission is located at 1,266 meV, corresponding to 952.5 nm and 979.3 nm, respectively. We therefore use a 960 nm short pass filter for the excitation and a 960 nm long pass filter for the detection. Interference cut-off filters are ideal for this purpose since they can be tuned by adjusting the angle.</li>
	</ol>
	</li>
	<li>Set the laser to excite at the transition energy of interest, which is done simply by entering the desired laser wavelength value in the appropriate input field on the front panel of the laser control software.</li>
	<li>Set the center wavelength to the predetermined value to monitor the -1 LO photon emission by entering the desired center wavelength value in the appropriate input field on the front panel of the spectrometer control software. 
	Note: The -1 LO photon emission for InAs/GaAs is approximately 36 meV below the exciton transition that will be excited.</li>
	<li>Using the camera software, start collection with the CCD by running the spectrum acquisition software in continuous mode by clicking the focus mode button. A signal should be visible or could still be hidden by the laser scatter.</li>
	<li>Maximize the signal. CRITICAL STEP: Tune the excitation short pass filter, by slightly adjusting its angle so that it has the proper wavelength cutoff. 
	Note: The optimal angle is established by monitoring the signal while adjusting the angle of the short pass filter. By changing the angle of the filter this changes the cut-off wavelength. The key is to make sure that as much laser light as possible is suppressed from the collection.</li>
</ol>
3. Quasi-resonance Measurement Setup
<ol>
	<li>Set experimental parameters on the computer controls using the custom software&#8217;s main screen. To do so, start the collection program and click on the Step Polarization, Temp, or WL tab. This sets all the experimental values and once run, collects data through the various parameters. 
	Note: For our experiment&#8217;s data taking all computer controls were custom programmed. The key is to have a software or program able to set the center wavelength of the spectrometer, control the CCD and source meter, and step the laser energy through the transition while collecting a range of spectrums at different biases.&#160;&#160;
	<ol>
		<li>Input the established laser energy range that the laser will scan through: from about 50 &#181;eV above the neutral exciton transition energy of 1,301.7 meV, to 50 &#181;eV below.&#160; Set the initial Wavelength to start the scan using the &#8220;Desired WL (nm)&#8221; field. Set the end range of the laser to scan over (&#8220;End Motor Unit&#8221;).</li>
		<li>Set the bias range that the source meter will scan through by clicking on the &#8220;Voltage Settings&#8221; tab. Set the starting bias value (&#8220;Voltage Start (V)&#8221;), the end bias value &#8220;Voltage End (V)&#8221; and the bias step size &#8220;Voltage Step (V)&#8221;. Here, the bias range scanned was 1.68 to 1.82 V.</li>
		<li>Input the integration time chosen by clicking on the &#8220;Camera Settings&#8221; tab. Set the integration time for the CCD under &#8220;Exposure (s)&#8221; (Refer to step 3.3). Select a reasonable integration time for the CCD. Experiment with the integration time to get a good signal. The larger the integration time the better signal averaging that is obtainable.20 
		Note: Integration times used for the experiment were 10 sec. But, integration times can be as low as 0.5 sec, depending on the strength of the PL signal. At times it is not even necessary to see the signal in individual scans but upon stitching the spectra together the data summation plus the capability of the eye to interpolate will reveal the transitions in the photoluminescence excitation (PLE) map.</li>
	</ol>
	</li>
</ol>
4. Data Acquisition
<ol>
	<li>Once the experimental parameters are established begin the experiment. Start collection by clicking the run button. 
	Note: &#160;At each laser energy the software varies the bias taking a spectrum and a background spectrum. This is done for each bias step. Then the laser energy is varied and the process continues until the entire range chosen is complete.</li>
	<li>Post-process the data.
	<ol>
		<li>Take the additional background scans that are taken at the end of every bias map and subtract the average from each of the bias columns. Use a background subtraction program, or write a program that takes the background columns, averages them together and subtract this averaged background spectra from each bias column in the data matrix. Note: See Supplemental Code File for the program used in our lab. 
		Note: This removes other spurious signals caused by any remaining scattered laser light, greatly improving the bias map.</li>
	</ol>
	</li>
	<li>Analyze the data.
	<ol>
		<li>For example, to extract the characteristic parameters of the spectral line use a mathematical fitting software to run a Lorentzian fitting at each bias slice of the PL map.21 Once the fitting procedure has been completed all information will be in the coefficients of the fitting such as the maximum intensity, spectral location and FWHM.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The above instructions demonstrate the phonon-assisted quasi-resonance spectroscopy method. By exciting into a QD discrete state, one can monitor the phonon emission line, achieving high resolutions. In the example provided, by using phonons it is even possible to resolve the lifetime-limited linewidth of the neutral exciton visible in experiments. The method is easy to incorporate into existing PL spectroscopy setups. As mentioned, once the energy of the desired transition line is identified via non-resonant spectroscop...

High resolution is the key to unlocking new knowledge. With this knowledge, new technologies can be developed such as better sensors, more precise manufacturing tools, and more efficient computational devices. Generating this key, however, often comes at a high cost of resources, time or both. This issue is omnipresent across all scales from the atomic physics of resolving the lifted degeneracies of electron spins to astronomy where a small spectral shift can lead to detection of planets next to distant stars.1,2,3 
The focus of this work is on using a standard spectrometer setup and showing how it can resolve spectral features below its resolution limit, especially with regard to the field of semiconductor optics. The example presented is that of anisotropic electron-hole (e-h) exchange splitting in InAs/GaAs quantum dots (QDs), which is on the order of a few &#181;eV.4 The resolution limit of the spectrometer can be overcome by combining standard PL and laser spectroscopy techniques. This method of quasi-resonance fluorescence has the added benefit of achieving laser limited resolution using a commonplace single-stage spectrometer.
A standard optical spectroscopy system for single QD PL spectroscopy consists of a single-stage 0.3-0.75 m monochromator and a charge coupled device (CCD) detector along with an excitation laser source and optics. Such a system is at best capable of resolving 50 &#181;eV in the near-infrared spectrum around 950 nm. Even with the use of statistical and deconvolution techniques, such a single monochromator setup is not capable of resolving less than 20 &#181;eV in PL measurements.5 This resolution can also be improved by using a triple spectrometer, in triple additive mode, where the spectrum is successively dispersed by all three gratings. The triple spectrometer has the advantage of increased resolution, capable of resolving around 10 &#181;eV. In an alternative configuration, triple subtractive mode, the first two gratings behave as a band pass filter, giving the added feature of being able to separate the excitation and detection by less than 0.5 meV. The drawback of the triple spectrometer is that it is a costly system.
Before presenting the method of interest, we briefly discuss other experimental approaches that, with added complexity, achieve better spectral resolution and are able to resolve the fine structure of single QDs. Elements of these methods are relevant to the presented method. One such method is adding a Fabry-Perot interferometer (FPI) in the detection path of a single spectrometer setup.6 Using this method the resolution is set by the finesse of the FPI. Thus, the spectrometer&#39;s resolution is improved to 1 &#181;eV, at the cost of added complexity and lower signal intensity.7 The interferometer method also changes the general operation of the spectrometer with the CCD camera, effectively becoming a single point detector, and the tuning through various energies is achieved by adjusting the FPI cavity itself.
Resonance fluorescence (RF) spectroscopy, another method where a single optical transition is both excited and monitored also offers the promise of high-resolution spectroscopy. The spectral resolution is only limited by the laser linewidth and keeps the CCD as a multi-channel detector, where not just one sensor is detecting the signal but a number of CCD pixels. This multichannel detection is advantageous in terms of signal averaging. The challenge in RF spectroscopy is separating the PL signal from the larger background of the scattered laser light, especially when measuring at the single QD level. A number of techniques can be used to lower the ratio of signal to scattered laser light, which involve either polarization8, spatial9 or temporal separation10 of the excitation and detection. The first is to use high extinction polarizers to suppress the scattered light, but this method has the unfavorable outcome of losing polarization information from the PL.8 Another possible method to obtain resonance fluorescence is to engineer semiconductor systems that are coupled to optical cavities where the excitation and detection paths are spatially separated. This eliminates the issue of having to resolve the PL signal from the large laser background. However, this method is limited to intricate sample fabrication which is in general resource intensive.9
Another class of methods that is also able to resolve minute energy differences is that of pure laser spectroscopy, such as differential transmission, which has the benefit of achieving laser-limited resolution with complete polarization information. This method typically requires lock-in detection to observe miniscule changes in the transmission signal compared to that of the large laser background.11 Lately, advances in nanofabrication have led to a boost of the fraction of laser light that interacts with the QD(s) to values up to 20%, by either using index-matched solid immersion lenses or embedding the dots in photonic crystal waveguides.12
Even though these methods have the capability of achieving high energy resolution, they come at the cost of expensive equipment, complex sample fabrication and loss of information. The method in this work combines elements from these three methods without adding complexity in instrumentation or sample fabrication to a regular PL setup.
Recent work has shown that with a triple spectrometer system in subtractive mode, it is possible to visualize the singlet-triplet fine structure in the two-photon transition spectrum of a quantum dot molecule (QDM).13 The involved energy splitting on the order of a few to tens of &#956;eV were resolved using a triple subtractive mode, which allowed to excite the transitions resonantly and detect within less than a meV. The spectral information was extracted by monitoring below the transition using acoustic phonons and other lower-lying exciton transitions. This method can also be applied to resolve the anisotropic e-h exchange splitting and even the lifetime-limited linewidth of the exciton transition of 8 &#181;eV and 4 &#181;eV, respectively as seen in Figure 1. Similar to this result, this paper will focus on a simple spectrometer setup that will incorporate many of the advantages that the other high resolution methods possess. Additionally the CCD will remain as a multi-channel detector. The experimental setup can also be kept fairly inexpensive relative to other high-resolution spectroscopy methods and has the added benefit of being easily modified to achieve single point correlation measurements. Unlike the result using acoustic phonons and a triple spectrometer, the underlying key is to make use of the LO-phonon satellite associated with the semiconductors and related alloys that make up semiconductor samples. The energy separation between LO-phonon satellite and the zero-phonon line (ZPL) is on the order of tens of meV for such samples, allowing the use of a single-stage spectrometer.14 This energy separation allows for use of the proposed quasi-resonance spectroscopy method by resonantly driving a transition and monitoring below the excitation by an energy equal to one LO phonon. This technique is analogous to that of PL excitation where one excites into an excited transition and monitors the ground state transition.15 The separation between the transition being excited and that of the LO-phonon satellite allows for the use of edge pass filters to suppress the elastically scattered light. This method of using the phonon satellite allows for laser linewidth limited resolution, since resonantly exciting the transition is typically the only time that the LO-phonon satellite emission becomes visible.

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high resolution phonon-assisted quasi-resonance fluorescence spectroscopy

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we demonstrate an optical spectroscopy method that is capable of resolving spectral features beyond that of the spin fine structure and homogeneous linewidth of single quantum dots (QDs) using a standard, easy-to-use spectrometer setup. This method incorporates both laser and photoluminescence spectroscopy, combining the advantage of laser line-width limited resolution with multi-channel photoluminescence detection. Such a scheme allows for considerable improvement of resolution over that of a common single-stage spectrometer. The method uses phonons to assist in the measurement of the photoluminescence of a single quantum dot after resonant excitation of its ground state transition. The phonon&#39;s energy difference allows one to separate and filter out the laser light exciting the quantum dot. An advantageous feature of this method is its straight forward integration into standard spectroscopy setups, which are accessible to most researchers.

The results presented in the figures show the high resolution capabilities of using phonons to assist in the PL measurement. The schematic (Figure 2) shows that, with the exception of the edge pass filters on both excitation and detection, the experimental setup remains a standard spectroscopy setup, with the optional addition of polarization control. Comparison with a single and triple spectrometer (Figure. 3) portrays the phonon-assisted method&#39;s gr...

Watch this Scientific Journal Video about High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy at JoVE.com

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we ...

high-resolution-phonon-assisted-quasi-resonance-fluorescence

Research

JoVE Journal

Engineering

7.5K Views.  University of California, Merced. The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup. 

Video: High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to &lt; 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

We discuss the construction and operation of a complex nonlinear optical
system that uses ultrafast all-optical switching to isolate Raman from fluorescence
signals. Using this system we are able to successfully separate Raman and fluorescence
signals utilizing pulse energies and average powers that remain biologically safe.

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ...

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 ...

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or &#8220;tapping mode&#8221; AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.
PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.
The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.

A method for investigating the structure of a protein photoreceptor using atomic force microscopy (AFM) is described in this paper. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) reveals intact protein dimers on a mica surface.

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

Genetics

Resonance Fluorescence of an InGaAs Quantum Dot in a Planar Cavity Using Orthogonal Excitation and Detection

The ability to perform simultaneous resonant excitation and fluorescence detection is important for quantum optical measurements of quantum dots (QDs). Resonant excitation without fluorescence detection &#8211; for example, a differential transmission measurement &#8211; can determine some properties of the emitting system, but does not allow applications or measurements based on the emitted photons. For example, the measurement of photon correlations, observation of the Mollow triplet, and realization of single photon sources all require collection of the fluorescence. Incoherent excitation with fluorescence detection &#8211; for example, above band-gap excitation &#8211; can be used to create single photon sources, but the disturbance of the environment due to the excitation reduces the indistinguishability of the photons. Single photon sources based on QDs will have to be resonantly excited to have high photon indistinguishability, and simultaneous collection of the photons will be necessary to make use of them. We demonstrate a method to resonantly excite a single QD embedded in a planar cavity by coupling the excitation beam into this cavity from the cleaved face of the sample while collecting the fluorescence along the sample&#39;s surface normal direction. By carefully matching the excitation beam to the waveguide mode of the cavity, the excitation light can couple into the cavity and interact with the QD. The scattered photons can couple to the Fabry-Perot mode of the cavity and escape in the surface normal direction. This method allows complete freedom in the detection polarization, but the excitation polarization is restricted by the propagation direction of the excitation beam. The fluorescence from the wetting layer provides a guide to align the collection path with respect to the excitation beam. The orthogonality of the excitation and detection modes enables resonant excitation of a single QD with negligible laser scattering background.

Resonant excitation of a single self-assembled quantum dot can be achieved using an excitation mode orthogonal to the fluorescence collection mode. We demonstrate a method using the waveguide and Fabry-Perot modes of a planar microcavity surrounding the quantum dots. The method allows complete freedom in the detection polarization.

The ability to perform simultaneous resonant excitation and fluorescence detection is important for quantum optical measurements of quantum dots (QDs). ...

Ultrafast Time-resolved Near-IR Stimulated Raman Measurements of Functional &#960;-conjugate Systems

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near infrared (near-IR) transitions, because it can overcome the low sensitivity of spontaneous Raman spectrometers in the near-IR region. Here, we describe technical details of a femtosecond time-resolved near-IR multiplex stimulated Raman spectrometer that we have recently developed. A description of signal generation and optimization, measurement, data acquisition, and calibration and correction of recorded data is provided as well. We present an application of our spectrometer to analyze the excited-state dynamics of &#946;-carotene in toluene solution. A C=C stretch band of &#946;-carotene in the second lowest excited singlet (S2) state and the lowest excited singlet (S1) state is clearly observed in the recorded time-resolved stimulated Raman spectra. The femtosecond time-resolved near-IR stimulated Raman spectrometer is applicable to the structural dynamics of &#960;-conjugate systems from simple molecules to complex materials.

Details of signal generation and optimization, measurement, data acquisition, and data handling for a femtosecond time-resolved near-IR stimulated Raman spectrometer are described. A near infrared stimulated Raman study on the excited-state dynamics of &#946;-carotene in toluene is shown as a representative application.

Femtosecond time-resolved stimulated Raman spectroscopy is a promising method of observing the structural dynamics of short-lived transients with near ...

Chemistry

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 ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

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