Measurement of rapid changes in cell volume by forward light scattering

Srinivas, Sangly P.; Bonanno, Joseph A.; Larivière, Els; Jans, Danny; Driessche, Willy Van

doi:10.1007/s00424-003-1145-5

Cited by 27 publications

(25 citation statements)

References 30 publications

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“…Adding a small amount of fluorescent dye to the medium rather than into the cells is a possible alternative to intracellular staining [11] but suffers from similar limitations. Confocal reflection from the cellmedium interface [35] or forward light scatter [13,47,50] appears to work best for statistical analysis of cell populations rather than for individual cells.…”

Section: Introductionmentioning

confidence: 98%

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

Gregg

McGuire

Focht³

et al. 2010

Pflugers Arch - Eur J Physiol

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Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cells by a strongly absorbing dye present in the extracellular medium. Cells are placed in a shallow compartment, and a nontoxic and cell-impermeant dye, such as acid blue 9, is added to the medium. Transmission images are collected at the wavelength of maximum dye absorption (630 nm). Where the cell body displaces the dye, the thickness of the absorbing layer is reduced; thus, an increase in cell thickness produces brighter images and vice versa. The absolute values for cell thickness and volume can be easily extracted from the image by computing the logarithm of intensity and dividing it by the absorption coefficient. The method is fast, impervious to instability of the light source, and has a high signal-to-noise ratio; it can be realized either on a laser scanning or a conventional microscope equipped with a bandpass filter. For long-term experiments, we use a Bioptechs perfusion chamber fitted with a 0.03-mm spacer and an additional port to enable rapid switching of solutions. To show possible applications of this method, we investigated the kinetics of the cell volume response to a hypotonic buffer and to the apoptotic agents staurosporine and ionomycin.

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Section: Introductionmentioning

confidence: 98%

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

Gregg

McGuire

Focht³

et al. 2010

Pflugers Arch - Eur J Physiol

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“…In the last few years, other physiological processes at the cellular and subcellular level such as membrane depolarization (12), cell swelling (13), and altered metabolism have been found to cause small changes in the local optical properties of neuronal tissue that are detectable by measurement of light-scattering signals. In studies carried out in isolated retinas (14) and retinal-slice preparations (15), changes of NIR transmission and scattering have been recorded from retinal layers, including the inner retina before and after light stimulation.…”

mentioning

confidence: 99%

Optophysiology: Depth-resolved probing of retinal physiology with functional ultrahigh-resolution optical coherence tomography

Bizheva

Pflug

Hermann

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

204

140

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Noncontact, depth-resolved, optical probing of retinal response to visual stimulation with a <10-m spatial resolution, achieved by using functional ultrahigh-resolution optical coherence tomography (fUHROCT), is demonstrated in isolated rabbit retinas. The method takes advantage of the fact that physiological changes in dark-adapted retinas caused by light stimulation can result in local variation of the tissue reflectivity. fUHROCT scans were acquired from isolated retinas synchronously with electrical recordings before, during, and after light stimulation. Pronounced stimulusrelated changes in the retinal reflectivity profile were observed in the inner͞outer segments of the photoreceptor layer and the plexiform layers. Control experiments (e.g., dark adaptation vs. light stimulation), pharmacological inhibition of photoreceptor function, and synaptic transmission to the inner retina confirmed that the origin of the observed optical changes is the altered physiological state of the retina evoked by the light stimulus. We have demonstrated that fUHROCT allows for simultaneous, noninvasive probing of both retinal morphology and function, which could significantly improve the early diagnosis of various ophthalmic pathologies and could lead to better understanding of pathogenesis.electroretinogram ͉ functional optical coherence tomography ͉ inner plexiform layer ͉ photoreceptors ͉ retinal imaging T he vertebrate retina consists of several distinct layers: nuclear layers containing cell bodies can be differentiated from plexiform layers with axons and dendrites forming the neuronal network that preprocesses light-evoked signals before transmission to the brain. Early stages of retinal disorders are often confined to one of these layers and are manifested by both morphological abnormalities and impaired physiological responses. Detection of such pathologies requires high-resolution imaging methods. Various imaging modalities such as fundus photography, ultrasound imaging, and optical coherence tomography (OCT) are clinically used for imaging retinal morphology. OCT is an emerging imaging technique that allows for noncontact, in vivo visualization of biological tissue morphology with a micrometer-scale resolution at imaging depths of 1-2 mm (1-3). Currently, electrophysiological tests such as electroretinography (ERG) (4) and multifocal ERG (5) are used for clinical assessment of retinal function.More then 25 years ago, it was observed that the isolated retina when stimulated with visible light changes the amount of transmitted near-infrared light (NIR) (6, 7). Photoreceptors (PRs) were determined to be the main source of this effect, and in the following years, this method was used for investigation and quantitative evaluation of the activation of the PR G protein transducin and the time course of transduction events (8-10 and reviewed in ref. 11). In the last few years, other physiological processes at the cellular and subcellular level such as membrane depolarization (12), cell swelling (13), and altered metabolism...

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“…1). After the main illuminating beam already scattered emerges from the sample, the intensity of light scattered at small angles carries information on cell volume as previously described [30,36,37]. This scattered light is captured through a lens endowed with a black plastic (1 mm wide) central band to block the laser main beam, and is measured with a photomultiplier (Hamamatsu R 928) at a fixed gain (-140 V dynode potential).…”

Section: Methodsmentioning

confidence: 99%

“…The method has been described and validated in the literature [30,31,33,34,35,36,37]. For illumination, a small laser-diode was used (model L52-265, Edmund Scientific, 4.2 mw, λ = 670 nm), generating a beam of rectangular profile reduced to a width of 1.5 mm by a variable adjustable slit.…”

Section: Methodsmentioning

confidence: 99%

Regulatory Volume Increase and Regulatory Volume Decrease Responses in HL-1 Atrial Myocytes

Cacace

Finkelsteyn

Tasso

et al. 2014

Cell Physiol Biochem

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Background/Aims: we have investigated whether cultured cardiomyocytes of the cell line HL-1 have the ability to perform regulatory volume responses both in hypotonic and hypertonic conditions. Furthermore, we characterized those regulatory responses and studied the effects of bumetanide and DIDS in volume regulation of HL-1 cells. Methods: we used a light scattering system to measure the transient volume changes of HL-1 cells when subjected to osmotic challenge. Results: We found that HL-1 cells correct for their volume excess by undergoing regulatory volume decrease (RVD), and also respond to hypertonic stress with a regulatory volume increase (RVI). Rate of RVD was 0.08 ± 0.04 intensity/min, and rate of RVI was 0.09 ± 0.01 intensity/min. Volume recovery was 83.68 ± 5.73 % for RVD and 92.3 ± 2.3 % for RVI. Bumetanide 50 µM inhibited volume recovery, from 92.3 ± 2.3 % (control) to 24.6 ± 8.8 % and reduced the rate of RVI from 0.070 ± 0.020 intensity/min (control) to 0.010 ± 0.005 intensity/min. 50 µM DIDS reduced volume recovery to 42.93 ± 7.7 % and rate of RVI, to 0.03 ± 0.01 intensity/min. Conclusions: these results suggest that bumetanide- and DIDS-sensitive mechanisms are involved in the RVI of HL-1 cells, which points to the involvement of the Na⁺/K⁺/2Cl^- cotransporter and Cl^-/bicarbonate exchanger in RVI, respectively.

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Measurement of rapid changes in cell volume by forward light scattering

Cited by 27 publications

References 30 publications

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy

Optophysiology: Depth-resolved probing of retinal physiology with functional ultrahigh-resolution optical coherence tomography

Regulatory Volume Increase and Regulatory Volume Decrease Responses in HL-1 Atrial Myocytes

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