Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.

The Angular Relationships Between the Coracohumeral Ligament and Adjacent Shoulder Structures Are Variable.

PURPOSE: To describe the arthroscopic anatomy of the coracohumeral ligament (CHL) in relation to visible anatomic reference points to aid in the execution of a more effective arthroscopic medial-lateral rotator interval closure. METHODS: Detailed dissection to identify the CHL was performed in 4 shoulders from 2 fresh-frozen donor cadavers with a deltopectoral approach. The angular relationship between the CHL and the superior border of the subscapularis tendon was determined via gross dissection. Arthroscopic images were used to determine the angular position of the CHL in relation to both the glenoid articular surface and the intraarticular segment of the tendon of the long head of the biceps brachii (LHB). RESULTS: Analysis of 4 cadaveric shoulders via gross dissection demonstrated the CHL to subtend a mean angle of 29° (range 16° to 39°) with respect to the superior border of the subscapularis tendon. Arthroscopic analysis of 4 cadaveric shoulders demonstrated the CHL to subtend a mean angle of 59° (range 38° to 77°) with respect to the glenoid articular surface. Additionally, arthroscopic analysis of 2 cadaveric shoulders demonstrated the CHL to subtend a mean angle of 29° (range 11° to 47°) with respect to the LHB tendon. CONCLUSION: Although the position of the CHL in relation to the subscapularis tendon, glenoid articular surface, and LHB tendon demonstrates a moderate degree of anatomic variability, these structures provide valuable anatomic reference points for the identification of the course of this significant static shoulder stabilizer. CLINICAL RELEVANCE: Comprehensive understanding of the angular relationships between the CHL and adjacent shoulder structures may assist with the execution of a more effective arthroscopic rotator interval closure.

The Comma Sign: The Coracohumeral Ligament and Superior Glenohumeral Ligament Exhibit Similar Quantitative Characteristics With Terminal Confluence at the Subscapularis Insertion.

PURPOSE: The purpose of this morphologic cadaveric study was to quantitatively define the composition of the previously described comma tissue along with its relation to the subscapularis tendon insertion. METHODS: Fresh frozen cadaveric shoulder specimens were included for analysis. The coracohumeral ligament (CHL) was exposed at its origin along the base of the coracoid process and freed laterally along its course to the lesser tuberosity adjacent to the bicipital groove. The superior glenohumeral ligament (SGHL) was identified and traced along its course deep to the CHL within the rotator interval with insertion onto the superior aspect of the lesser tuberosity. The midpoint diameters of the SGHL and CHL and their composite insertional diameters on the subscapularis tendon insertion and lesser tuberosity were measured with digital calipers. The mean diameter was determined from 3 measurements taken of each ligament. RESULTS: Eight specimens were included. With the use of digital calipers, the mean midpoint diameters of the SGHL and CHL were identified as 5.99 mm (range, 5.25-6.91 mm) and 5.13 mm (range, 4.28-5.72 mm), respectively. The composite insertional diameter of the SGHL and CHL on both the lesser tuberosity and humeral insertion of the subscapularis tendon was 9.93 mm (range, 6.69-12.05 mm). At its insertion, the SGHL and CHL comprised 54% and 46% of the comma tissue, respectively. Additionally, all specimens were identified as showing a confluence of the SGHL and CHL composite insertion with the subscapularis tendon at the point of its humeral head insertion. CONCLUSIONS: The comma tissue is a pivotal structure for the identification, mobilization, and repair of retracted subscapularis tendon tears. Therefore, quantitative knowledge of the midpoint diameter, insertional diameter, and composite distribution of the CHL and SGHL provided by this morphologic cadaveric analysis may aid surgeons in their efforts to restore the native anatomy. CLINICAL RELEVANCE: Subscapularis tendon tears have often been under-addressed during rotator cuff repair. The comma tissue has been described as an anatomic structure that can aid in the identification, mobilization, and repair of retracted subscapularis tendon tears. Therefore, quantitative knowledge of this important arthroscopic landmark may aid surgeons in their efforts to restore the native anatomy.

Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope.

We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity, and long-timescale decays. We demonstrate our approach using a three-dimensional tracking microscope to study genomic lambda-phage DNA molecules with various fluorescence label configurations.

Precise characterization of the conformation fluctuations of freely diffusing DNA: beyond Rouse and Zimm.

We studied the dynamics of single freely diffusing fluorescence-labeled double-stranded lambda-phage DNA molecules using dual-color 3-dimensional feedback tracking microscopy and intramolecular fluorescence correlation spectroscopy. Our technique is independently sensitive to the molecule's diffusion coefficient D and radius of gyration R(g) and is concentration insensitive, providing greater precision for characterizing the molecule's intramolecular motion than other methods. We measured D = 0.80 +/- 0.05 microm(2)/s and R(g) approximately 420 nm, consistent with the Kirkwood-Riseman prediction for a flexible polymer with strong hydrodynamic interactions (HI), but we find the statistics of intramolecular motion inconsistent with the Zimm model for such a polymer. We address a dispute in the experimental literature, finding that previous measurements on double-stranded DNA likely lacked the sensitivity to distinguish between the Zimm model and the HI-free Rouse model. Finally, we observe fluorescence fluctuations with a correlation time of over 2 s that cannot be explained by either model and propose that they may be signatures of excluded volume interactions.

Outcome of a second two-stage reimplantation for periprosthetic knee infection.

UNLABELLED: Recurrent or persistent infection after two-stage exchange arthroplasty for previously infected total knee replacement is a challenging clinical situation. We asked whether a second two-stage procedure could eradicate the infection and preserve knee function. We evaluated 18 selected patients with failed two-stage total knee arthroplasty implantation treated with a second two-stage reimplantation between 1999 and 2005. Failure of treatment was defined as recurrence or persistence of infection. The minimum followup was 24 months (mean, 40 months; range, 24-83 months). Recurrent or persistent infection was diagnosed in four of 18 patients, two of whom were successfully treated with a third two-stage exchange arthroplasty. Knee Society score questionnaires administered at the last followup showed an average Knee Society knee score of 73 points (range, 24-100 points) and an average functional score of 49 points (range, 20-90 points). The data suggest repeat two-stage exchange arthroplasty is a reasonable option for eradicating periprosthetic infection, relieving pain, and achieving a satisfactory level of function for some patients. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Fluctuations in closed-loop fluorescent particle tracking.

We present a comprehensive theory of closed-loop particle tracking for calculating the statistics of a diffusing fluorescent particle's motion relative to the tracking lock point. A detailed comparison is made between the theory and experimental results, with excellent quantitative agreement found in all cases. A generalization of the theory of (open-loop) fluorescence correlation spectroscopy is developed, and the relationship to previous results is discussed. Two applications of the statistical techniques are given: a method for determining a tracked particle's localization and an algorithm for rapid particle classification based on real-time analysis of the tracking control signal.

Mini-Open Posterior Compartment Release for Chronic Exertional Compartment Syndrome of the Leg.

Chronic exertional compartment syndrome (CECS) is a well-recognized cause of leg pain in endurance athletes. Surgical fasciotomy for posterior leg CECS historically has inferior clinical results compared with anterior and lateral compartment release. Poor surgical technique with inadequate release may contribute to less reliable outcomes. In this Technical Note with accompanying video, we describe a mini-open approach for posterior CECS of the leg.

Epidemiology and Outcomes of Lisfranc Injuries Identified at the National Football League Scouting Combine.

BACKGROUND: Lisfranc injuries are challenging to treat and may have a detrimental effect on athletic performance. PURPOSE: (1) Determine the epidemiological characteristics of Lisfranc injuries at the annual National Football League (NFL) Scouting Combine, (2) define player positions at risk for these injuries, and (3) evaluate the impact that these injuries and radiographic findings have on NFL draft position and performance. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: All players who sustained a Lisfranc injury prior to Combine evaluation between 2009 and 2015 were evaluated. The epidemiological characteristics, player positions affected, treatment methods, and number of missed collegiate games were recorded. Radiographic outcomes were analyzed via Combine radiograph findings, while NFL performance outcomes were assessed for all Lisfranc injuries (2009-2013) compared with matched controls in the first 2 years of play. RESULTS: A total of 41 of 2162 (1.8%) Combine participants were identified with Lisfranc injuries, of whom 26 of 41 (63.4%) were managed operatively. Players who underwent surgery were more likely to go undrafted compared with players managed nonoperatively (38.5% vs 13.3%, operative vs nonoperative management, respectively; P = .04) and featured a worse NFL draft pick position (155.6 vs 109; P = .03). Lisfranc-injured players when compared with controls were noted to have worse outcomes in terms of NFL draft position (142 vs 111.3, Lisfranc-injured players vs controls, respectively; P = .04), NFL career length 2 years or longer (62.5% vs 69.6%; P = .23), and number of games played (16.9 vs 23.3; P = .001) and started (6.8 vs 10.5; P = .08) within the first 2 years of their NFL career. Radiographs demonstrated that 17 of 41 (41.5%) athletes had residual Lisfranc joint displacement greater than 2 mm compared with the contralateral foot. Lisfranc-injured athletes with greater than 2 mm residual displacement, when compared with matched controls, had worse draft position (156.9 vs 111.2 for Lisfranc-injured players vs controls, respectively; P = .009) and fewer games played (14.4 vs 23.3; P = .001) and started (3.1 vs 10.5; P = .03). Moreover, athletes with greater than 2 mm residual displacement featured worse outcomes across all assessed NFL variables versus athletes with residual displacement of 2 mm or less. CONCLUSION: Lisfranc injuries identified at the NFL Combine have an adverse effect on an NFL athlete's draft status, draft position, and overall play during initial NFL seasons. In particular, residual displacement of the Lisfranc joint has a detrimental effect on the first 2 seasons of NFL play and may lead to long-lasting negative effects on the athlete's career.

Arthroscopic Superior Capsule Reconstruction Technique in the Setting of a Massive, Irreparable Rotator Cuff Tear.

Massive, irreparable rotator cuff tears are challenging to treat and associated with pain and severe limitation in shoulder elevation due to the proximal migration of the humeral head and, consequently, subacromial impingement. Furthermore, retraction of the tendons in combination with fat infiltration and muscular weakness results in unpredictable treatment outcomes. While conservative treatment may be warranted for low-demand patients, surgical treatment is often indicated for a successful return to activities of daily living as well as an improved quality of life. The reported failure rate of rotator cuff repair for the treatment of a massive rotator cuff tear varies. However, this failure is often found at the interface between the tendon and tendon footprint. Several techniques have been reported to address this pathology, including muscular transfer, arthroscopic debridement, augmentation procedures, and superior capsule reconstruction. In particular, superior capsule reconstruction has been reported as a safe and effective method to treat a massive, irreparable rotator cuff tear. The purpose of this Technical Note is to describe our preferred technique of a superior capsule reconstruction for the treatment of a massive, irreparable rotator cuff tear.

Epidemiology of Navicular Injury at the NFL Combine and Their Impact on an Athlete's Prospective NFL Career.

BACKGROUND: Navicular injuries can result in persistent pain, posttraumatic osteoarthritis, and diminished performance and function. PURPOSE: To determine the epidemiology of navicular fracture in players participating in the National Football League (NFL) Scouting Combine and evaluate the impact of a navicular injury on the NFL draft position and NFL game play compared with matched controls. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Data were collected on players who previously sustained a navicular injury and participated in the NFL Combine between 2009 and 2015. The epidemiology of navicular injury was determined through an evaluation of the number of injuries, surgeries, and collegiate games missed as well as the position played, a physical examination, the surgical technique, and imaging findings. Players with a previous navicular injury (2009-2013) were compared with a set of matched controls. NFL performance outcomes included the draft position, career length ≥2 years, and number of games played and started within the first 2 years. RESULTS: Between 2009 and 2015, 14 of 2285 (0.6%) players were identified as having sustained a navicular injury. A total of 11 of 14 (79%) athletes had sustained an overt navicular fracture, while 3 of 14 (21%) were diagnosed with stress reactions on magnetic resonance imaging. Eight patients who sustained a navicular fracture underwent surgery. There was evidence of ipsilateral talonavicular arthritis in 75% of players with a navicular fracture versus only 60% in the uninjured foot (odds ratio, 1.3; P = .04). Fifty-seven percent of players with navicular injury (72.7% of fractures) were undrafted versus 30.9% in the control group (P = .001). Overall, 28.6% of players with navicular fracture played ≥2 years in the NFL compared with 69.6% in the control group (P = .02). CONCLUSION: A previous navicular fracture results in a greater risk of developing posttraumatic osteoarthritis. Although only a low prevalence of navicular injury in prospective NFL players was noted, players with these injuries had a greater probability of not being drafted and not competing in at least 2 NFL seasons when compared with matched controls without an injury history to the NFL Combine.

Latarjet Technique for Treatment of Anterior Shoulder Instability With Glenoid Bone Loss.

Anterior glenohumeral instability is a common clinical entity, particularly among young athletic patient populations. Nonoperative management and arthroscopic treatment of glenohumeral instability have been associated with high rates of recurrence, particularly in the setting of glenohumeral osseous defects. Coracoid transfer, particularly the Latarjet procedure, has become the treatment of choice for recurrent anterior glenohumeral instability in the setting of osseous deficiencies greater than 20% to 30% of the glenoid surface area and may also be considered for the primary treatment of recurrent instability in the high-risk contact athlete, even in the setting of limited osseous deficiency. The following Technical Note provides a diagnostic approach for suspected glenohumeral instability, as well as a detailed description of the congruent-arc Latarjet procedure, performed with a deltoid split, with its postoperative management.

Epidemiology of Injuries Identified at the NFL Scouting Combine and Their Impact on Performance in the National Football League: Evaluation of 2203 Athletes From 2009 to 2015.

BACKGROUND: At the annual National Football League (NFL) Scouting Combine, the medical staff of each NFL franchise performs a comprehensive medical evaluation of all athletes potentially entering the NFL. Currently, little is known regarding the overall epidemiology of injuries identified at the combine and their impact on NFL performance. PURPOSE: To determine the epidemiology of injuries identified at the combine and their impact on initial NFL performance. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: All previous musculoskeletal injuries identified at the NFL Combine from 2009 to 2015 were retrospectively reviewed. Medical records and imaging reports were examined. Game statistics for the first 2 seasons of NFL play were obtained for all players from 2009 to 2013. Analysis of injury prevalence and overall impact on the draft status and position-specific performance metrics of each injury was performed and compared with a position-matched control group with no history of injury or surgery. RESULTS: A total of 2203 athletes over 7 years were evaluated, including 1490 (67.6%) drafted athletes and 1040 (47.2%) who ultimately played at least 2 years in the NFL. The most common sites of injury were the ankle (1160, 52.7%), shoulder (1143, 51.9%), knee (1128, 51.2%), spine (785, 35.6%), and hand (739, 33.5%). Odds ratios (ORs) demonstrated that quarterbacks were most at risk of shoulder injury (OR, 2.78; P = .001), while running backs most commonly sustained ankle (OR, 1.39; P = .040) and shoulder injuries (OR, 1.55; P = .020) when compared with all other players. Ultimately, defensive players demonstrated a greater negative impact due to injury than offensive players, with multiple performance metrics significantly affected for each defensive position analyzed, whereas skilled offensive players (eg, quarterbacks, running backs) demonstrated only 1 metric significantly affected at each position. CONCLUSION: The most common sites of injury identified at the combine were (1) ankle, (2) shoulder, (3) knee, (4) spine, and (5) hand. Overall, performance in the NFL tended to worsen with injury history, with a direct correlation found between injury at a certain anatomic location and position of play. Defensive players tended to perform worse compared with offensive players if injury history was present.

Ankle impingement.

Ankle impingement is a syndrome that encompasses a wide range of anterior and posterior joint pathology involving both osseous and soft tissue abnormalities. In this review, the etiology, pathoanatomy, diagnostic workup, and treatment options for both anterior and posterior ankle impingement syndromes are discussed.

Outcomes of Lisfranc Injuries in the National Football League.

BACKGROUND: Tarsometatarsal (Lisfranc) joint injuries commonly occur in National Football League (NFL) competition; however, the career effect of these injuries is unknown. PURPOSE: To define the time to return to competition for NFL players who sustained Lisfranc injuries and to quantify the effect on athletic performance. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: Data on NFL players who sustained a Lisfranc injury between 2000 and 2010 were collected for analysis. Outcomes data included time to return to competition, total games played after season of injury, yearly total yards and touchdowns for offensive players, and yearly total tackles, sacks, and interceptions for defensive players. Offensive power ratings (OPR = [total yards/10] + [total touchdowns × 6]) and defensive power ratings (DPR = total tackles + [total sacks × 2] + [total interceptions × 2]) were calculated for the injury season and for 3 seasons before and after the injury season. Offensive and defensive control groups consisted of all players without an identified Lisfranc injury who competed in the 2005 season. RESULTS: The study group was composed of 28 NFL athletes who sustained Lisfranc injuries during the study period, including 11 offensive and 17 defensive players. While 2 of 28 (7.1%) players never returned to the NFL, 26 (92.9%) athletes returned to competition at a median of 11.1 (interquartile range [IQR], 10.3-12.5) months from time of injury and missed a median of 8.5 (IQR, 6.3-13.0) regular-season games. Analysis of pre- and postinjury athletic performance revealed no statistically significant changes after return to sport after Lisfranc injury. The magnitude of change in median OPR and DPR observed in offensive and defensive Lisfranc-injured study groups, -34.8 (IQR, -64.4 to 1.4) and -13.5 (-30.9 to 4.3), respectively, was greater than that observed in offensive and defensive control groups, -18.8 (-52.9 to 31.5) and -5.0 (-22.0 to 14.0), respectively; however, these differences did not reach statistical significance (P = .33 and .21, respectively). Evaluation of the durability of injured players after the season of injury revealed no statistically significant difference in career length compared with controls. CONCLUSION: More than 90% of NFL athletes who sustained Lisfranc injuries returned to play in the NFL at a median of 11.1 months from time of injury. Offensive and defensive players experienced a decrease in performance after return from injury that did not reach statistical significance compared with their respective control groups over a similar time period.

Single-molecule fluorescence experiments determine protein folding transition path times.

The transition path is the tiny fraction of an equilibrium molecular trajectory when a transition occurs as the free-energy barrier between two states is crossed. It is a single-molecule property that contains all the mechanistic information on how a process occurs. As a step toward observing transition paths in protein folding, we determined the average transition-path time for a fast- and a slow-folding protein from a photon-by-photon analysis of fluorescence trajectories in single-molecule Förster resonance energy transfer experiments. Whereas the folding rate coefficients differ by a factor of 10,000, the transition-path times differ by a factor of less than 5, which shows that a fast- and a slow-folding protein take almost the same time to fold when folding actually happens. A very simple model based on energy landscape theory can explain this result.

Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma.

Prolactin receptor signaling can modulate proliferation, survival, motility, angiogenesis, and differentiation in breast cancer. Increased serum prolactin is associated with a significantly increased risk of breast cancer in post-menopausal women. The purpose of this study was to examine the expression of prolactin receptor-associated signaling proteins in breast cancer vs benign breast tissue. Breast tissue microarrays representing 40 cases of benign and malignant pathologies were obtained from the Cooperative Human Tissue Network. Standard immunohistochemistry for prolactin and prolactin receptor-associated proteins was performed. Both positive regulators (c-Myb, Nek3, Vav2) and negative regulators (PIAS3, SIRP) of prolactin receptor signaling were examined. Virtual slides were created from the stained breast tissue microarrays. Labels were scored by region of interest and labeling indices incorporating percent target labeled and label intensity were created. Quantitative determinations of labels were made using the Clarient image system. The unpaired t-test was used to compare labels from benign and malignant tissues. Visual scoring data showed upregulation of Nek3 (P=0.000377), PIAS3 (P=0.000257), and prolactin (P=0.002576) in breast cancer vs normal/hyperplastic epithelium. c-Myb showed a trend toward upregulation, but this did not achieve statistical significance (P=0.107374). SIRP (P=0.002060) was downregulated. Vav2 showed a trend toward downregulation (P=0.107456), but this did not achieve statistical significance. Clarient analysis corroborated upregulation in cancer of Nek3 (P=0.000013), PIAS3 (P=0.000067), and prolactin (P=0.017569). In conclusion, regulators of prolactin receptor signaling show heterogeneity in their expression in benign vs malignant breast tissue. Since these species are known to regulate prolactin-mediated actions, these results suggest multiple targets for modulating prolactin receptor-mediated growth and differentiation in breast cancer.

Feedback localization of freely diffusing fluorescent particles near the optical shot-noise limit.

We report near-optimal tracking of freely diffusing fluorescent particles in a quasi-two-dimensional geometry via photon counting and real-time feedback. We present a quantitative statistical model of our feedback network and find excellent agreement with the experiment. We monitor the motion of a single fluorescent particle with a sensitivity of 15 nm/sqrt Hz while collecting fewer than 5000 fluorescence photons/s. Fluorescent microspheres (diffusion coefficient 1.3 microm2/s) are tracked with a root-mean-square tracking error of 170 nm, within a factor of 2 of the theoretical limit set by photon counting shot noise.

Quantum dot photon statistics measured by three-dimensional particle tracking.

We present an instrument for performing correlation spectroscopy on single fluorescent particles while tracking their Brownian motion in three dimensions using real-time feedback. By tracking CdSe/ZnS quantum dots in water (diffusion coefficient approximately 20 microm2/s), we make the first measurements of photon antibunching (at approximately 10 ns) on single fluorophores free in solution and find fluorescence lifetime heterogeneity within a quantum dot sample. In addition, we show that 2-mercaptoethanol suppresses short time-scale intermittency (1 ms to 1 s) in quantum dot fluorescence by reducing time spent in the off-state.