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1.
Philos Trans R Soc Lond B Biol Sci ; 368(1611): 20120035, 2013 Feb 05.
Article in English | MEDLINE | ID: mdl-23267185

ABSTRACT

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.


Subject(s)
Electrons , Microscopy, Fluorescence/methods , Molecular Imaging/instrumentation , Photons , Computational Biology , Diffusion , Equipment Design , Fluorescence , Molecular Conformation , Molecular Imaging/methods , Sensitivity and Specificity , Time Factors
2.
Proc SPIE Int Soc Opt Eng ; 82282012 Jan 21.
Article in English | MEDLINE | ID: mdl-24382989

ABSTRACT

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

3.
Proc SPIE Int Soc Opt Eng ; 8033: 1350904, 2011 May 12.
Article in English | MEDLINE | ID: mdl-23833700

ABSTRACT

Cross strip and cross delay line readout microchannel plate detectors in 18 mm, 25 mm and 40 mm active area formats including open face (UV/particle) and sealed tube (optical) configurations have been constructed. These have been tested with a field programmable gate array based electronics for single event encoding. Using small pore MCPs (6 µm) operated in a pair, we achieve gains of >1 × 106 which is sufficient to provide spatial resolution of ~17 µm FHWM with the 18 mm and 40 mm cross strip readouts. New cross strip electronics can process high output event rates (> 4 MHz) with high spatial resolution, and self triggered event timing accuracy of ~1.5 ns for sealed tube XS optical sensors. A peak quantum efficiency of between 13% and 19% at 500 nm has been achieved with SuperGenII photocathodes with response from 400 nm to 900 nm for both cross strip and cross delay line sealed tubes. Local area counting rates of up to 40 kHz (100µm spot) have been attained with XS sealed tubes, along with image linearity and stability to better than 50 µm. 25mm cross delay line tubes achieve ~50 µm resolution and > 2 MHz output event rates.

4.
Proc SPIE Int Soc Opt Eng ; 8033: 803316, 2011 May 13.
Article in English | MEDLINE | ID: mdl-24729836

ABSTRACT

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain.

5.
Proc SPIE Int Soc Opt Eng ; 7608(76082D)2010 Jan 24.
Article in English | MEDLINE | ID: mdl-21625288

ABSTRACT

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors.

6.
Curr Pharm Biotechnol ; 10(5): 543-58, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19689323

ABSTRACT

The expanding spectrum of applications of single-molecule fluorescence imaging ranges from fundamental in vitro studies of biomolecular activity to tracking of receptors in live cells. The success of these assays has relied on progress in organic and non-organic fluorescent probe developments as well as improvements in the sensitivity of light detectors. We describe a new type of detector developed with the specific goal of ultra-sensitive single-molecule imaging. It is a wide-field, photon-counting detector providing high temporal and high spatial resolution information for each incoming photon. It can be used as a standard low-light level camera, but also allows access to a lot more information, such as fluorescence lifetime and spatio-temporal correlations. We illustrate the single-molecule imaging performance of our current prototype using quantum dots and discuss on-going and future developments of this detector.


Subject(s)
Photons , Quantum Dots , Electrons , Equipment Design , Microscopy, Fluorescence , Nanotechnology , Radiographic Image Interpretation, Computer-Assisted , Spectrometry, Fluorescence
7.
Clin Orthop Relat Res ; (298): 75-9, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8118998

ABSTRACT

Successful treatment of the infected hip prosthesis demands careful planning for infection control and reimplantation. Inadequate surgical debridement dooms well-executed revision surgery to infection failure. Reimplantation is difficult after resection arthroplasty, and this difficulty is increased with longer intervals between resection and reimplantation. Older patients will rehabilitate better if the interval is shorter. The reported protocol successfully compromises between one-stage exchanges and reported two-stage exchanges.


Subject(s)
Hip Prosthesis , Prosthesis-Related Infections/surgery , Aged , Debridement , Female , Follow-Up Studies , Humans , Male , Middle Aged , Prosthesis-Related Infections/microbiology , Recurrence , Reoperation , Staphylococcal Infections/microbiology , Staphylococcus aureus , Staphylococcus epidermidis
9.
Skeletal Radiol ; 20(6): 465-7, 1991.
Article in English | MEDLINE | ID: mdl-1925683

ABSTRACT

We have presented a 23-year follow-up of a patient with left atrial myxoma with continuing slowly growing, skeletal metastases. The relatively indolent nature of the metastatic disease and the lack of mitotic figures suggest that the metastatic lesions may be treated locally. The patient underwent therapeutic tumor embolization and local resection of the pelvic lesion, with good results as she continues to do well with close follow-up.


Subject(s)
Bone Neoplasms/secondary , Heart Neoplasms/pathology , Myxoma/secondary , Pubic Bone , Bone Neoplasms/diagnostic imaging , Female , Follow-Up Studies , Heart Atria , Humans , Middle Aged , Myxoma/diagnostic imaging , Radiography , Time Factors
11.
Curr Probl Cancer ; 10(3): 117-68, 1986 Mar.
Article in English | MEDLINE | ID: mdl-3514130

ABSTRACT

The most important factor to consider in deciding between treatment options in the management of metastatic bone disease is the level of the patient's dysfunction and pain. Severe dysfunction or pain demands a treatment that predictably leads to a quick resumption of the painless activities of daily living. A treatment that predictably will restore function in months may seem reasonable in patients with a normal remaining life span, but is untenable if those months represent a high percentage of remaining life span, as they do in metastatic disease afflicted patients. The treating physician needs also to understand the basis for the patient's dysfunction. A destroyed joint will not return to painless function even if the metastasis responsible is totally eliminated. A bone that has lost its structural integrity, even though not grossly fractured, will not support weight bearing for months even if the metastasis is eliminated. Control of the metastatic tumor does not always equate with return to function. Treatment options in the management of metastatic bone disease are not mutually exclusive. In many patients treatment options are combined. Surgical stabilization may best return the patient's function while he is being treated postoperatively with radiotherapy or chemotherapy for good neoplasm control. Neoplasm control should not be such an overriding concern that function is not addressed. Function can almost always be returned to the patient, but neoplasm "cure" is rarely achieved in this group of patients. It is a reasonable goal to avoid allowing bone metastasis to progress to pathological fracture. Routine periodic examinations and bone scans should commonly alert the treating physician to the presence of metastatic bone disease well before fracture occurs. Pathological fracture narrows the range of treatment options, mitigates against full functional restoration, demands a rehabilitation hiatus, and acutely frightens the patient who does not have time to participate fully in treatment decisions. An impending pathological fracture can be treated with surgery, radiotherapy, chemotherapy, or hormonal manipulation. The options are basically operative or nonoperative. Lesions that predictably will fracture short term, involve joints, or will cause catastrophic consequences if fracture occurs should be strongly considered for surgical stabilization. Other factors to consider are the location of the metastasis, the primary tumor, and the expected response to nonoperative therapy. The patient becomes a surgical candidate for the above reasons and not because of any estimated life span.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Bone Neoplasms/complications , Fracture Fixation , Fractures, Spontaneous/surgery , Adolescent , Adult , Aged , Bone Neoplasms/diagnostic imaging , Bone Neoplasms/secondary , Bone Neoplasms/therapy , Breast Neoplasms , Carcinoma, Renal Cell , Female , Femoral Fractures/etiology , Femoral Fractures/surgery , Fracture Fixation, Internal , Fracture Fixation, Intramedullary , Fractures, Spontaneous/diagnostic imaging , Fractures, Spontaneous/etiology , Hip Fractures/etiology , Hip Fractures/surgery , Humans , Humeral Fractures/etiology , Humeral Fractures/surgery , Kidney Neoplasms , Lung Neoplasms , Male , Middle Aged , Multiple Myeloma , Prostatic Neoplasms , Radiography , Spinal Injuries/etiology , Spinal Injuries/surgery , Tibial Fractures/etiology , Tibial Fractures/surgery
12.
Comput Radiol ; 10(2-3): 99-106, 1986.
Article in English | MEDLINE | ID: mdl-3709131

ABSTRACT

Twenty-seven adults with extremity masses were examined by magnetic resonance imaging (MRI). In 26/27 cases, computed tomography (CT) scans were available for comparison. Imaging with multiple pulse sequences is necessary to optimize diagnosis by MRI. Advantages of MRI include direct sagittal and coronal imaging, demonstration of vessels without contrast and superior soft tissue contrast. CT better demonstrates cortical destruction and small calcifications, which may be important in some cases. For noncalcified masses without bone involvement, MRI is equivalent or superior to CT. In those cases with calcification or bone involvement, MRI plays a role complementary to CT.


Subject(s)
Arm , Bone Neoplasms/diagnosis , Leg , Magnetic Resonance Spectroscopy , Soft Tissue Neoplasms/diagnosis , Adolescent , Adult , Aged , Bone Neoplasms/diagnostic imaging , Female , Humans , Male , Middle Aged , Muscles/diagnostic imaging , Soft Tissue Neoplasms/diagnostic imaging , Tomography, X-Ray Computed
13.
Am Surg ; 51(7): 363-6, 1985 Jul.
Article in English | MEDLINE | ID: mdl-4014878

ABSTRACT

Osteomyelitis in the adult patient has been associated with failure of eradication, late recurrence, nonunion, and prolonged hospitalization. A staged aggressive approach has been used for the past seven years to treat 53 patients with adult osteomyelitis. This approach includes: evaluation of bone necrosis and identification of the etiologic organisms by deep bone culture; radical surgical debridement of devascularized tissue; intensive systemic antibiotics; and early bone and soft tissue reconstruction. All patients have been followed at least 1 year (mean, 33 months). Lower extremity bones predominated in the series (24 tibias, 13 femurs); and 19 patients had bony instability. Thirty-seven patients had initial successful eradication of their infections with 26 of these returning to full activity status. The remaining 16 patients developed recurrent infection; however, 11 patients totally responded to further aggressive treatment. Of the five failures in the total series, three patients required amputation and two patients have persistent infection. Fifteen of the 19 patients with bony instability healed with initial treatment, and the remaining four patients healed with subsequent treatment. Six patients had primary muscle flap soft tissue reconstruction, and an additional two patients had reconstruction as a secondary procedure. In all these patients with tibial instability, bony union was accelerated compared to those patients with tibial instability not receiving muscle flaps (4 months vs 12 months). The muscle coverage provided by either pedicled flaps or transferred by microvascular anastomoses.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Osteomyelitis/surgery , Adult , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/etiology , Bacterial Infections/pathology , Bone and Bones/microbiology , Bone and Bones/pathology , Combined Modality Therapy , Extremities/pathology , Gram-Positive Bacteria/isolation & purification , Humans , Middle Aged , Osteomyelitis/drug therapy , Osteomyelitis/microbiology , Osteonecrosis/microbiology , Osteonecrosis/pathology , Postoperative Complications , Recurrence , Surgical Wound Dehiscence/etiology , Time Factors
14.
15.
J Bone Joint Surg Am ; 63(3): 376-9, 1981 Mar.
Article in English | MEDLINE | ID: mdl-7204434

ABSTRACT

Eight patients with tetraplegia at the sixth cervical level had ten flexor pollicis longus tenodeses performed to create a key grip. Two of the patients also had tendon transfers to create active finger flexion; these failed. The remaining six patients were objectively evaluated by the hand-function test of Jebsen et al. to measure gains in hand function. All patients improved postoperatively, the average improvement being 31 per cent. Although these patients did not add many new hand activities to their repertoire, they showed increased speed and ease of pre-existing hand functions. Three patients did gain the ability to self-catheterize, enabling two of them to live totally independent life-styles. Fixation of the thumb interphalangeal joint with Kirschner wires did not provide sufficient long-term stability, and initial arthrodesis of this joint is recommended in conjunction with the flexor pollicis longus tenodesis.


Subject(s)
Quadriplegia , Tendons/surgery , Thumb/surgery , Activities of Daily Living , Humans , Life Style , Prospective Studies
16.
Am J Surg Pathol ; 4(4): 391-9, 1980 Aug.
Article in English | MEDLINE | ID: mdl-7425205

ABSTRACT

Tumors that were identical in morphology to a previously excised atrial myxoma were found in the choroid plexus and the scapula of a 46-year-old woman 8 and 10.5 years, respectively, after cardiac surgery. Ultrastructurally the scapular lesion resembled previously reported atrial myxomas. This case demonstrates that atrial myxoma is a true neoplasm capable of metastatic spread despite its slow growth and innocuous histologic appearance.


Subject(s)
Bone Neoplasms/secondary , Cerebral Ventricle Neoplasms/secondary , Choroid Plexus/pathology , Heart Neoplasms/pathology , Myxoma/secondary , Bone Neoplasms/pathology , Cerebral Ventricle Neoplasms/pathology , Female , Heart Atria/pathology , Heart Neoplasms/secondary , Humans , Male , Middle Aged , Myxoma/pathology , Myxoma/ultrastructure , Scapula/pathology
18.
Arthritis Rheum ; 23(1): 83-91, 1980 Jan.
Article in English | MEDLINE | ID: mdl-7352948

ABSTRACT

In osteoarthritis a net increase in proteoglycan synthesis has been noted until the disease is far advanced and presumably reflects an attempt by the chondrocyte to repair the defect in the cartilage matrix. Because salicylates are the agents most commonly employed in treatment of osteoarthritis and because we recently showed that 10(-3) M sodium salicylate (i.e., approximately 20 mg%) suppresses proteoglycan synthesis in normal canine knee cartilage in vitro, we have studied the effects of this compound on osteoarthritis knee cartilage from dogs whose anterior cruciate ligament had been transected 9 weeks previously. The data indicated that the augmented synthesis of glycosaminoglycans in the degenerating cartilage was suppressed to a much greater degree by 10(-3) M sodium salicylate than the lower level of glycosaminoglycan synthesis in control cartilage from the contralateral knee of the same animal. Uptake of 14C-acetylsalicylic acid was increased about 35% in osteoarthritic cartilage, suggesting that the drug permeated it more readily than normal cartilage. The salicylate-induced suppression of proteoglycan synthesis in the osteoarthritic cartilage was not accompanied by reversal of the defect in proteoglycan aggregation or by improvement in the (presumed) defect in proteoglycan-collagen interaction in the matrix, as reflected by the abnormally high proportion of 35S-proteoglycans present in the culture medium.


Subject(s)
Cartilage, Articular/metabolism , Osteoarthritis/metabolism , Proteoglycans/biosynthesis , Sodium Salicylate/pharmacology , Animals , Aspirin/metabolism , Body Water/analysis , Cartilage, Articular/pathology , Cells, Cultured , Dogs , Glycosaminoglycans/biosynthesis , Knee , Osteoarthritis/pathology , Uronic Acids/analysis
19.
J Bone Joint Surg Am ; 62(4): 557-60, 1980.
Article in English | MEDLINE | ID: mdl-7380856

ABSTRACT

External compressive fixators were used for fixation after a femoral trochanteric osteotomy for severe slipped capital femoral epiphysis in two large teen-aged boys. The method was found to simplify both the operative fixation and the postoperative management. However, the healing time seemed to be prolonged.


Subject(s)
Epiphyses, Slipped/therapy , Femur , Orthopedic Fixation Devices , Adolescent , Epiphyses, Slipped/surgery , Femur/surgery , Humans , Male , Osteotomy/methods , Postoperative Care , Time Factors , Wound Healing
20.
Johns Hopkins Med J ; 145(3): 131-5, 1979 Sep.
Article in English | MEDLINE | ID: mdl-288942

ABSTRACT

A brother and sister with osteogenic sarcoma are reported. A review of the literature reveals thirty additional cases of osteogenic sarcoma in familial aggregation, sixteen of sibling relationship. Genetic and environmental etiologic factors of osteogenic sarcoma are discussed.


Subject(s)
Bone Neoplasms/genetics , Humerus , Osteosarcoma/genetics , Adolescent , Bone Neoplasms/diagnosis , Child , Female , Femoral Neoplasms/diagnosis , Humans , Male , Osteosarcoma/diagnosis
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