Name: a simple dewar/cryostat for thermally equilibrating samples at known temperatures for accurate cryogenic luminescence measurements
Uploaded: 2016-07-19T15:00:00.000+00:00
Duration: 6 min 6 s
Description: Watch this Scientific Journal Video about A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements at JoVE.com

It is a pleasure to acknowledge the Office of the Dean of the School of Arts and Sciences and the Office of the Provost at Concordia University for support of this research.&#160;&#160;The authors wish to thank G. A. Crosby for his many contributions to this investigation.

1. Sample Preparation and Loading for Cryogenic Spectroscopy
<ol>
	<li>Prepare ~3 ml of a ~10-3-10-4 M solution of luminescent chromophore in an appropriate solvent. 
	Note: While many solvents can be used, water and various alcohol solvents (e.g., ethanol, ethanol/methanol mixtures, ethylene glycol, and glycerol) provide an excellent combination of solubility and surface tension characteristics for cryogenic work.</li>
	<li>Prepare a sample loop by twisting a length of bare copper wire ~0.4 mm in diameter around a nail or screw to give a single loop ~3 mm in diameter followed by an ~30 mm straight length of braided copper wire.</li>
	<li>Rinse the sample loop with 95% ethanol to remove finger oils and other contaminants and allow to dry. To ensure cleanliness of the loop, rinse first with nitric acid, second with distilled water, and finally with ethanol.</li>
	<li>Load the sample solution into the sample loop by dipping the loop into the sample solution (Step 1.1), letting surface tension of the solution hold a thin film of the solution in the loop.</li>
	<li>Immediately dip the loaded sample loop into liquid nitrogen to freeze and stabilize the thin film sample solution in the loop taking care to use eye protection (safety goggles) and hand protection (gloves).</li>
</ol>
2. Thermocouple Preparation, Alignment and Setup
<ol>
	<li>Prepare a copper-Constantan (Type T) thermocouple from two lengths of uninsulated 0.4 mm diameter copper wire and one length of 0.4 mm diameter uninsulated constantan (copper-nickel alloy) wire by forming two copper-Constantan junctions: a sample junction and a standard reference junction (0 &#176;C ice/water mixture). 
	Note: While the junctions can be silver soldered together, it is completely satisfactory to twist the copper and Constantan wires together to form the junctions.
	<ol>
		<li>Clamp the copper and Constantan wires together with needle-nose pliers at a ~90&#176; angle. While pulling firmly on the two wires, snugly braid them together for five-six turns.</li>
	</ol>
	</li>
	<li>Arrange all thermocouple wires so they do not touch at any point other than at the two junctions. If the thermocouple wires are covered with a thin insulation coating, they may touch and points other than the junctions.
	<ol>
		<li>When using insulated thermocouple wires, scrape away the insulation at the ends of the wires where the junctions and the electrical contacts with the voltmeter terminals are formed. Be careful to check that the insulation material on the wires does not luminesce. Test the thermocouple wire insulation as a potential source of spurious luminescence by placing a small piece of insulated wire in the sample loop, exciting the sample with light of the chosen wavelength, and looking for an emission signal when no sample is present.</li>
	</ol>
	</li>
	<li>Connect the two copper thermocouple wires from the sample and reference junctions to the input terminals of a high-impedance 5&#189; digit digital voltmeter.</li>
	<li>Place both the sample and reference junctions in the 0 &#176;C water/ice bath and zero the voltmeter.</li>
	<li>Align the loaded sample loop and the sample junction of the thermocouple in the liquid-nitrogen-filled optical Dewar to coincide with appropriate height and direction of the excitation light beam. Keep the thermocouple junction and the sample loop as close together as possible and at exactly the same height in the Dewar.</li>
</ol>
3. General Mechanical Support and Alignment of Sample Loop and Thermocouple Junction in the Optical Dewar
<ol>
	<li>To align and hold the sample loop in the desired height in the optical Dewar, prepare an adjustable-height holding clamp by affixing a small electrical alligator clip to a 5 mm diameter x 30 mm length wood dowel.
	<ol>
		<li>Bore a hole slightly smaller than 5 mm diameter in a cork that fits snugly into the top of the optical Dewar. Clamp the braided wire section of the sample loop in the alligator clip, then slide the dowel up or down in the cork to achieve the desired sample height in the Dewar.</li>
	</ol>
	</li>
	<li>To align and hold the thermocouple junction at the desired height in the optical Dewar, use another 5-mm diameter wood dowel. Align the thermocouple junction so that it sticks out 10-20 mm below the bottom of the dowel. Align the copper and constantan wires on opposite sides of the dowel and wrap tightly with 12.5 mm-width polytetrafluoroethylene (PTFE) plumber&#39;s tape to hold these wires firmly in place.</li>
	<li>Bore another hole slightly smaller than 5 mm in diameter in the cork at the top of the Dewar to accommodate this second dowel and allow for vertical height adjustment of the thermocouple junction near the sample loop.</li>
	<li>Bore a third small hole in the cork at the top of the Dewar to serve as a nitrogen gas boil off vent hole.</li>
</ol>
4. Temperature-dependent Cryogenic Spectroscopy in the Optical Dewar
<ol>
	<li>Turn on all electronics on the CCD spectrograph at least 1 hr in advance to allow the electronics to warm up and the Peltier-cooled CCD camera to reach a stable operating temperature.</li>
	<li>After the warm up period is over, wavelength calibrate the CCD spectrograph against a series known atomic emission lines or bands. Measure the spectrum of a low-pressure atomic emission lamp emitting bands of known wavelengths and correlate CCD pixel numbers activated by the bands with the known band wavelengths. 
	Note: In most modern CCD spectrographs, including the Andor CCD spectrograph used in this research, the wavelength calibration process is automated in the software.
	<ol>
		<li>Intensity calibrate the spectrograph by comparing the measured the spectrum of a quartz-halogen lamp operating at 3,200 K to the known spectral profile of the lamp, which closely approximates a 3,200 K black body.</li>
	</ol>
	</li>
	<li>Pre-align the excitation and emission optics to ensure that the excitation light is hitting the sample in the loop and the emitted light from the sample is being collected and impinged upon the entrance slit of the CCD-spectrograph. 
	Note: This is two-step mechanical process. The first step is an initial coarse alignment of the optics to get the emitted light onto the entrance slit of the spectrograph such that a luminescence signal is detected by the CCD. The second step consists of a careful optimization of the sample&#39;s luminescence signal strength by systematic adjustments of the positions of the exciting light beam, the Dewar, the sample itself in the loop, and the emission collection optical elements.</li>
	<li>Once the optics are aligned and optimized as described in 4.3, measure a 77 K liquid nitrogen reference luminescence spectrum of the sample. For this spectrum, ensure that the frozen thin-film sample in the copper wire loop, the thermocouple adjacent to the loop, the Dewar, the excitation beam, and the emission collection optics are all in their final optimized positions and the sample is completely covered with liquid nitrogen.</li>
	<li>Set up the reference junction of the thermocouple on a wood dowel with the wires wrapped in PTFE tape just as the sample junction is. This protects the reference junction and prevents undesirable wire contact.</li>
	<li>Verify that the 0 &#176;C reference thermocouple junction is immersed in a water-ice slush. Check the ice level in the reference junction frequently. Set up the reference junction in a small, wide-mouth laboratory Dewar to reduce the rate at which the ice melts.</li>
	<li>Check the measured thermocouple voltage at 77 K against the literature voltage at 77 K listed for a Type T copper-Constantan thermocouple. The two thermocouple voltages should be in very close agreement. Make appropriate corrections for pressure (e.g., using the Clausius-Clapeyron equation) if the atmospheric pressure is less than 1 atm.</li>
	<li>Allow the liquid nitrogen to boil off slowly. 
	Note: The temperature of the sample rises slowly (over a period of several hours) as the distance h between the sample and the liquid nitrogen level slowly increases as a consequence of liquid nitrogen boil off. The digital voltmeter responds to this temperature rise, providing an accurate temperature measurement since the sample thermocouple junction is fully immersed in cold nitrogen vapor.</li>
	<li>Momentarily turn on or unblock the excitation light and use the CCD spectrograph to acquire a luminescence spectrum using manufacturer&#39;s protocol. The spectral data acquisition process should be optimized so that it only takes a few seconds.
	<ol>
		<li>Turn off or re-block the excitation light just as soon as the data acquisition process finishes to minimize errors that arise from unwanted excitation-light-induced sample photochemistry and/or excitation-light-induced sample heating.</li>
		<li>Record the thermocouple voltage at the beginning and end of the spectral data acquisition interval. The thermocouple voltage should not change appreciably (i.e., associated temperature change &#916;T within 0.0-0.1 K) during the very short time interval (~1-10 sec) over which the spectral data are acquired at a given temperature. Convert voltage readings to temperatures by referencing a Type T copper-Constantan temperature vs. voltage table.</li>
	</ol>
	</li>
	<li>Allow the liquid nitrogen boil off to continue, and measure another spectrum when the sample temperature has increased by ~5 K. Make no changes in the optics, electronics or excitation light intensity during the course of these temperature-dependent spectral measurements. 
	Note: Depending upon the quality of the optical Dewar, this warm up process takes from 5 to 15 min per ~5 K interval.</li>
	<li>Intensity-correct the CCD spectral data sets by subtracting off the dark frame intensities (i.e., the spectral intensity recorded by the CCD when the entrance slit is blocked) and accounting for wavelength-dependent spectral response. Subtract the baseline dark count intensity count level from each sample spectrum.
	<ol>
		<li>Correct for the wavelength dependent spectral response by measuring the intensity of a standard of known intensity, such as a 3200 K tungsten halogen standard lamp, and using the ratio of the known intensity to the measured intensity to correct the measured intensity of the sample at each wavelength.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Baker, D. C., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+and+magnetic+evidence+for+multiple-state+emission+from+tris(2,2'-bipyridine)ruthenium(II)+sulfate.">Spectroscopic and magnetic evidence for multiple-state emission from tris(2,2'-bipyridine)ruthenium(II) sulfate.</a> Chem. Phys. 4 (3), 428-433 (1974).</li><li>Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+investigations+of+excited+states+of+transition+metal+complexes.">Spectroscopic investigations of excited states of transition metal complexes.</a> Acc. Chem. Res. 8 (7), 231-238 (1975).</li><li>Hager, G. D., Watts, R. J., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+transfer+excited+states+of+ruthenium(II)+complexes+II.+Relationships+of+level+parameters+to+molecular+structure.">Charge transfer excited states of ruthenium(II) complexes II. Relationships of level parameters to molecular structure.</a> J. Am. Chem. Soc. 97 (24), 7037-7042 (1975).</li><li>Elfring, W. H., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excited+states+of+mixed-ligand+chelates+of+ruthenium(II)+and+rhodium(III).">Excited states of mixed-ligand chelates of ruthenium(II) and rhodium(III).</a> J. Phys. Chem. 80 (20), 2206-2211 (1976).</li><li>Elfring, W. H., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excited+states+of+mixed-ligand+chelates+of+ruthenium(II).+Quantum+yield+and+decay+time+measurements.">Excited states of mixed-ligand chelates of ruthenium(II). Quantum yield and decay time measurements.</a> J. Am. Chem. Soc. 103 (10), 2683-2687 (1976).</li><li>Fordyce, W. A., Rau, H., Stone, M. L., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+state+emission+from+rhodium(I)+and+iridium(I)+complexes.">Multiple state emission from rhodium(I) and iridium(I) complexes.</a> Chem. Phys. Lett. 77 (2), 405-408 (1981).</li><li>Pankuch, B., Crosby, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+measurements+of+solutions+and+rigid+glasses.">Spectroscopic measurements of solutions and rigid glasses.</a> Chem. Instrum. 2 (3), 329-335 (1970).</li><li>Landee, C. P., Greeney, R. E., Lamas, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+helium+cryostat+for+a+vibrating+sample+magnetometer.">Improved helium cryostat for a vibrating sample magnetometer.</a> Rev. Sci. Instrum. 58 (10), 1957-1958 (1987).</li><li>Fairman, R. A., Spence, K. V. N., Kahwa, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+liquid+nitrogen+gas-flow+cryostat+for+variable+temperature+laser+luminescence.">A simple liquid nitrogen gas-flow cryostat for variable temperature laser luminescence.</a> Rev. Sci. Instrum. 65 (2), 503-504 (1994).</li><li>Meyer, G. D., Ortiz, T. P., Costello, A. L., Kenney, J. W., Brozik, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+fiber-optic+coupled+luminescence+cryostat.">A simple fiber-optic coupled luminescence cryostat.</a> Rev. Sci. Instrum. 73 (12), 4369-4374 (2002).</li></ol>

The authors have nothing to disclose.

The development of this apparatus for low temperature luminescence spectroscopy arose out of necessity. It was critical that solutions containing the chromophore of interest and also supersaturated with oxygen could be loaded, frozen, and positioned for spectroscopy all in an instant in a Dewar/cryostat design in which sample temperature was well defined, stable, and slowly changeable. Virtually all commercial cryostats take much more time to load with sample than these experimental constraints would allow. It was also i...

Within the cryogenic temperature domain, temperature-dependent investigations of the electronic luminescence spectra and luminescence lifetimes of light emitting molecules provide a wealth of information about the excited electronic states of these molecules and the photochemical and photophysical phenomena that arise from these states. The pioneering temperature-dependent photophysical investigations of Crosby and co-workers on ruthenium(II), rhodium(I), and rhodium(III) complexes of 1,10-phenanthroline, 2,2&#39;-bipyridine, and other ligands illustrate well the inherent power of temperature-dependent spectroscopy to elucidate the structures, symmetries, energetics, and chemical behaviors of a manifold of emissive excited electronic states.1-6 
However, to do temperature-dependent cryogenic spectroscopy well is not a trivial matter. It is all too easy for the sample under spectroscopic interrogation not to be thermally equilibrated and thus to manifest a wide range of temperatures across a thermal gradient. The resulting measured spectrum is, in effect, a superposition of emissions over a range of temperatures. Moreover, even the average temperature over this range of temperatures might be quite different from the readout of the temperature probe (e.g., a thermocouple or resistance temperature device) placed on or near to the sample. Thus, to do temperature-dependent cryogenic spectroscopy correctly requires the establishment of experimental conditions under which the sample temperature is known, stable, uniform, and, when the time comes, adjustable. These conditions can be achieved with extremely modest apparatus comprised of a CCD spectrograph, excitation source, optical Dewar, and thermocouple operating under simple, straightforward experimental protocols (see Figure 1).
<img alt="Figure 1" fo:display-align="center" src="/files/ftp_upload/54267/54267fig1.jpg" /> 
Figure 1. Luminescence Spectrograph Setup for Low Temperature Spectroscopy. The system as shown in this top view includes: (a.) CCD detector, (b.) spectrograph, (c.) entrance slit and filters, (d.) luminescence collection optics, (e.) laser or arc lamp excitation source, (f.) excitation beam, (g.) a fused-silica optical Dewar on xyz translation mount, (h.) thermocouple sample junction, (i.) sample, (j.) thermocouple reference junction: 0 &#176;C = 273.15 K ice/water bath, (k.) digital voltmeter. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54267/54267fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Undesirable thermal gradients in the sample and erroneous average sample temperatures are almost sure to result if one side of a sample is placed in physical contact with a cryogenic &#34;cold finger&#34; surface while the other side of the sample is in vacuum. The most practical way to ensure that the complete sample is at uniform measurable temperature T is to totally immerse the sample and the temperature probe in a cryogenic liquid at temperature T (e.g., liquid nitrogen or liquid helium) or in a cryogenic vapor at temperature T (e.g., cold nitrogen or cold helium vapor). Variable-temperature cryostats achieve a constant temperature sample environment by balancing cryogen flow with electrical resistance heating to achieve the desired cryogenic sample temperature.7-9 A thermal exchange gas may be employed to ensure the sample temperature is uniform. The idea is to have the sample in thermal equilibrium with the exchange gas which in turn is in thermal equilibrium with the cryostat. Cryostat designs have emerged that achieve thermal equilibration of the sample at various temperatures simply by adjusting the sample height h above the liquid level of the cryogen in a storage Dewar.10 Samples are excited and luminescence is detected via fiber optic cables or lens systems. At a given sample/probe height h, the cryogen vapor temperature is T(h) and this temperature increases as h increases (i.e., the Dewar provides a smooth thermal gradient dT/dh &#62; 0 in the vapor). The cryogen gas above the liquid in effect becomes the exchange gas. Positioning a small sample and temperature probe at h ensures thermal equilibration of the sample at T(h). To increase sample temperature, h is increased. To decrease sample temperature, h is decreased. The low temperature limit of such a cryostat is the temperature of the liquid cryogen at h = 0. This low temperature limit can be decreased further by reducing the pressure. In a large storage Dewar (e.g., a 100-L liquid helium Dewar or a 10-L liquid nitrogen Dewar), the cryogen boil-off rate is negligible during the time frame of a series of spectroscopic measurements thus allowing an adjustment in sample height h above the liquid cryogen to become a known adjustment in sample temperature.
Spectroscopic investigations in this laboratory of the temperature dependence of oxygen-induced quenching of the luminescence from transition metal complexes led to the adaptation of a small fused silica optical Dewar for variable-temperature spectroscopic investigations with liquid nitrogen in the 77-300 K range (see Figure 2).
<img alt="Figure 2" fo:display-align="middle" src="/files/ftp_upload/54267/54267fig2.jpg" /> 
Figure 2. Fused Silica Optical Dewar Setup for Variable Temperature (77-300 K) Cryogenic Luminescence Spectroscopy. This diagram of the optical Dewar illustrates the complete variable temperature system. (a.) liquid nitrogen, (b.) transparent (4.0 cm) unsilvered optical access region of Dewar, (c.) copper sample loop, (d.) thermocouple junction, (e.) silvered region of Dewar, (f.) alligator clip, (g.) wood dowel, (h.) distance between liquid nitrogen level and sample, (i.) evacuated region between inner and outer Dewar walls, (j.) cork stopper, (k.) nitrogen gas vent hole, (l.) thermocouple wires, (m.) thermocouple wires separated and secured to wood dowel with PTFE tape. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54267/54267fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Fused silica is non-emissive and provides high optical transmission from the near ultraviolet, through the visible, and out to the near infrared (~200-2,000 nm). The basic concepts operative in the large storage Dewar system described previously10, where sample height above the liquid cryogen determines sample temperature, were successfully carried over on a small scale using this small optical Dewar. However, instead of mechanically adjusting the sample height h above a stationary liquid cryogen level to adjust the sample temperature T, the sample position with respect to the Dewar itself is fixed (Figure 2). The slow boil off of the liquid nitrogen in the optical Dewar over a period of several hours gradually increases the distance h of the sample above the falling liquid nitrogen level (Figure 3).
<img alt="Figure 3" fo:display-align="middle" src="/files/ftp_upload/54267/54267fig3.jpg" /> 
Figure 3. Close up of Sample Region of Optical Dewar. Temperatures: sample immersed in liquid nitrogen to level h0, to give T0 = 77 K; sample immersed in cold nitrogen vapor at levels h1 &#60; h2 &#60; h3 above the liquid nitrogen level to give sample temperatures T1 &#60; T2 &#60; T3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54267/54267fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This allows for a slow, controlled increase in the sample temperature over time (up to several hours) while maintaining both the sample and the temperature probe, a copper-Constantan thermocouple junction, in thermal equilibrium with the cold nitrogen vapor. Luminescence spectra spanning the visible and near-infrared regions are acquired in just a few milliseconds per spectrum (or hundreds of spectra per second) with a CCD-equipped luminescence spectrograph during which sample temperature is virtually constant (&#916;T &#60; 0.1 K) as each spectral dataset is acquired. Typical wait times between spectra at temperatures ~5 K apart are ~5-15 min. Moreover, the effects of sample heating or photochemical degradation of the sample by the exciting light are minimized since the excitation light is only allowed to strike the sample a few seconds per spectrum. In the interest of simplicity, portability, and quickness of sample loading, fiber optic cables are not employed. Samples are excited directly with either the 365 nm band from a mercury arc lamp or the 405 nm line of a diode laser. Emitted light from the samples is picked up directly from the emitting sample in the Dewar by a collection lens and impinged on the entrance slit of the spectrograph by a focusing lens. Samples of the ruthenium and rhodium complexes under investigation are prepared for spectroscopic study as thin films of ~10-3-10-4 M solute in oxygen-saturated solutions. The solutions are held by surface tension in small copper wire loops (~3 mm loop diameter formed out of 0.0150 in. dia. copper wire). The thermocouple junction height is then adjusted so it is equal to the sample height (hthermocouple = hsample) and in close proximity to the sample loop as shown in Figures 2 and 3. Temperatures are determined by measuring the voltage difference between the thermocouple sample junction and a 0 &#176;C water/ice thermocouple reference junction using a high-impedance digital voltmeter and comparing to a temperature vs. voltage table for a Type T copper/Constantan thermocouple. The thin-film sample solutions captured in the wire loops are flash frozen by quick immersion in liquid nitrogen in the optical Dewar. Then the frozen solutions are allowed to warm up very gradually over time, remaining frozen, while their luminescence spectra are measured as a function of temperature. The luminescence intensity versus temperature data are analyzed according to the following model.
The total luminescence intensity of the sample at temperature T is given as the sum of the intensities arising from oxygenated and unoxygenated complexes:
<img alt="Equation 2" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq2.jpg" />.&#160;&#160;&#160; (1)
The luminescence intensity from the complexes without oxygen is assumed to be temperature independent. However, the luminescence intensity of the oxygenated complexes will decrease exponentially with increasing temperature due to oxygen quenching. This can be described by an Arrhenius equation of the form
<img alt="Equation 3" fo:vertical-align="middle" src="/files/ftp_upload/54267/54267eq3.jpg" />.&#160;&#160;&#160;&#160; (2)
In Equation (2), Ea is the quenching activation energy and k is the Boltzmann constant. The maximum luminescence intensity will be observed in the low temperature region (see Figure 5), where there is insufficient thermal energy to overcome the quenching activation barrier (i.e., energy transfer from the complex to oxygen). If Equation (2) is substituted into Equation (1), the expression
<img alt="Equation 5" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq5.jpg" />&#160;&#160;&#160;&#160; (3)
is obtained. In Equation (3), <img alt="Equation 6" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq6.jpg" /> is the intensity arising from oxygenated complexes in the low temperature region. Rearrangement of Equation (3) yields
<img alt="Equation 7" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq7.jpg" />.&#160;&#160;&#160;&#160; (4)
Taking the natural logarithm of the both sides of Equation (4) gives the expression
<img alt="Equation 8" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq8.jpg" />.&#160;&#160;&#160;&#160; (5)
From Equation (5) it is evident that a plot of <img alt="Equation 9" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq9.jpg" />versus&#160;<img alt="Equation 10" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq10.jpg" /> will give a straight line with <img alt="Equation 11" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq11.jpg" />, from which the luminescence quenching activation energy is obtained as
<img alt="Equation 12" fo:display-align="middle" src="/files/ftp_upload/54267/54267eq12.jpg" />.&#160;&#160;&#160;&#160; (6)

<table><tbody><tr><td>Diode laser 405 nm</td><td>Generic</td><td></td><td>Generic pencil-type laser pointer for luminescence excitation: 5 mW at 405&amp;nbsp;nm</td></tr><tr><td>Quartz optical dewar</td><td>Custom fabrication</td><td></td><td>3.5 cm id. x 25.0 cm length with 4.5 cm unsilvered region for optical access</td></tr><tr><td>Programmable 5 1/2 digit DMM</td><td>Keithley</td><td>Model 192</td><td>High impedence DMM for reading thermocouple voltages</td></tr><tr><td>Copper thermocouple wire</td><td>Omega Engineering</td><td>SPCP-010</td><td>0.010 in. diameter bare copper thermocouple wire</td></tr><tr><td>Constantan thermocouple wire</td><td>Omega Engineering</td><td>SPCC-010</td><td>0.010 in. diameter bare Constantan (copper/nickel) thermocouple wire</td></tr><tr><td>Polychromator/Spectrograph</td><td>Jarrell-Ash</td><td>82-415</td><td>0.25 m Ebert monochromator with back slit assembly removed to enable operation as a polychromator</td></tr><tr><td>CCD camera</td><td>Andor</td><td>DV-401-UV</td><td>Thermoelectrically cooled (-35 &amp;#176;C) CCD camera for detecting emitted light</td></tr><tr><td>Copper wire for sample loop</td><td>Generic</td><td></td><td>0.0150 in. diameter bare copper wire for sample loop</td></tr></tbody></table>

a simple dewar/cryostat for thermally equilibrating samples at known temperatures for accurate cryogenic luminescence measurements

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and a CCD spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, fast sample freezing, fast alignment of the sample, accurate and stable sample temperatures, and small size and portability of the Dewar/cryostat cryogenic unit. When coupled with the fast data acquisition rates of the CCD spectrograph, this Dewar/cryostat is capable of supporting cryogenic luminescence spectroscopic measurements on luminescent samples at a series of known, stable temperatures in the 77-300 K range. A temperature-dependent study of the oxygen quenching of luminescence in a rhodium(III) transition metal complex is presented as an example of the type of investigation possible with this Dewar/cryostat. In the context of this apparatus, a stable temperature for cryogenic spectroscopy means a luminescent sample that is thermally equilibrated with either liquid nitrogen or gaseous nitrogen at a known measureable temperature that does not vary (&#916;T &#60; 0.1 K) during the short time scale (~1-10 sec) of the spectroscopic measurement by the CCD. The Dewar/cryostat works by taking advantage of the positive thermal gradient dT/dh that develops above liquid nitrogen level in the Dewar where h is the height of the sample above the liquid nitrogen level. The slow evaporation of the liquid nitrogen results in a slow increase in h over several hours and a consequent slow increase in the sample temperature T over this time period. A quickly acquired luminescence spectrum effectively catches the sample at a constant, thermally equilibrated temperature.

Representative results obtained in the above described apparatus for a temperature-dependent luminescence quenching study in the 77-200 K region of the luminescent compound Tris(4,7-dimethyl-1,10-phenanthroline)rhodium(III), [Rh(4,7-Me2-1,10-phen)3]3+, dissolved in oxygen-saturated glycerol are listed in Table 1 and plotted in Figures 4, 5, and 7.
<table border="1" fo:keep-together.within-page...

Watch this Scientific Journal Video about A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements at JoVE.com

A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and ...

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Universidad San Sebastian

Research

JoVE Journal

Engineering

9.5K Views.  Concordia University. A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

Video: A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Bioengineering

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient optical micro-imaging technique which can be used to map and quantify such behavior. Europium thenoyltrifluoroacetonate (EuTFC) has a 612 nm luminescence line whose activation efficiency drops strongly with increasing temperature, due to T-dependent interactions between the Eu3+ ion and the organic chelating compound. This material may be readily coated on to a sample surface by thermal sublimation in vacuum. When the coating is excited with ultraviolet light (337 nm) an optical micro-image of the 612 nm luminescent response can be converted directly into a map of the sample surface temperature. This technique offers spatial resolution limited only by the microscope optics (about 1 micron) and time resolution limited by the speed of the camera employed. It offers the additional advantages of only requiring comparatively simple and non-specialized equipment, and giving a quantitative probe of sample temperature.

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

Measuring the Densities of Aqueous Glasses at Cryogenic Temperatures

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid cooling, to prepare the desired cryogenic temperature phase. Microliter to picoliter size drops are cooled by projection into a liquid nitrogen-argon (N2-Ar) mixture. The cryogenic temperature phase of the drop is evaluated using a visual assay that correlates with X-ray diffraction measurements. The density of the liquid N2-Ar mixture is adjusted by adding N2 or Ar until the drop becomes neutrally buoyant. The density of this mixture and thus of the drop is determined using a test mass and Archimedes principle. With appropriate care in drop preparation, management of gas above the liquid cryogen mixture to minimize icing, and regular mixing of the cryogenic mixture to prevent density stratification and phase separation, densities accurate to &#60;0.5% of drops as small as 50 pL can readily be determined. Measurements on aqueous cryoprotectant mixtures provide insight into cryoprotectant action, and provide quantitative data to facilitate thermal contraction matching in biological cryopreservation.

A protocol for determining the vitreous phase densities of micro- to pico-liter size drops of aqueous mixtures at cryogenic temperatures is described.

We demonstrate a method for determining the vitreous phase cryogenic temperature densities of aqueous mixtures, and other samples that require rapid ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Biochemistry

Biological Samples Preparation for Speciation at Cryogenic Temperature using High-Resolution  X-Ray Absorption Spectroscopy

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample preparation is a key and often complex procedure, particularly for biological samples. Although X-ray speciation techniques are widely used, no detailed protocol has been yet disseminated for users of the technique. Further, chemical state modification is of concern, and cryo-based techniques are recommended to analyze the biological samples in their near-native hydrated state to provide the maximum preservation of chemical integrity of the cells or tissues. Here, we propose a cellular preparation protocol based on cryo-preserved samples. It is demonstrated in a high energy resolution fluorescence detected X-ray absorption spectroscopy study of selenium in cancer cells and a study of iron in phytoplankton. This protocol can be used with other biological samples and other X-ray techniques that can be damaged by irradiation.

This protocol presents a detailed procedure to prepare biological cryosamples for synchrotron-based X-ray absorption spectroscopy experiments. We describe all the steps required to optimize sample preparation and cryopreservation with examples of the protocol with cancer and phytoplankton cells. This method provides a universal standard of sample cryo-preparation.

The study of elements with X-ray absorption spectroscopy (XAS) is of particular interest when studying the role of metals in biological systems. Sample ...

Chemistry

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

Biology

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than conventional fluorescence microscopy. This super-resolution (SR) technique enables visualization of molecular processes in whole cells and has the potential to be used in conjunction with electron microscopy and X-ray tomography to correlate structural and functional information. A SIM microscope for cryogenically preserved samples (cryoSIM) has recently been commissioned at the correlative cryo-imaging beamline B24 at the UK synchrotron. 
It was designed specifically for 3D imaging of biological samples at cryogenic temperatures in a manner compatible with subsequent imaging of the same samples by X-ray microscopy methods such as cryo-soft X-ray tomography. This video article provides detailed methods and protocols for successful imaging using the cryoSIM. In addition to instructions on the operation of the cryoSIM microscope, recommendations have been included regarding the choice of samples, fluorophores, and parameter settings. The protocol is demonstrated in U2OS cell samples whose mitochondria and tubulin have been fluorescently labelled.

This protocol demonstrates how to image biological cryo-preserved samples using cryo-structured illumination microscopy. We demonstrate the methodology by imaging the cytoskeleton of U2OS cells.

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

Regulating Temperature in the Lab: Preserving Samples Using Cold

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs. This video will discuss some of the methods for keeping laboratory samples cold and will explain the correct cooling method to use for each experimental requirement.
For example, cooling agents, such as ice and dry ice, are typically used when keeping samples cold during experiments. This video discusses the physical properties of the most commonly used cooling agents, as well as safety precautions for working with them.
When it comes to keeping samples cold in between experiments, cooling equipment, including laboratory grade refrigerators and freezers can be used to preserve samples for extended period of time. Also discussed in this video are types of samples and reagents that can be stored in the commonly-available laboratory cooling equipment.
Finally, the concept of cryopreservation is introduced as a process through which tissues, cells, and biomolecules are cooled to sub-zero temperatures, thereby effectively stopping all sample-degrading biological activity. Several methods of cryopreservation are discussed that minimize or eliminate the formation of damaging ice crystals.

Preserving Samples Using Cold: Cooling Agents and Instruments, and Cryopreservation

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs.  This video will ...