A Leica microscope was used as the gold standard and compared to the designed brightfield and polarized brightfield optical instruments that were attached to an iPhone 5s cell-phone as described in the experimental methods section below and as shown in Fig. 1.

Figure 1 (A) Leica DMLM polarized white light microscope used as reference for comparison in this study; and (B) a microscope lens combination implemented into a 3D-printed fitting to allow similar function to a traditional polarized laboratory microscope. The MOPID system was configured in transmission mode with a magnification designed for 40X when using a mobile phone camera. An iPhone 5s was used with polarizer sheets added and a 3D-printed fitting to hold the light source, diffuser, sample slide and microscope attachment. Full size image

Resolution and Field of View Performance Testing

Individual United States Air-Force (USAF) resolution target images (Edmund Optics, Barrington, NJ) are shown in Fig. 2, using both the reference microscope and constructed MOPID device, each equipped for transmission mode imaging. The approximate magnification achievable by the MOPID at the camera face was determined to range from 40X to 100X depending on the sample, illumination settings and the FOV for the individual images acquired with the mobile phone camera. The overall full-width half maximum (FWHM) was calculated by averaging over the total number of FWHM values individually determined for the sample based on different Group and Element measurements for each image.

Figure 2 USAF resolution target images were utilized to determine FOV, resolution and other optical system parameters with a reference Leica microscope that included (A) a 20x magnification; and (B) a 40x magnification. The USAF target images on the bottom were acquired using the polarized mobile platform with (C) full zoom with microscope attachment for cell-phone on iPhone 5s and (D) Digitally zoomed image taken from the cell-phone image shown (C). Full size image

Using Group 7 Element 6, the smallest group on the USAF resolution target, without using the devices digital zoom function, the FOV for each configuration was determined. From the acquired images, shown in Fig. 2B–D and 3, the FOVs were calculated to be 0.42 mm × 0.24 mm for the reference microscope with a 40x objective and 0.78 mm × 0.79 mm for the MOPID configuration shown in Fig. 1B that was attached to an iPhone 5s cellular phone. Next, calculations for spatial resolution were determined using the FWHM measurement of a fit function for the derivative of the boundary line intensity value between light and dark regions on the USAF target. Calculating the total effective system magnification occurred after careful interpretation of acquired USAF target image features with known distance measurements. This magnification varied from the approximate magnification value at the camera face because of digital enlargement capabilities of the acquired image via the camera software settings on the iPhone 5s before capturing the photograph.

Figure 3 IPhone 5 s (left) configuration USAF target image and (right) the calculated derivative for a line spread across Group 7 Element 6. FOV was 0.78 mm × 0.79 mm. Full size image

Using the highest resolvable Group (Group 7 for iPhone 5s configuration) from the USAF resolution target, the systems spatial (lateral) resolution was determined to be ~1.05 μm and the reference Leica determined to have a resolution of ~0.47 μm. The measured resolution is a factor of 2.6 larger than the nominal Rayleigh resolution limit of 0.4 μm for the portable system. An expected increase in measured resolution as compared to nominal Rayleigh resolution limit occurs because the optical components utilized in the construction of the MOPID consist of low-cost plastic lenses. Poor lens selection results in improper correction for field of curvature and additional aberrations that are present in the system, resulting in reduced resolution away from the field radius of best focus. In addition to the plastic microscope lens components, the mobile phone camera lens assembly also contributes to reduced system resolution observed, resulting in non-diffraction limited performance. However, as previously reported with many brightfield cell-phone microscope designs5,41,44, the system limitations did not hinder the mobile phone camera from being able to capture high-definition (HD) images of malaria infected blood smear samples and additional non-malaria samples allowing for useful diagnosis and comparison between reference images utilizing a commercially available laboratory microscope.

In this study, determination of FOV and resolution for the acquired images was important for comparing the acquired images using the MOPID device with the reference images and to verify a minimum metric can be achieved when employing traditional parameters such as shape of parasites present or ratio of malaria infections to RBCs present within a sample for determining if an infection is present. These parameters are additionally useful in determining malaria strain type, parasitemia level and if an infection is present.

While many parameters can be used to compare the two images, the main constraints in the design described are that the overall system resolution needs to be adequate to measure within the individual red blood cells (RBCs) (<5 microns) in order to see some instances of the presence of hemozoin within an infected blood smear sample. Thus, better resolution of the system provides an increased likelihood to observe the presence of individual malaria infected birefringent clusters within a given sample volume. The FOV measurement is important not only for comparative purposes with the gold standard images but also for determining the number of total fields that would be required to image in order to provide relevant diagnostic capability with a limit of detection less than 30 parasites/μl. For example, current microscopy experts determine the number of malaria parasites compared to the number of red blood cells in up to 100 field-of-views to provide a final determination as to whether the sample is considered infected or uninfected with malaria. In the proposed setup, a larger imaging FOV, without sacrificing the ability to diagnose individual malaria parasites, allows for less individual images needed to properly diagnose parasitemia levels in the sample.

Non-Malaria Polarized Light Comparative Sample Images

Prior to characterizing the MOPID towards the clinically relevant malaria application, non-polarized and cross-polarized images were evaluated from the same area of a slide containing wheat starch. The resolution of the images acquired with each system were compared in addition to evaluating if the classic Maltese cross could be depicted from the polarization changes as light transmits through the wheat starch molecules. Indeed, in Fig. 4B,C the starch molecules exhibit a Maltese cross configuration. Additionally, using a 40X objective with an NA of 0.65 on the Leica DMLM microscope comparable FOV and resolution were achieved for the MOPID images acquired over the same area.

Figure 4 Images of a microscope slide coated with wheat starch acquired using a Leica microscope with a 40Xobjective with (A) no polarizers present in the imaging plane and (B) with a polarizer and analyzer crossed at 90 degrees in the imaging plane. For comparison, the reference an iPhone 5s utilized to acquire images of the same location on the wheat starch slide were acquired with (C) no polarizers present; and (D.) with polarizer and analyzer crossed at 90 degrees. In both setups, the polarized images illustrate the presence of a Maltese cross for each starch molecule. Full size image

The image acquisition conditions for the cell-phone images and the Leica images are listed in Tables 1 and 2. For the iPhone setup used to obtain the wheat starch images, integration times of 1/255 seconds and 1/30 seconds were used. These were based on the mobile phones auto integration/exposure setting with ISO speeds of 32 and 80 for the non-polarized 4C and polarized 4D images, respectively. The need for longer integration time in image 4D is based on the low light present in the cross-polarized configuration versus the normal light configuration. Additionally, the corrected focal length for each image was 30 mm and the magnification was set to 0.25.

Table 1 Figures 4–6 image settings. Full size table

Table 2 iPhone 5 s settings. Full size table

Brightfield and Polarized Imaging of Malaria

Comparative brightfield images acquired to analyze a specific zoomed in section on the infected thin blood smear with the reference microscope in non-polarized transmission mode and the MOPID in non-polarized mode are shown in Fig. 5. To compare the respective images acquired from each system, to further illustrate the minimal resolvability of single cell characteristics utilizing the MOPID, each of the images acquired in Fig. 5A–B were enlarged and cropped, as shown with the respective images above Fig. 5A–B. These cropped images represent a smaller region within the total image for evaluating individual RBC resolution. Figure 5 illustrates that the low-cost high resolution MOPID is capable of achieving <2 μm resolution. The Leica reference design resolution was calculated to be ~0.47 μm.

Figure 5 Images acquired of mouse malaria strain blood smear without polarized light using (A) a Leica microscope with a 40X magnification objective and (B) the same area of the slide imaged utilizing the iPhone 5s mobile phone based design. Above each of the respective images is a zoomed in image of the same region for each photo to better illustrate the comparable resolution of the two microscopes. Full size image

Images shown in Fig. 5 (Enlarged Image A and B) illustrate that the MOPID has the high resolution of the Leica microscope reference and it is capable of resolving single RBC boundaries in many cases where overlap of the individual RBCs is not extensive. Although, this could potentially be a problem in traditional microscopic histological examination of the malaria-infected blood smears, it is not an issue in the proposed polarized light cell-phone based setup because of the fact that enhanced contrast is achieved when examining the cross-polarized images in the presence of birefringent variation caused by the hemozoin in the sample.

To show the contrast polarized light microscopy provides from thin smears of Plasmodium chabaudi malaria-infected blood samples, the images in Fig. 6 are presented. Specifically in Fig. 6A,B, brightfield non-polarized and polarized thin Giemsa-stained blood smear samples of malaria-infected RBCs at 40X magnification images were obtained via a digital SLR camera mounted onto a Leica DMLM polarized microscope. As indicated by the presence of birefringent changes in the polarized reference image, Fig. 6B, the sample had positive infected areas with the malaria-parasite. The presence of hemozoin particles in the sample cause the polarized transmitted illumination light to vary in intensity and wavelength due to variation in the light as it transmits through the birefringent hemozoin particles. The result of this change is represented by seven bright white dots that appear in the cross-polarized reference image. It should be noted that it is very challenging to detect these hemozoin particles in the original non-polarized reference imaging system without being a highly trained technician. This confirms previous reports, by Maude et al. and others, that the use of polarized microscopy in observing the presence of malaria-infected RBCs has shown to improve diagnostic capability up to two fold in some instances7,26. Following the acquisition of the two reference images, two additional images, a non-polarized and polarized image, were capture from the same malaria-infected sample and sample region of the blood smear with the MOPID and are shown in Fig. 6C,D. It is clear from the non-polarized images in Figs 5 and 6A,C that the mobile platform has a reduced system resolution as compared to the reference microscope in polarized mode. However, in examining the polarized images from both systems it is clear that the presence of birefringence appears at the same spots within the sample. This indicates that the results obtained with the MOPID are capable of determining the presence of malaria with lower system resolution and with less user expertise than traditional microscopy requires.

The main advantages of the system described in this report are the reduction in cost and complexity associated with conducting polarized microscopy for malaria detection on a mobile platform as well as a reduction in the need for a trained microscopy expert to diagnose the presence of malaria in a blood smear sample in the field. This significantly increases the potential application for the approach in addition to increasing the likelihood of adoption of the technique in developing countries where cost, complexity and lack of expertly trained technicians can often prohibit the use of a polarized microscopy technique or even traditional laboratory microscopy as the standard of diagnosis.

The image acquisition settings for the images acquired with the cell-phone and Leica microscopes are listed in Tables 1 and 2. The iPhone blood smear images, Figs 5B and 6C–D, had integration times of 1/1580 seconds, 1/1642 seconds and 1/30 seconds based on the mobile phones auto integration/exposure setting with ISO speeds of 32, 32 and 80 for the non-polarized Figs 5B and 6C and polarized 6D images respectively. The difference in value is determined based on the low light setting in the cross polarized configuration versus the normal light configuration. Additionally, the corrected focal length for each image was 42 mm, 150 mm and 150 mm with the magnification set to 0.17, 0.25, 0.25. Additionally, Fig. 5B had a digital zoom ratio of 1.4 while Fig. 6C,D having a 5x digital zoom ratio.

An independent validation of the presumed malaria infected birefringent areas for the sample area provided in the report was performed utilizing the reference microscope configured in a traditional white light microscopy orientation. With this setup, polarized and non-polarized images were evaluated and compared within the region suspected of infection. Each birefringent area was observed closely to determine if the birefringent area occurred within a cell body that appeared to be infected based on traditional metrics such as shape and color properties. Further, the birefringent area was evaluated to determine if the change occurred in plane with the cellular components of the sample. Often, contaminants that may be present from the preparation process will exist out of plane with the cellular components of the sample. From reported literature, dust or dirt is the primary component that can often contaminate a sample during the preparation procedure. If present, the dust or dirt can often appear very similar to changes observed from the presence of hemozoin in the sample. Although, the dust also generates changes in the state of polarization, the changes primarily occur out of plane from the sample RBCs2,7. To determine if the birefringent area was in fact generated from the presence of hemozoin the following criteria were utilized in each polarized image: (1) a comparison of the non-polarized and polarized image verifying the birefringent area occurs in the same region of a cell in the non-polarized image. (2) determination if the birefringent area occurred in the same image plane or close to the image plane of the cellular components and (3) the coloring and shape of the cellular components in the proximity of the birefringent area were evaluated to determine if they are consistent with reported changes due to the presence of a malaria infection.