**4. Clinical applications**

Lately, DH microscopy is being developed for clinical applications in widely different areas of medicine such as transmembrane water flux, cancer screening, sperm motility, blood cell analysis, and inflammation.

### **4.1. Transmembrane water flux**

In some cases, current imaging techniques are not very well developed. One such example is the measurement of transmembrane water fluxes in epithelial cells directly linked to the activity of a protein involved in cystic fibrosis [41]. DH microscopy was used to quantify the transmembrane water fluxes in situ by determining the phase shift associated with activation of chloride channels. This opens up for the usage of DH microscopy to screen drugs acting on water transporter molecules.

#### **4.2. Cancer screening**

Recently, Benzerdjeb et al. reported a preliminary study with DH microscopy as a screening tool for cervical cancer. The study is based on materials from three randomly chosen laboratories, which was analyzed and subjected to DH microscopy. The sensitivity and specificity of DH microscopy was calculated for the detection of neoplasia. The results demonstrated for the first time that the DH microscopy technique is suitable for the processing of gynecologic cervical samples [42].

#### **4.3. Sperm analysis**

DH has been used to characterize sperm cells, supplying data for both morphology, motility, and the concentration of the sperm cells, without affecting the sperm reviewed in Ref. [43]. The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection [44, 45]. Indeed, DH microscopy generates useful information on the dimensions and structure of human sperm, not revealed by conventional phase-contrast microscopy, in particular the volume of vacuoles. This suggests its use as an additional prognostic tool in assisted reproduction technology to better underline the differences between normal and abnormal sperm morphology.

#### **4.4. Blood cell screening**

The function of RBCs is strongly connected to their shape, related to different diseases [46]. Therefore, a robust classification method would be of great advantage when analyzing RBC for medical diagnosis and therapeutics. DH microscopy has allowed several investigators to determine vital erythrocyte parameters including morphology and cell counting [15, 47, 48]. Indeed, a DH microscopy-based automated RBC classification method could have the potential for use in drug testing and the diagnosis of RBC-related diseases. Malaria parasites induce morphological, biochemical, and mechanical changes in RBC. Main clinical diagnostics of malaria is based on microscopic inspection of blood smears, treated with reagents, which stains the malarial parasites. In developing countries, visual identification of malarial RBCs may become unreliable due to lack of sufficiently trained technicians and poor-quality microscopes and reagents. Anand et al. describe the use of quantitative DH microscopy for automatic identification of malaria-infected RBCs by comparing their shape profiles at different axial planes [49]. A correlation algorithm discriminates between the shapes of the cells and determines whether the cell is infected by malaria parasite. Shape comparison is fast and was found to yield fairly accurate discrimination. This technique is mostly advantageous for healthcare personnel working in developing countries. Similarly, other RBC infecting microbes can be investigated, such as *Babesia microti,* which is an obligate parasite in humans, hamster, and mouse. To find alternatives to methods requiring experience of professional technicians, usually based on optical microscope with Giemsa-stained blood smears, DH methodology was exploited [50]. The authors found the technique to be useful for determining morphological modifications in host RBCs, quantifying contents, and concentration of the cellular dry mass, as well as dynamic membrane fluctuations measured at the individual cell level, which are in turn strongly correlated with the mechanical deformability of cell membrane. An automatic compact diagnostic tool will be advantageous especially for healthcare personnel working in developing countries, which lack trained professionals and high-quality equipments.

Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D-structure of platelet aggregates. Boudejltia et al. used DH microscopy to develop a convenient method to characterize the platelet and aggregate 3D shapes [51]. This is the first report on analysis of platelets aggregates by DH microscopy. According to the authors, the method is particularly well suited for the study of the platelet physiology, the physiopathology in clinical practice, and the development of new drugs.

#### **4.5. Quantification of inflammation**

**4. Clinical applications**

326 Holographic Materials and Optical Systems

analysis, and inflammation.

**4.1. Transmembrane water flux**

water transporter molecules.

**4.2. Cancer screening**

cervical samples [42].

**4.3. Sperm analysis**

abnormal sperm morphology.

**4.4. Blood cell screening**

Lately, DH microscopy is being developed for clinical applications in widely different areas of medicine such as transmembrane water flux, cancer screening, sperm motility, blood cell

In some cases, current imaging techniques are not very well developed. One such example is the measurement of transmembrane water fluxes in epithelial cells directly linked to the activity of a protein involved in cystic fibrosis [41]. DH microscopy was used to quantify the transmembrane water fluxes in situ by determining the phase shift associated with activation of chloride channels. This opens up for the usage of DH microscopy to screen drugs acting on

Recently, Benzerdjeb et al. reported a preliminary study with DH microscopy as a screening tool for cervical cancer. The study is based on materials from three randomly chosen laboratories, which was analyzed and subjected to DH microscopy. The sensitivity and specificity of DH microscopy was calculated for the detection of neoplasia. The results demonstrated for the first time that the DH microscopy technique is suitable for the processing of gynecologic

DH has been used to characterize sperm cells, supplying data for both morphology, motility, and the concentration of the sperm cells, without affecting the sperm reviewed in Ref. [43]. The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection [44, 45]. Indeed, DH microscopy generates useful information on the dimensions and structure of human sperm, not revealed by conventional phase-contrast microscopy, in particular the volume of vacuoles. This suggests its use as an additional prognostic tool in assisted reproduction technology to better underline the differences between normal and

The function of RBCs is strongly connected to their shape, related to different diseases [46]. Therefore, a robust classification method would be of great advantage when analyzing RBC for medical diagnosis and therapeutics. DH microscopy has allowed several investigators to determine vital erythrocyte parameters including morphology and cell counting [15, 47, 48]. Indeed, a DH microscopy-based automated RBC classification method could have the potential for use in drug testing and the diagnosis of RBC-related diseases. Malaria parasites induce Lenz et al. have utilized DH microscopy for investigation of inflammatory bowel diseases including Crohn's disease and ulcerative colitis [52]. Dextran sodium sulfate-induced colitis was performed and colonic sections were subsequently examined by histological analyses and by DH microscopy. With DH microscopy, optical path length delay including refractive index was monitored, and thereby tissue density assessment could be performed. Indeed, the average refractive index was an accurate marker to distinguish between different layers of the intestinal wall of the colitis induced murine model.

Furthermore, DH microscopy reliably detects inflamed colonic segments with a strong correlation between the severity of inflammation and the refractive index. In conclusion, DH microscopy analysis opens a novel diagnostic option for optical quantification of inflammation in murine models of colitis. Moreover, the same research group assessed cellular growth and motility in epithelial wound healing in vitro using DH microscopy [53]. Interestingly, phase images quantifying cell thickness, dry mass, and tissue density were demonstrated. The study concluded that the technique can assist in the evaluation of potential therapeutics in, for example, helping to elucidate the specific role of certain cytokines for wound healing.
