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

Abstract

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.

Swelling rather than shrinkage precedes apoptosis in serum-deprived vascular smooth muscle cells

“…7). This observation is consistent with significant reduction of mean volume in staurosporine-treated HeLa, U937 and NG108-15 cells measured by Coulter-type cell size analyzer [14,28] as well as decrease of single cell volume estimated from the cross-sectional area of cerebellar granule neurones [29] and by recently developed 3D reconstruction technique using negative transmission contrast rendered to T24 cells by strongly colored membrane-impermeable dye [30]. Third, decreased cell volume detected in cells undergoing apoptosis can be at least partially explained by secondary events, such as plasma membrane budding and the formation of apoptotic bodies rather than by primary shrinkage triggered by outwardly directed movements of intracellular osmolytes and osmotically obliged water.…”

Integrated automated nanomanipulation and real-time cellular surface imaging for mechanical properties characterization

“…6 A fast and resistant to instability of the light source technique is developed to obtain the volume and thickness of the adherent cells. 7 It is an essential need to have access to a comprehensive model of the mechanical probe to study the dynamic behavior of the AFM system and having a precise control scheme for acquiring high-resolution image of the ultra-small materials. For this aim, there are various research works studying different models for the AFM system.…”

Thickness Profiling of Formaldehyde-Fixed Cells by Transmission-Through-Dye Microscopy

“…While we have evidence (11) that AB9 does not enter living cells other than by endocytosis (which can be a slow process), the question becomes critical for chemically fixed samples. Indeed, cells treated with cross-linking fixatives paraformaldehyde (PFA) and glutaraldehyde become partially leaky, and the extent of membrane damage may be sensitive to fixation conditions (14).…”

“…When the absorption is too strong, parts of cells lying deep inside the dye may become too dark to be imaged. For imaging of live cells, food colorants Acid Blue 9 (AB9) or Patent Blue V at 510 mg/mL appear to be compatible with normal cell functioning (11); at these concentrations, the absorption at 630 nm is 0.10.2 m 1 , allowing sufficiently deep light penetration to obtain complete images of cell monolayers while keeping the vertical resolution below the diffraction limit (about 0.1 m/pixel).…”

“…Holographic (6,7) or interferometric (8,9) microscopy require highly specialized instrumentation. In an attempt to overcome these problems, we have developed transmission-through-dye (TTD) imaging, a technique in which all the thickness information is contained in a single image (10,11). Knowing cell thickness at every point enables calculation of volumea parameter that plays an important role in many normal and pathological conditions (12).…”