Advantages of digital holographic microscopy for real-time full field absolute phase imaging

Abstract

Different interferometric techniques were developed last decade toobtain full field, quantitative, and absolute phase imaging, suchas phase-shifting, Fourier phase microscopy, Hilbert phase microscopyor digital holographic microscopy (DHM). Although, these techniquesare very similar, DHM combines several advantages. In contrast, tophase shifting, DHM is indeed capable of single-shot hologram recordingallowing a real-time absolute phase imaging. On the other hand, unliketo Fourier phase or Hilbert phase microscopy, DHM does not requireto record in focus images of the specimen on the digital detector(CCD or CMOS camera), because a numerical focalization adjustmentcan be performed by a numerical wavefront propagation. Consequently,the depth of view of high NA microscope objectives is numericallyextended. For example, two different biological cells, floating atdifferent depths in a liquid, can be focalized numerically from thesame digital hologram. Moreover, the numerical propagation associatedto digital optics and automatic fitting procedures, permits vibrationsinsensitive full- field phase imaging and the complete compensationfor a priori any image distortion or/and phase aberrations introducedfor example by imperfections of holders or perfusion chamber. Examplesof real-time full-field phase images of biological cells have beendemonstrated.