Advertisement

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

  • 531 Accesses

  • 20 Citations

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Aagaard-Tillery KM, Jelinek DF (1995) Differential activation of a calcium-dependent endonuclease in human B lymphocytes. J Immunol 155:3297–3307

  2. 2.

    Agero U, Monken CH, Ropert C, Gazzinelli RT, Mesquita ON (2003) Cell surface fluctuations studies with defocusing microscopy. Phys Rev 67:051904-1–051904-9

  3. 3.

    Berninghausen O, Leippe M (1997) Necrosis versus apoptosis as the mechanism of target cell death induced by Entamoeba histolytica. Infect Immun 65:3615–3621

  4. 4.

    Bertrand R, Solary E, O’Connor P, Kohn KW, Pommier Y (1994) Induction of a common pathway of apoptosis by staurosporine. Exp Cell Res 211:314–321

  5. 5.

    Bettega D, Calzolari P, Doglia SM, Dulio B, Tallone L, Villa AM (1998) Cell thickness measurements by confocal fluorescence microscopy on C3H10T1/2 and V79 cells. Int J Radiat Biol 74:397–403

  6. 6.

    Boudreault F, Grygorczyk R (2004) Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging. J Microsc 215:302–312

  7. 7.

    Boudreault F, Grygorczyk R (2004) Cell swelling-induced ATP release is tightly dependent on intracellular calcium elevations. J Physiol 561:499–513

  8. 8.

    Bullard TA, Borg TK, Price RL (2005) The expression and role of protein kinase C in neonatal cardiac myocyte attachment, cell volume, and myofibril formation is dependent on the composition of the extracellular matrix. Microsc Microanal 11:224–234

  9. 9.

    Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE (1998) Micropatterned surfaces for control of cell shape, position, and function. Biotechnol Prog 14:356–363

  10. 10.

    Crowe WE, Altamirano J, Huerto L, Alvarez-Leefmans FJ (1995) Volume changes in single N1E-115 neuroblastoma cells measured with a fluorescent probe. Neuroscience 69:283–296

  11. 11.

    Droste MS, Biel SS, Terstegen L, Wittern K-P, Wenck H, Wepf R (2005) Noninvasive measurement of cell volume changes by negative staining. J Biomed Opt 10:064017-1–064017-10

  12. 12.

    Dutta AK, Korchev YE, Shevchuk AI, Hayashi S, Okada Y, Sabirov RZ (2008) Spatial distribution of maxi-anion channel on cardiomyocytes detected by smart-patch technique. Biophys J 94:1646–1655

  13. 13.

    Fischbarg J, Kuangt K, Hirsch J, Lecuona S, Rogozinski L, Silverstein SC, Loike J (1989) Evidence that the glucose transporter serves as a water channel in J774 macrophages. Proc Natl Acad Sci USA 86:8397–8401

  14. 14.

    Frederick B, Matskevich I, Lang F (2006) Cell volume regulatory mechanisms. In: Lang F (ed) Mechanisms and significance of cell regulation. Contributions to nephrology, vol 152. Karger, Basel, pp 1–8

  15. 15.

    Fujimoto S, Katsuki H, Kume T, Akaike A (2006) Thrombin-induced delayed injury involves multiple and distinct signaling pathways in the cerebral cortex and the striatum in organotypic slice cultures. Neurobiol Dis 22:130–142

  16. 16.

    Gervais P, de Marañón IM, Evrard C, Ferret E, Moundanga S (2003) Cell volume changes during rapid temperature shifts. J Biotechnol 102:260–279

  17. 17.

    Gil-Parrado S, Fernández-Montalván A, Assfalg-Machleidt I, Popp O, Bestvater F, Holloschi A, Knoch TA, Auerswald EA, Welsh K, Reed JC, Fritz H, Fuentes-Prior P, Spiess E, Salvesen GS, Machleidt W (2002) Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members. J Biol Chem 277:27217–27226

  18. 18.

    Grinstein S, Cohen S (1987) Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. J Gen Physiol 89:185–213

  19. 19.

    Gunaratnam M, Grant MH (2004) Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes. Toxicol In Vitro 18:245–253

  20. 20.

    Gwag BJ, Canzoniero LMT, Sensi SL, DeMaro JA, Koh JY, Goldberg MP, Jacquin M, Choi DW (1999) Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons. Neuroscience 90:1339–1348

  21. 21.

    Häussinger D, Reinehr R, Schliess F (2006) The hepatocyte integrin system and cell volume sensing. Acta Physiol 187:249–255

  22. 22.

    Hamann S, Kiilgaard JF, Litman T, Alvarez-Leefmans FJ, Winther BR, Zeuthen T (2002) Measurement of cell volume changes by fluorescence self-quenching. J Fluorescence 12:139–145

  23. 23.

    Hansen WR, Tulyathan O, Dawson SC, Cande WZ, Fletcher DA (2006) Giardia lamblia attachment force is insensitive to surface treatments. Eukaryot Cell 5:781–783

  24. 24.

    Hill DA, Chiosea S, Jamaluddin S, Roy K, Fischer AH, Boyd DD, Nickerson JA, Imbalzano AN (2004) Inducible changes in cell size and attachment area due to expression of a mutant SWI/SNF chromatin remodeling enzyme. J Cell Sci 117:5847–5854

  25. 25.

    Hosoi K, Min K-Y, Iwagaki A, Murao H, Hanafusa T, Shimamoto C, K-i K, Kato M, Fujiwara S, Nakahari T (2004) Delayed shrinkage triggered by the Na+–K+ pump in terbutaline-stimulated rat alveolar type II cells. Exp Physiol 89:373–385

  26. 26.

    Huang S, Ingber DE (2000) Shape-dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks. Exper Cell Res 261:91–103

  27. 27.

    Hurley PT, Ferguson CJ, Kwon TH, Andersen ML, Norman AG, Steward MC, Nielsen S, Case RM (2001) Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas. Am J Physiol Gastrointest Liver Physiol 280:G701–G709

  28. 28.

    Ito Y (1999) Surface micropatterning to regulate cell functions. Biomaterials 20:2333–2342

  29. 29.

    Katsura T, Verbavatz JM, Farinas J, Ma T, Ausiello DA, Verkman AS, Brown D (1995) Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells. Proc Natl Acad Sci USA 92:7212–7216

  30. 30.

    Korchev YE, Gorelik J, Lab MJ, Sviderskaya EV, Johnston CL, Coombes CR, Vodyanoy I, Edwards CR (2000) Cell volume measurement using scanning ion conductance microscopy. Biophys J 78:451–457

  31. 31.

    Krumschnabel G, Maehr T, Nawaz M, Schwarzbaum PJ, Manzl C (2007) Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling. Apoptosis 12:1755–1768

  32. 32.

    Lang F, Gulbins E, Szabo I, Lepple-Wienhues HSM, Duranton C, Lang KS, Wieder T (2004) Cell volume and the regulation of apoptotic cell death. J Mol Recognit 17:473–480

  33. 33.

    Liu J, Lee P, Galbiati F, Kitsis RN, Lisanti MP (2001) Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation. Am J Physiol Cell Physiol 280:C823–C835

  34. 34.

    Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y (2000) Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci 97:9487–9492

  35. 35.

    Maric K, Wiesner B, Lorenz D, Klussmann E, Betz T, Rosenthal W (2001) Cell volume kinetics of adherent epithelial cells measured by laser scanning reflection microscopy: determination of water permeability changes of renal principal cells. Biophys J 80:1783–1790

  36. 36.

    Mège JL, Capo C, Benoliel AM, Foa C, Bongrand P (1985) Study of cell deformability by a simple method. J Immunol Methods 82:3–15

  37. 37.

    Model MA, Khitrin AK, Blank JL (2008) Measurement of the absorption of concentrated dyes and their use for quantitative imaging of surface topography. J Microsc 231:156–167

  38. 38.

    Morgan AJ, Jacob R (1994) Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by direct action at the plasma membrane. Biochem J 300:665–672

  39. 39.

    Nehrke K, Melvin JE (2002) The NHX family of Na + –H + exchangers in Caenorhabditis elegans. J Biol Chem 277:29036–29044

  40. 40.

    Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S (2001) Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532:3–16

  41. 41.

    Pasantes-Morales H, Sánchez Olea R, Miranda D, Morán J (1997) Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease. Am J Physiol 272:C1798–C1803

  42. 42.

    Plettenberg S, Weiss EC, Lemor R, Wehner F (2008) Subunits alpha, beta and gamma of the epithelial Na+ channel (ENaC) are functionally related to the hypertonicity-induced cation channel (HICC) in rat hepatocytes. Pflugers Arch 455:1089–1095

  43. 43.

    Porcelli AM, Ghelli A, Zanna C, Valente P, Ferroni S, Rugolo M (2003) Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells. Ann NY Acad Sci 1010:342–346

  44. 44.

    Raat NJH, De Smet P, Van Driessche W, Bindels RJM, Van Os CH (1996) Measuring volume perturbation of proximal tubular cells in primary culture with three different techniques. Am J Physiol 271:C235–C241

  45. 45.

    Shiba Y, Kanno Y (1990) Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum. Cell Mol Life Sci 46:492–495

  46. 46.

    Shimuzu T, Maeno E, Okada Y (2007) Prerequisite role of persistent shrinkage in apoptosis of human epithelial cells. Acta Physiol Sinica 59:512–516

  47. 47.

    Srinvas SP, Bonanno JA, Larivière E, Jans D, Van Driessche W (2003) Measurement of rapid changes in cell volume by forward light scattering. Pflugers Arch 447:97–108

  48. 48.

    Stamer WD, Peppel K, O’Donnell ME, Roberts BC, Wu F, Epstein DL (2001) Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume. Invest Ophthalmol Vis Sci 42:1803–1811

  49. 49.

    Van Elburg HJ, Kuypers LC, Decreamer WF, Dirckx JJJ (2007) Improved correction of axial geometrical distortion in index-mismatched fluorescent confocal microscopic images using high-aperture objective lenses. J Microsc 228:45–54

  50. 50.

    Verkman AS (2005) Water permeability measurement in living cells and complex tissues. J Membrane Biol 173:73–87

  51. 51.

    Vogel V, Sheetz M (2006) Local force and geometry sensing regulate cell functions. Nature Rev 7:265–275

  52. 52.

    Waldegger S, Steuer S, Risler T, Heidland A, Capasso G, Massry S, Lang F (1998) Mechanisms and clinical significance of cell volume regulation. Nephrol Dial Transplant 13:867–874

  53. 53.

    Wei L, Xiao AY, Jin C, Yang A, Lu ZY, Yu SP (2004) Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons. Pflugers Arch 448:325–334

  54. 54.

    Wöll E, Ritter M, Scholz W, Häussinger D, Lang F (1993) The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells. FEBS Lett 322:261–265

  55. 55.

    Zelenina M, Brismar H (2000) Osmotic water permeability measurements using confocal laser scanning microscopy. Eur Biophys J 29:165–171

Download references

Acknowledgments

We are grateful to Dr. Stephen Fisher for discussions and for osmolarity measurements. We also thank Mr. Michael Sulak and Dr. James Jamison for providing cell cultures. The work was supported by NIH grant 1R15GM186816.

Author information

Correspondence to Michael A. Model.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(10.1 MB)

ESM 1

(10.1 MB)

ESM 2

(1.64 MB)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Gregg, J.L., McGuire, K.M., Focht, D.C. et al. Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy. Pflugers Arch - Eur J Physiol 460, 1097–1104 (2010) doi:10.1007/s00424-010-0869-2

Download citation

Keywords

  • Aquaporins
  • Cell volume
  • Microscopy
  • Osmotic stress
  • Imaging