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Biophysical Journal Feb 2015Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set...
Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.
Topics: Animals; Baltimore; Biomarkers; Biophysics; Congresses as Topic; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; History, 20th Century; History, 21st Century; Humans; Light; Mice; Microscopy, Fluorescence
PubMed: 25650914
DOI: 10.1016/j.bpj.2014.12.012 -
Cytometry. Part a : the Journal of the... Mar 2012Proper illumination is essential for light microscopy. Whereas in early years incandescent light was the only illumination, today, more and more specialized light... (Review)
Review
Proper illumination is essential for light microscopy. Whereas in early years incandescent light was the only illumination, today, more and more specialized light sources, such as lasers or arc lamps are used. Because of the high efficiency and brightness that light-emitting diodes (LED) have reached today, they have become a serious alternative for almost all kinds of illumination in light microscopy. LED have a high durability, do not need expensive electronics, and they can be switched in nanoseconds. Besides this, they are available throughout the UV/Vis/NIR-spectrum with a narrow bandwidth. This makes them ideal light sources for fluorescence microscopy. The white LED, with a color temperature ranging from 2,600 up to 5,000 K is an excellent choice for bright-field illumination with the additional advantage of simple brightness adjustments without changing the spectrum. This review discusses the different LED types, their use in the fluorescence microscope, and discusses LED as specialized illumination sources for Förster resonance energy transfer and fluorescent lifetime imaging microscopy.
Topics: Equipment Design; Fluorescence Resonance Energy Transfer; Light; Lighting; Microscopy, Confocal; Microscopy, Fluorescence
PubMed: 22290727
DOI: 10.1002/cyto.a.22023 -
Indian Journal of Ophthalmology Oct 2019To evaluate light exposure from microscope versus intracameral illuminations to patient's and surgeon's retina during cataract surgery. (Comparative Study)
Comparative Study
PURPOSE
To evaluate light exposure from microscope versus intracameral illuminations to patient's and surgeon's retina during cataract surgery.
METHODS
Thirty consecutive patients who had cataract surgery using microscope and intracameral illuminations. At the point of the ocular of an operating microscope, optical illuminance and irradiance from the microscope illumination (60, 40, 20% intensity) and the intracameral illumination (60% intensity) were measured using a light meter and a spectrometer at a pause after lens capsule polishing in cataract surgery.
RESULTS
Average illuminance (lux) was 1.46, 0.66, 0.27, and 0.1 from 60%, 40%, 20% intensity microscope illuminations and 60% intracameral illumination. Average total spectral irradiance (μW/cm2) was 1.25, 0.65, 0.26, and 0.03 from 60%, 40%, 20% intensity microscope illuminations and 60% intracameral illumination.
CONCLUSION
Microscope ocular illuminance and irradiance during cataract surgery were higher in the microscope illumination than in the intracameral illumination. It suggests that light exposure reaching patient's and surgeon's retina during cataract surgery is lower in the intracameral illumination than in the microscope illumination.
Topics: Aged; Aged, 80 and over; Female; Humans; Lens Implantation, Intraocular; Light; Lighting; Male; Microscopy; Middle Aged; Operative Time; Phacoemulsification; Prospective Studies; Radiation Dosage; Radiation Injuries; Retina
PubMed: 31546495
DOI: 10.4103/ijo.IJO_316_19 -
Nature Communications Jul 2022Ultra-violet (UV) light has still a limited scope in optical microscopy despite its potential advantages over visible light in terms of optical resolution and of...
Ultra-violet (UV) light has still a limited scope in optical microscopy despite its potential advantages over visible light in terms of optical resolution and of interaction with a wide variety of biological molecules. The main challenge is to control in a robust, compact and cost-effective way UV light beams at the level of a single optical spatial mode and concomitantly to minimize the light propagation loss. To tackle this challenge, we present here photonic integrated circuits made of aluminum oxide thin layers that are compatible with both UV light and high-volume manufacturing. These photonic circuits designed at a wavelength of 360 nm enable super-resolved structured illumination microscopy with conventional wide-field microscopes and without modifying the usual protocol for handling the object to be imaged. As a biological application, we show that our UV photonic chips enable to image the autofluorescence of yeast cells and reveal features unresolved with standard wide-field microscopy.
Topics: Light; Lighting; Microscopy; Photons
PubMed: 35896536
DOI: 10.1038/s41467-022-31989-8 -
Biosensors Dec 2022Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low...
Three-dimensional imaging of live processes at a cellular level is a challenging task. It requires high-speed acquisition capabilities, low phototoxicity, and low mechanical disturbances. Three-dimensional imaging in microfluidic devices poses additional challenges as a deep penetration of the light source is required, along with a stationary setting, so the flows are not perturbed. Different types of fluorescence microscopy techniques have been used to address these limitations; particularly, confocal microscopy and light sheet fluorescence microscopy (LSFM). This manuscript proposes a novel architecture of a type of LSFM, single-plane illumination microscopy (SPIM). This custom-made microscope includes two mirror galvanometers to scan the sample vertically and reduce shadowing artifacts while avoiding unnecessary movement. In addition, two electro-tunable lenses fine-tune the focus position and reduce the scattering caused by the microfluidic devices. The microscope has been fully set up and characterized, achieving a resolution of 1.50 μm in the x-y plane and 7.93 μm in the z-direction. The proposed architecture has risen to the challenges posed when imaging microfluidic devices and live processes, as it can successfully acquire 3D volumetric images together with time-lapse recordings, and it is thus a suitable microscopic technique for live tracking miniaturized tissue and disease models.
Topics: Lighting; Microscopy, Fluorescence; Imaging, Three-Dimensional; Microscopy, Confocal; Lab-On-A-Chip Devices
PubMed: 36551076
DOI: 10.3390/bios12121110 -
BMC Ophthalmology Jan 2023To evaluate the effect of the light intensity of the surgical microscope and illuminated chopper on the anterior chamber temperature.
BACKGROUND
To evaluate the effect of the light intensity of the surgical microscope and illuminated chopper on the anterior chamber temperature.
STUDY DESIGN
Experimental study.
METHODS
A model eye (Kitaro WetLab System; FCI Ophthalmics, Pembroke, MA, USA) was used in this experimental study. The illuminance of a surgical microscope (Leica M300; Leica Microsystems, Wetzlar, Germany) and illuminated chopper (iChopper NAM-25 GB; Oculight, Korea) with a light source (iVision; Oculight) was measured using an illuminometer. In addition, the temperature in the anterior chamber of the model eye filled with balanced salt solution when using the surgical microscope with a light intensity from level 1 to level 6 and the illuminated chopper at 99% light intensity was measured for 10 min.
RESULTS
The anterior chamber temperature was increased by 0.2, 0.5, 1.0, and 1.4 ℃ when using the surgical microscope at level 3 (10050 lux), 4 (16490 lux), 5 (24900 lux), and 6 (32500 lux), respectively, for 10 min. The illuminated chopper at 99% light intensity (14893 lux) positioned in the anterior chamber increased the anterior chamber temperature by 0.2° C after 10 min, which was equal to the increase in the temperature caused by the surgical microscope at level 3.
CONCLUSION
The photothermal effect of the illuminated chopper directly positioned in the anterior chamber appeared to be similar to that of a microscope with similar illuminance. Therefore, the illuminated chopper is safe in terms of anterior chamber temperature changes in cataract surgery.
Topics: Humans; Temperature; Light; Cataract Extraction; Microscopy; Anterior Chamber
PubMed: 36690966
DOI: 10.1186/s12886-023-02784-w -
Scientific Reports Nov 2022A recurring issue with microstructure studies is specimen lighting. In particular, microscope lighting must be deployed in such a way as to highlight biological elements...
A recurring issue with microstructure studies is specimen lighting. In particular, microscope lighting must be deployed in such a way as to highlight biological elements without enhancing caustic effects and diffraction. We describe here a high frequency technique due to address this lighting issue. First, an extensive study is undertaken concerning asymptotic equations in order to identify the most promising algorithm for 3D microstructure analysis. Ultimately, models based on virtual light rays are discarded in favor of a model that considers the joint computation of phase and irradiance. This paper maintains the essential goal of the study concerning biological microstructures but offers several supplementary notes on computational details which provide perspectives on analyses of the arrangements of numerous objects in biological tissues.
Topics: Algorithms; Lighting; Imaging, Three-Dimensional
PubMed: 36380079
DOI: 10.1038/s41598-022-24176-8 -
The Journal of Investigative... Aug 1998Since its introduction over a decade ago, confocal microscopy has found wide applicability in the microscopy of thick specimens and living tissue because of its ability... (Comparative Study)
Comparative Study Review
Since its introduction over a decade ago, confocal microscopy has found wide applicability in the microscopy of thick specimens and living tissue because of its ability to obtain images from deep inside the sample without interference from scattered or out-of-focus light. Three new instruments that are capable of imaging optically dense specimens such as the skin are considered here: a modified tandem-scanning confocal, the video-rate laser-scanning confocal microscope (both of which were developed specifically to examine skin in vivo), and the two-photon laser-scanning microscope, a design which is "inherently confocal." The tandem-scanning and video-rate confocals use visible and infra-red light, respectively, to acquire reflection images, whereas the two-photon scanner is a fluorescence microscope. The advantages and drawbacks of each of these instruments is considered.
Topics: Dermatology; Equipment Design; Humans; Microscopy, Confocal; Photons; Research
PubMed: 9734833
DOI: 10.1038/jidsymp.1998.33 -
Plant Physiology Oct 2017In vivo variable chlorophyll fluorescence measurements of photosystem II (PSII) quantum yields in optically dense systems are complicated by steep tissue light gradients...
In vivo variable chlorophyll fluorescence measurements of photosystem II (PSII) quantum yields in optically dense systems are complicated by steep tissue light gradients due to scattering and absorption. Consequently, externally measured effective PSII quantum yields may be composed of signals derived from cells differentially exposed to actinic light, where cells located deeper inside tissues receive lower irradiance than cells closer to the surface and can display distinct photophysiological status. We demonstrate how measured distributions of PSII quantum yields in plant tissue change under natural tissue light gradients as compared with conventionally measured quantum yields with even exposure to actinic light. This was achieved by applying actinic irradiance perpendicular to one side of thallus cross sections of the aquatic macrophyte with laser light sheets of defined spectral composition, while imaging variable chlorophyll fluorescence from cross sections with a microscope-mounted pulse amplitude-modulated imaging system. We show that quantum yields are highly affected by light gradients and that traditional surface-based variable chlorophyll fluorescence measurements result in substantial underestimations and/or overestimations, depending on incident actinic irradiance. We present a method for using chlorophyll fluorescence profiles in combination with integrating sphere measurements of reflectance and transmittance to calculate depth-resolved photon absorption profiles, which can be used to correct apparent PSII electron transport rates to photons absorbed by PSII. Absorption profiles of the investigated aquatic macrophyte were different in shape from what is typically observed in terrestrial leaves, and based on this finding, we discuss strategies for optimizing photon absorption via modulation of the structural organization of phytoelements according to in situ light environments.
Topics: Chlorophyll; Electron Transport; Fluorescence; Fucus; Light; Microscopy; Optical Imaging; Photons; Photosynthesis; Photosystem II Protein Complex
PubMed: 28821593
DOI: 10.1104/pp.17.00820 -
Scientific Reports Jul 2019An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A...
An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A standard benchtop microscope platform, equipped with e.g., bright-field and phase-contrast modes, is of importance and convenience for various users because the wide-field and label-free properties allow for morphological imaging without the need for specific sample preparation. However, these microscopes never have capability of acquiring molecular contrast in a label-free manner. Here, we develop a simple add-on optical unit, comprising of an amplitude-modulated mid-infrared semiconductor laser, that is attached to a standard microscope platform to deliver the additional molecular contrast of the specimen on top of its conventional microscopic image, based on the principle of photothermal effect. We attach this unit, termed molecular-contrast unit, to a standard phase-contrast microscope, and demonstrate high-speed label-free molecular-contrast phase-contrast imaging of silica-polystyrene microbeads mixture and molecular-vibrational spectroscopic imaging of HeLa cells. Our simple molecular-contrast unit can empower existing standard microscopes and deliver a convenient accessibility to the molecular world.
Topics: HeLa Cells; Humans; Lasers, Semiconductor; Light; Microscopy, Phase-Contrast; Microspheres; Molecular Imaging; Polystyrenes; Silicon Dioxide; Spectrophotometry, Infrared
PubMed: 31316091
DOI: 10.1038/s41598-019-46383-6