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1.
J Microsc ; 292(2): 64-77, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37616077

ABSTRACT

'openFrame' is a modular, low-cost, open-hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open-source and proprietary hardware components or software enables low-cost, yet research-grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long-term time-lapse image data acquisition, slide-scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single-shot closed-loop focus lock, which we have demonstrated to accommodate defocus up to ±37 µm with <200 nm accuracy, and a two-step autofocus mode which we have shown can operate with defocus up to ±68 µm. We have used this to implement automated single molecule localisation microscopy (SMLM) in a relatively low-cost openFrame-based instrument using multimode diode lasers for excitation and cooled CMOS cameras.

2.
Sci Rep ; 13(1): 3342, 2023 02 27.
Article in English | MEDLINE | ID: mdl-36849727

ABSTRACT

Improving cardiac function through stem-cell regenerative therapy requires functional and structural integration of the transplanted cells with the host tissue. Visualizing the electromechanical interaction between native and graft cells necessitates 3D imaging with high spatio-temporal resolution and low photo-toxicity. A custom light-sheet fluorescence microscope was used for volumetric imaging of calcium dynamics in co-cultures of adult rat left ventricle cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. Aberration-free remote refocus of the detection plane synchronously to the scanning of the light sheet along the detection axis enabled fast dual-channel 3D imaging at subcellular resolution without mechanical sample disturbance at up to 8 Hz over a ∼300 µm × 40 µm × 50 µm volume. The two cell types were found to undergo electrically stimulated and spontaneous synchronized calcium transients and contraction. Electromechanical coupling improved with co-culture duration, with 50% of adult-CM coupled after 24 h of co-culture, compared to 19% after 4 h (p = 0.0305). Immobilization with para-nitroblebbistatin did not prevent calcium transient synchronization, with 35% and 36% adult-CM coupled in control and treated samples respectively (p = 0.91), indicating that electrical coupling can be maintained independently of mechanotransduction.


Subject(s)
Induced Pluripotent Stem Cells , Humans , Adult , Animals , Rats , Coculture Techniques , Myocytes, Cardiac , Calcium , Imaging, Three-Dimensional , Mechanotransduction, Cellular , Microscopy, Fluorescence , Calcium, Dietary
3.
J Microsc ; 273(2): 148-160, 2019 02.
Article in English | MEDLINE | ID: mdl-30508256

ABSTRACT

Super-resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high-performance computing (HPC) cluster and quantify the speed advantage for a four-node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD-enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software. LAY DESCRIPTION: Optical microscopy is now able to provide images with a resolution far beyond the diffraction limit thanks to relatively new super-resolved microscopy (SRM) techniques, which have revolutionized the ability to study biological structures. One approach to SRM is to randomly switch on and off the emission of fluorescent molecules in an otherwise conventional fluorescence microscope. If only a sparse subset of the fluorescent molecules labelling a sample can be switched on at a time, then each emitter will be, on average, spaced further apart than the diffraction-limited resolution of the conventional microscope and the separate bright spots in the image corresponding to each emitter can be localised to high precision by finding the centre of each feature using a computer program. Thus, a precise map of the emitter positions can be recorded by sequentially mapping the localisation of different subsets of emitters as they are switched on and others switched off. Typically, this approach, described as single molecule localisation microscopy (SMLM), results in large image data sets that can take many minutes to hours to process, depending on the size of the field of view and whether the SMLM analysis employs a computationally-intensive iterative algorithm. Such a slow workflow makes it difficult to optimise experiments and to analyse large numbers of samples. Faster SMLM experiments would be generally useful and automated high throughput SMLM studies of arrays of samples, such as cells, could be applied to drug discovery and other applications. However, the time required to process the resulting data would be prohibitive on a normal computer. To address this, we have developed a method to run standard SMLM data analysis software tools in parallel on a high-performance computing cluster (HPC). This can be used to accelerate the analysis of individual SMLM experiments or it can be scaled to analyse high throughput SMLM data by extending it to run on an arbitrary number of HPC processors in parallel. In this paper we outline the design of our parallelised SMLM software for HPC and quantify the speed advantage when implementing it on four HPC nodes compared to a powerful desktop computer.


Subject(s)
Image Processing, Computer-Assisted/methods , Single Molecule Imaging/methods , Software , Algorithms
4.
Rev Sci Instrum ; 88(1): 013707, 2017 Jan.
Article in English | MEDLINE | ID: mdl-28147687

ABSTRACT

We report the characterisation of gated optical image intensifiers for fluorescence lifetime imaging, evaluating the performance of several different prototypes that culminate in a new design that provides improved spatial resolution conferred by the addition of a magnetic field to reduce the lateral spread of photoelectrons on their path between the photocathode and microchannel plate, and higher signal to noise ratio conferred by longer time gates. We also present a methodology to compare these systems and their capabilities, including the quantitative readouts of Förster resonant energy transfer.

5.
Oncogene ; 33(34): 4316-29, 2014 Aug 21.
Article in English | MEDLINE | ID: mdl-24362530

ABSTRACT

The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.


Subject(s)
Forkhead Transcription Factors/metabolism , Mitosis , SUMO-1 Protein/metabolism , Sumoylation , Antibiotics, Antineoplastic/pharmacology , Antigens, CD , Binding Sites , Cadherins/metabolism , Cell Proliferation/drug effects , Cytoplasm/metabolism , Drug Resistance, Neoplasm , Epirubicin/pharmacology , Forkhead Box Protein M1 , G2 Phase Cell Cycle Checkpoints , HeLa Cells , Humans , MCF-7 Cells , Nocodazole/pharmacology , Protein Transport , Proteolysis
6.
Phys Med Biol ; 58(18): 6447-58, 2013 Sep 21.
Article in English | MEDLINE | ID: mdl-23999099

ABSTRACT

Ultrasound (US) is a widely used clinical imaging modality that offers penetration depths in tissue of >10 cm. However, the spatial resolution in US imaging is fundamentally limited by diffraction to approximately half the wavelength of the sound wave employed. The spatial resolution of optical microscopy is limited by the same fundamental physics, but in recent years super-resolution imaging techniques have been developed that overcome the diffraction limit through the localization of many spatially separated photo-switchable or photo-activatable fluorophores. In this paper, we apply a related approach to demonstrate super-resolution imaging with US. We imaged dilute suspensions of microbubble contrast agents flowing through narrow tube-based phantoms. By spatially localizing multiple spatially isolated microbubbles, we constructed super-resolved microbubble location density maps that clearly resolve features 5.1-2.2 times smaller than the US system point spread function full width half maximum in the lateral and axial directions respectively. Our initial characterization experiment using a fixed 100 µm diameter brass wire and a US frequency of 2 MHz suggests that for an ideal stationary point scatterer the ultimate resolution of the unmodified clinical US system used could be in the range of 2-4 µm.


Subject(s)
Contrast Media/pharmacology , Microbubbles , Ultrasonography/methods , Acoustics , Cellulose/chemistry , Contrast Media/chemistry , Diagnostic Imaging/methods , Equipment Design , Fluorescent Dyes/chemistry , Microscopy/methods , Optics and Photonics , Phantoms, Imaging , Time Factors , Ultrasonics
7.
Proc Inst Mech Eng H ; 224(2): 291-306, 2010.
Article in English | MEDLINE | ID: mdl-20349820

ABSTRACT

Recently there have been significant advances in developing hybrid techniques combining electromagnetic waves with ultrasound for biomedical imaging, namely photoacoustic, thermoacoustic, and acousto-optic (or ultrasound modulated optical) tomography. All three techniques take advantage of tissue contrast offered by electromagnetic (EM) waves, while achieving good spatial resolution in deeper tissue facilitated by ultrasound. In this review the principles of the three techniques are introduced. A description of existing experimental and image reconstruction techniques is provided. Some recent key developments are highlighted and current issues in each of the areas are discussed.


Subject(s)
Elasticity Imaging Techniques/instrumentation , Elasticity Imaging Techniques/methods , Image Interpretation, Computer-Assisted/instrumentation , Microscopy, Acoustic/instrumentation , Microscopy, Acoustic/methods , Thermography/instrumentation , Thermography/methods , Equipment Design , Image Enhancement/instrumentation , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods
8.
Opt Express ; 16(25): 20306-16, 2008 Dec 08.
Article in English | MEDLINE | ID: mdl-19065169

ABSTRACT

This paper describes a new optically sectioning microscopy technique based on oblique selective plane illumination combined with oblique imaging. This method differs from previous selective plane illumination techniques as the same high numerical aperture lens is used to both illuminate and image the specimen. Initial results obtained using fluorescent pollen grains are presented, together with a measurement of the resolution of the system and an analysis of the potential performance of future systems. Since only the plane of the specimen that is being imaged is illuminated, this technique is particularly suited to time-lapse 3-D imaging of sensitive biological systems where photobleaching and phototoxicity must be kept to a minimum, and it could also be applied to image microfluidic technology for lab-on-a-chip, cytometry and other applications.


Subject(s)
Computer-Aided Design , Image Enhancement/instrumentation , Imaging, Three-Dimensional/instrumentation , Lenses , Microscopy, Fluorescence/instrumentation , Optical Devices , Equipment Design , Equipment Failure Analysis , Light , Scattering, Radiation
9.
Br J Dermatol ; 159(1): 152-61, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18460029

ABSTRACT

BACKGROUND: Fluorescence lifetime imaging (FLIM) is a novel imaging technique that generates image contrast between different states of tissue due to differences in fluorescence decay rates. OBJECTIVES: To establish whether FLIM of skin autofluorescence can provide useful contrast between basal cell carcinomas (BCCs) and surrounding uninvolved skin. METHODS: Unstained excision biopsies of 25 BCCs were imaged en face with FLIM following excitation of autofluorescence with a 355 nm pulsed ultraviolet laser. RESULTS: Using FLIM we were able to distinguish areas of BCC from surrounding skin in an ex vivo study. Significant reductions in mean fluorescence lifetimes between areas of BCC and areas of surrounding uninvolved skin were demonstrated (P < 0.0001). These differences were apparent irrespective of the decay model used to calculate the fluorescence lifetimes (single vs. stretched exponential) or the long-pass filter through which the emitted autofluorescence was collected (375 vs. 455 nm). Conversely, there was no significant difference between the BCC and uninvolved areas of each sample when mean autofluorescence intensities were examined. Moreover, wide-field false-colour images of fluorescence lifetimes clearly discriminated areas of BCC from the surrounding uninvolved skin. CONCLUSIONS: We therefore believe that FLIM has a potential future clinical role in imaging BCCs for rapid and noninvasive tumour delineation and as an aid to determine adequate excision margins with best preservation of normal tissue.


Subject(s)
Carcinoma, Basal Cell/diagnosis , Diagnostic Imaging/methods , Skin Neoplasms/diagnosis , Adult , Aged , Aged, 80 and over , Contrast Media , Female , Fluorescence , Humans , Male , Middle Aged , Neoplasm Staging/methods , Sensitivity and Specificity
10.
Rev Sci Instrum ; 78(12): 123101, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18163714

ABSTRACT

The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440 nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (<2 h) ex vivo skin lesions, illustrating its potential for skin cancer detection and diagnosis.


Subject(s)
Biomarkers, Tumor/analysis , Luminescent Measurements/instrumentation , Skin Neoplasms/diagnosis , Spectrometry, Fluorescence/instrumentation , Equipment Design , Equipment Failure Analysis , Humans , Molecular Probe Techniques , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Fluorescence/methods
11.
Opt Express ; 15(20): 12548-61, 2007 Oct 01.
Article in English | MEDLINE | ID: mdl-19550524

ABSTRACT

We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor.

12.
Opt Express ; 15(24): 15656-73, 2007 Nov 26.
Article in English | MEDLINE | ID: mdl-19550853

ABSTRACT

We present a time domain optically sectioned fluorescence lifetime imaging (FLIM) microscope developed for high-speed live cell imaging. This single photon excited system combines wide field parallel pixel detection with confocal sectioning utilizing spinning Nipkow disc microscopy. It can acquire fluorescence lifetime images of live cells at up to 10 frames per second (fps), permitting high-speed FLIM of cell dynamics and protein interactions with potential for high throughput cell imaging and screening applications. We demonstrate the application of this FLIM microscope to real-time monitoring of changes in lipid order in cell membranes following cholesterol depletion using cyclodextrin and to the activation of the small GTP-ase Ras in live cells using FRET.

14.
Opt Lett ; 30(24): 3353-5, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16389829

ABSTRACT

We demonstrate an optically sectioned fluorescence lifetime imaging microscope with a wide-field detector, using a convenient, continuously tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated in a microstructured fiber.

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