Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer

Benjamin Rappaz, Alexander Barbul, Yves Emery, Rafi Korenstein, Christian Depeursinge, Pierre Magistretti, Pierre Marquet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

190 Scopus citations


Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (λ = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 ± 0.012, volume 83 ± 14 fl, MCH = 29.9 pg, and MCHC 362 ± 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 ± 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

Original languageEnglish (US)
Pages (from-to)895-903
Number of pages9
JournalCytometry Part A
Issue number10
StatePublished - Oct 1 2008


  • Digital holographic microscopy
  • Impedance volume analyzer
  • Laser scanning confocal microscopy
  • MCHC
  • MCV
  • Mean corpuscular hemoglobin concentration
  • Red blood cell volume
  • Refractive index

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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