Phase Microscopy

DHM® is a phase microscopy technique that, additionally to providing an intensity contrast, provides Quantitative Phase Measurements (QPM).

Why does holography provide phase information ?

Holography derives from the Greek words “holos” meaning whole or entire, and “graphy” meaning a process of writing or recording. The whole information of a wave consists in its wavelength, its amplitude, its phase, and its polarization state. Dennis Gabor has received the Nobel prize for finding a way to record  both phase and intensity information on a substrate sensitive only to intensity (named hologram), nowadays digital cameras. Used in conjunction with coherent light sources, Digital Holography enables to record and retrieve both amplitude and phase wavefront information within the measured field of view.

Why is the phase of a wave so relevant ?

The phase can be considered as the position of the wave as a function of time. Knowing  the speed of the light enables to measure distances, and then to determine the 3D topography of the sample. It gives geometrical information  with the wavelength as a vertical reference. Sub-nanometer vertical resolution can then be achieved.

Moreover, the phase (and the amplitude) of the light propagating through and reflected by a sample is affected by its dielectric constant (refractive index). Interpretation of such change provides relevant information on material properties.

Why is the phase quantitative and absolute ?

The topography is related to the phase by a very well defined, precise, and calibrated length: the wavelength of the illumination source. Units are not arbitrary contrary to an intensity measurement which depends in particular on the illumination intensity, detection efficiency, and amplification of the recorded signal.

What is QPM (Quantitative Phase Measurement) ?

QPM is the difference of phase of the wavefront within the measurement field of view. Its  interpretation give access to sample geometry, sample material properties in material science, and on cellular morphology and content in life science. Application of QPM in both material sciences and life sciences is explained in the two tabs on this section. The output of QPM is also called OPD ( Optical Path Diffrence), especially in the life science field.

Is the periodicity of the wave a limitation ?

The phase is by definition periodic. Thus there is a potentially ambiguity when using it to measure a distance. Many people think that it cannot be used for measurements of heights larger than the wavelength. For many applications, this is actually not the case. A first mathematical technique called phase unwrapping  is supplied with the Koala software and can be used on samples with topography smooth enough. It enables to measure topography with height differences of more than hundred microns. A second technique using Multi-wavelengths DHM® enables to measure rougher surfaces and samples with steps.

DHM® provides an optical measurements of the samples. Its interpretation depends on DHM® configuration (reflection or transmission), but provides geometrical and material information.

Phase interpretation for DHMs in reflection configuration

Phase Microscopy Profilometry DHM principle reflection

figure 1 : measurement principle in reflection

Figure 1 illustrates the principle of phase measurement by a DHM® configured in reflection on a homogeneous reflective sample lit by a monochromatic plane wave of wavelength λ. The wavefront is deformed when reflected on the sample surface. This deformation with respect to the incident wave is called dephasing Δφ. For a homogeneous sample this dephasing is linearly related to the 3D topography of the sample  by the formula:

Δh = λΔφ / 4π n

where Δh is the height of the sample and n the refractive index of the immersion medium (n = 1 in air, 1.33 in water). For non-homogeneous samples the physical characteristics of the surface must be taken into account (Reflectometry analysis).

Phase interpretation for DHM® in transmission configuration

Phase Microscopy Profilometry DHM principle transmission

Figure 2. measurement principle in transmission

Figure 2 illustrates the principle of phase measurement by a DHM® configured in transmission on a homogeneous transmissive sample with a refractive index n2 lit by a monochromatic plane wave of wavelength λ. The wavefront is deformed when transmitted through the sample. This deformation with respect to the incident wave, measured in degree or in radian, is called dephasing Δφ. For a homogeneous sample this dephasing is directly connected to the 3D thickness variations Δh of the sample by the formula:

Δh = (λΔφ) /[2π (n2– n1)]

where Δh is the height of the sample and n1 the refractive index of the immersion medium (n1=1 in air, 1.33 in water).

Light traveling through a sample is in general modified both in amplitude and in phase. But cellular structures, which are mostly transparent and do not absorb light, do not affect the amplitude of the wave, but only its phase. Cells are mainly such so-called “phase objects”.  Therefore  they are not visible using classical bright field microscope. To overcome this issue, the first system of  phase contrast microscope has been invented in the early 1930s by Frits Zernike (Nobel prize 1953). It makes possible for biologists to visualize changes of the phase of the light induced by cellular structures.

As self-explained by their names, phase contrast microscopes (Zernike, Nomarski or differential interference contrast (DIC)or Hoffman modulation contrast microscopes)  provide contrast images revealing details, but do not provide absolute and quantitative measurements of the change of phase induced by the sample. In 1950, J. Dyson described well the limitations of such phase contrast approaches and proposed an interferometer microscope for Quantitative Phase Measurement (QPM). These first QPM microscopes were difficult to operate for cellular imaging, considering the intrinsic short coherence of  light sources available at that time and consequently the requirements for matching reference and object wavefronts. Moreover, at that time, the presently so efficient digital acquisition and processing hardware were not available. The first QPM for biological interpretation has been proposed by Barer system in 1952.

Considering in one hand the difficulty of operation of these early QPM microscopes and on the other hand the lack of experiments to interpret measurements in terms of underlying biological processes, the interest and use of QPM microscopes have decreased in favor of staining to make biological structures visible.

Early 1990s, the group of founders of Lyncée Tec worked on the development of a DHM® for fast QPM. They were the first ones to demonstrate QPM measurement from a single hologram based on off-axis digital holography and patented it. In parallel, the group worked intensively on the interpretation of the QPM in life Science. The major steps of this discovery is described here below. They have been achieved using the DHM® by Lyncée Tec.

Dyson phase contrast microscope

Intracellular content

The original experiment published by Barer has been repeated using DHM® by Lyncée Tec. The first series of experiments have been performed on culture of yeast. They confirm that QPM enables direct measurement of protein content in yeasts, and time-lapse measurements and therefore quantitative, label-free and strictly non-invasive time monitoring of protein production and concentration. A second series of experiments concern Red Blood Cells (RBCs) hemoglobin concentration measurements. The measurement procedure has been validated  with results obtained by alternative measurement systems.

Cellular morphology and intracellular concentration

As discussed in the introduction, the phase value measured by QPM systems depends on both the averaged intracellular refractive index and the cell thickness. The first research direction of scientists using DHM® by Lyncée Tec has been naturally the investigation of methods for separating cell thickness and averaged intracellular information. This has been achieved by several methods and published [Rappaz, Boss 2013]. It has been first applied for strictly non-invasive time-lapse cell morphology studies and cellular volume measurements.

One of the major outcome of this study is that for short periods of time, typically a few minutes, in absence of relevant protein production, QPM is much more sensitive to changes of intracellular component than to a change of morphology. This important result has been well demonstrated in a publication by applying a hypotonic solution on neurons of mice. The measurements show a strong decrease of the phase when it is well known that the cells volume increases.  All the examples of the following paragraph demonstrate the same important feature.

Mobility and growth of yeast cells

Membrane activity

As briefly explained, QPM variations can be directly interpreted in term of change of concentration. Seminal publications have demonstrated in recent years that the specification of the DHM® by Lyncée Tec enables measurement of:

  • Trans-membrane currents. Ions traveling through membranes are hydrated by a known stoichiometric amount of water molecules. Any ion exchange through the membrane increases or decreases the amount of water within the cell and changes the intracellular concentration. It has been shown in several publications[Pavillon 2010, Jourdain 2011, 2013]  that phase measurements enable to monitor ion channel activity.
  • Water fluxes. Water exchange through membranes changes directly the intracellular concentration and thus can be measured directly by DHM® as demonstrated in several publications [e.g. Jourdain 2013]
  • Co-transporters. Monitoring of co-transporters activity is demonstrated in several publications [Jourdain 2011]
  • Cell viability. Cell survival is strongly linked to the ability of cells to balance any external change by activating channels. Absence of such regulation process is synonym for cell death and usually associated with a non-reversible change of the intracellular concentration. This can be measured by DHM® as demonstrated in several publications [Kuhn 2011, Pavillon 2012, Rappaz 2013]

Innovative research

Many other innovative applications leading to underlying biological interpretation have been investigated by DHM®:

Lyncée Tec and the associated research teams are proud of being pioneers in the development of DHM® and in the interpretation of QPM in life science.