2.1.

Equipment and Chemicals

The $l\mathrm{PDMS}/\text{glycerol}$ interface is formed at an aperture, which is cut in a $120\text{-}\mu \mathrm{m}$-thick transparent poly(ethylene terephthalate) (PET) sheet using Nd:YAG solid-state laser (F3w-40, YUCO optics, New York). The aperture can also be created using commercially available punching machines. The meniscus imaging system is built using Leica $8\times$ objective ($\mathrm{NA}=0.18$), a $10\times$ wide-field microscope eye-piece (23 mm diameter) and an iBall^® ROBO K20 webcam. A 1-ml BD Tuberculin syringe is used for pressure control. Glycerol is purchased from Merck (Mumbai, India). PDMS (Sylgard^®184 silicone elastomer kit) is procured from Dow Corning (Mumbai, India). Xiaomi MI4 smartphone (13MP camera) and Carl Zeiss Axio Imager Z1 (conventional transmission microscope) are used in the study. The translational stage used to build the smartphone microscope was purchased from Holmarc, Kochi, India (Model: TS-65). The histopathology slide (thyroid gland tissue sample) is purchased from V. K. Scientific Industries (Mumbai, India). The malaria (P. falciparum) infected human blood sample is cultured in Molecular Parasitology Lab, IIT Bombay.

2.2.

Lens Fabrication

The block diagram in Fig. 1(a) describes the basic principle of lens fabrication. The system broadly consists of three components: an interface at two immiscible liquids, pressure control, and visual feedback. The curvature of the meniscus formed at the interface is tuned using the pressure control mechanism. The visual feedback assists in controlling the meniscus curvature to fabricate lenses of different curvatures reproducibly.

Fig. 1

Lens fabrication. (a) Basic principle, (b) schematic diagram showing various components of the setup, (c) lens terminology, (d) and (e) top view and side view (across RR′) of the pressure chamber, respectively, showing different parts before $l\mathrm{PDMS}$ is poured, and (f) photo of the actual setup.

Figure 1(b) shows a schematic of the setup used for lens fabrication with the three components. The lid of the chamber is designed to have a transparent PET sheet with a circular aperture ($\mathbf{A}$), where the $l\mathrm{PDMS}/\text{glycerol}$ interface forms. The chamber is filled with 60% glycerol and covered with the lid without air bubbles trapped inside. $l\mathrm{PDMS}$ is prepared by mixing the monomer and the curing agent in the ratio of $10:1$, and degassed. Since $l\mathrm{PDMS}$ and glycerol are immiscible liquids, when $l\mathrm{PDMS}$ is poured on $\mathbf{A}$, a meniscus is formed at the interface of the two liquids. The meniscus imaging system ($\mathbf{S}$) provides a real-time magnified view of the meniscus, which is subsequently displayed on a computer monitor. This is used as a visual feedback to adjust the hydrostatic pressure in glycerol and vary the meniscus curvature. The hydrostatic pressure in glycerol is manually controlled using the syringe.

A hot air gun is used to cure $l\mathrm{PDMS}$ (at $\sim 80°\mathrm{C}$ to 90°C for $\sim 20\min$). The lens fabrication is done in two steps. Step 1: in the first 10 min, the meniscus curvature is kept at minimum till $l\mathrm{PDMS}$ gets partially cured and becomes elastic. Step 2: in the next 10 min, the hydrostatic pressure in glycerol is adjusted to obtain the required curvature till $l\mathrm{PDMS}$ gets completely cured. The detailed protocol of lens fabrication is described in Appendix A.1. The partial curing of $l\mathrm{PDMS}$ prevents it from wetting the side of the PET sheet in contact with glycerol for higher meniscus curvatures. The cured PDMS is easily peeled off from the lid, resulting in a plano-convex PDMS lens [Fig. 1(c)]. The lens is rinsed with running deionized water and dried. The diameter of the aperture in the PET sheet determines the lens diameter. The two pedestals and a cover slip [Figs. 1(d) and 1(e)] are used to maintain a consistent lens base thickness ($\sim 1\mathrm{mm}$) across all the lenses fabricated using this setup. The imaging system ($\mathbf{S}$) was made of an $8\times$ objective ($\mathrm{NA}=0.18$) and a $10\times$ wide-field eye-piece (23 mm diameter) [Fig. 1(f)]. The eye-piece was attached to a webcam with variable focus (5 cm to infinity) to image the meniscus. The distance between the objective and the eye-piece was adjusted to achieve a working distance of $\sim 25\mathrm{mm}$ to focus on the meniscus.

Glycerol is chosen as it has the advantage of not evaporating from the chamber when the setup is not in use. PET is used to fabricate the aperture as it has an optimum contact angle ($\sim 47\mathrm{deg}$) with 60% glycerol. This is to have lenses with a range of curvatures for a given lens diameter. The provision of a variable focus webcam helps in using the same meniscus imaging system ($\mathbf{S}$) for lenses of the size range considered in this study.

2.2.1.

Meniscus curvature control

The reproducibility of the lens fabrication technique is ensured by the visual feedback and a meniscus guiding system. The meniscus guiding system defines guide points ($C_k$) on the axis normal to the plane of $\mathbf{A}$. The circular aperture appears as an ellipse since $\mathbf{S}$ is placed at an angle with respect to the plane of $\mathbf{A}$. As shown in Fig. 2(a), $\mathbf{A}$ lies in the POQ plane and ON is the axis normal to the plane. Plane $V$ is the viewing plane of $\mathbf{S}$, which is at an angle $\phi$ with respect to ON. $\mathbf{A}$ is projected as an ellipse on $V$ with $L_{\text{major}}$ and $L_{\text{minor}}$ as the length of the major and minor axes, respectively. $L_{\text{major}}$ is equal to the diameter of $\mathbf{A}$. $L_{\text{minor}}$ is obtained from the live view of the meniscus. The tilt angle ($\phi$) of $\mathbf{S}$ is computed as

Eq. (1)

$$\phi ={\sin }^{-1}\left(\frac{L_{\text{minor}}}{L_{\text{major}}}\right).$$

Fig. 2

Computation of guide points. (a) Projection of a guide point $C_k$ on the viewing plane (plane $V$) of $\mathbf{S}$. $C_k$ is on the axis normal to plane POQ. (b) Location of the guide points $C_1$ to $C_5$. (c) Live image of a 1-mm-diameter meniscus displayed on the GUI with guide points superimposed.²⁵ $P_1$ to $P_4$ are points provided by the user (Fig. 3, Video 1).

Fig. 3

The video shows the fabrication of a lens as seen by the graphic user interface of the meniscus guiding system. Meniscus at air/glycerol interface followed by PDMS/glycerol interface is shown. The red points indicate the user defined points. The blue and green points indicate the computed guide points. The green guide point specifically indicates that the lens fabricated using this guide point is hemispherical. In this video, the aperture diameter used is 2 mm and the lens produced is hemispherical. (Video 1, mp4, 6446 KB [URL: https://doi.org/10.1117/1.JBO.23.2.025002.1].)

$C_k$ is a guide point on ON at a distance $x$ from the center ($O$) of the aperture. The guide point seen from $\mathbf{S}$ will be the projection of $C_k$ on the line ${\mathrm{ON}}^{\prime }$ at a distance $x\cos \phi$ from $O$. Five guide points ($C_1$ to $C_5$) are defined by Eq. (2), where $k$ is the guide point number ranging from 1 to 5 and $a$ is the radius of $\mathbf{A}$ [Fig. 2(b)]

Eq. (2)

$$x=\frac{k*a}{3}.$$

In this study, the number and position of the guide points are arbitrarily chosen to demonstrate the control on curvature. There can be more guide points at different positions.

A graphic user interface (GUI) is developed in MATLAB R2009a to (1) acquire and display the visual feedback from $\mathbf{S}$, (2) compute the guide points, and (3) superimpose them on the live image as shown in Fig. 2(c). In the GUI, the user defines the plane of $\mathbf{A}$ by selecting the four points $P_1$ to $P_4$. Subsequently, the GUI computes the guide points $C_1$ to $C_5$, and superimposes them on the visual feedback display. The user defined points are used to compute: ellipse major axis ($P_1$ and $P_2$), ellipse minor axis ($P_3$), and the direction of the meniscus with respect to the plane of $\mathbf{A}$ ($P_4$). Thus, the user should define a minimum of four points to describe the interface. In this study, PDMS lenses with different curvatures corresponding to the guide points are fabricated using two apertures ($\mathbf{A}$) of diameters 1 and 5 mm, respectively.

2.3.

Microaperture Fabrication

Microaperture was fabricated using capillary encapsulation technique as reported by Cybulski et al.¹² for glass ball lenses. This technique is low cost and allows self-assembly of the microaperture with the lens. We modified this technique for PDMS lenses (Fig. 4). A glass slide with PDMS ($\sim 1\text{-}\mathrm{mm}$ thick) was used to flatten a 1-mm-diameter PDMS lens [Fig. 4(a)]. The PDMS side of the glass slide was in contact with the curved side of the lens. A capillary was created between the two surfaces into which an opaque polymer solution was poured [Figs. 4(b) and 4(c)]. Once the opaque solution cures, the glass slide was removed. The lens gets back to its original shape due to the elastic nature of PDMS [Fig. 4(d)]. Thus, the microaperture was self-assembled with the lens in the resultant structure. The resultant aperture diameter was measured as $\sim 613\mu \mathrm{m}$ [Fig. 4(e)].

Fig. 4

Microaperture fabrication using capillary encapsulation technique. (a) Two glass slides $G_1$ and $G_2$ in parallel with a PDMS lens mounted on $G_1$. (b) Capillary formation due to lens flattening. (c) Encapsulation of the lens by the opaque plastic solution (Smooth-on Smooth-cast ONYX^®). (d) Final structure in which the microaperture is self-assembled on the lens after the opaque solution cures. $G_1$ and $G_2$ are separated and the lens resumes its original shape. (e) The fabricated microaperture under a transmission microscope.

3.1.

Lens Surface Roughness

The bulk and surface characterization of the fabricated PDMS lenses is performed using X-ray microtomography (microCT) and atomic force microscopy (AFM) studies (Fig. 5). The microCT images demonstrate that (1) the lens surface is nearly free of defects or ripples [Figs. 5(a) and 5(b)], and (2) the bulk of the lens is also defect free [Figs. 5(c) and 5(d)]. The root mean square surface roughness is measured as 628 pm using AFM [Fig. 5(e)]. This value is much less than $\frac{\lambda }{20}$ (assuming $\lambda =550\mathrm{nm}$ for white light illumination). The images demonstrate that the fabrication technique produces high quality, almost defect-free lenses that are suitable for optical imaging. The planar face of the fabricated lens is formed with the help of a glass coverslip of $\sim 2\text{-}\mathrm{nm}$ root mean square surface roughness.

Fig. 5

The bulk and surface characterization of a 1-mm PDMS lens. (a–d) X-ray microtomography (microCT) images. (a) and (b) The side and top-view, respectively. (c) and (d) The lens at two different sectional planes (Fig. 6, Video 2). The images show that the lenses are nearly free of defects or ripples on the surface, and have no defects in the bulk. (e) AFM image across $1\mu \mathrm{m}\times 1\mu \mathrm{m}$ area on the lens surface. The root mean square surface roughness is measured as 628 pm.

Fig. 6

MicroCT of a fabricated lens. The lens is fabricated using 1-mm-diameter aperture. The movie shows the side and top-view of the lens. Also, the bulk of the lens is shown across two different sectional planes. The microCT analysis shows that the fabricated lenses are nearly free of defects or ripples on the surface, and have no defects in the bulk (Video 2, mp4, 618 KB [URL: https://doi.org/10.1117/1.JBO.23.2.025002.2].)

3.2.

Characterization of Lens Radius of Curvature (R) and Focal Length (f)

The fabricated PDMS lenses using 1- and 5-mm aperture diameters are characterized for radius of curvature and focal length as shown in Fig. 7. Figures 7(a) and 7(d) show the side-view of the lenses with various curvatures for 1 and 5 mm diameters, respectively. The nature of the lens profile is determined by fitting a circle to the side-view image of the lens using least squares approximation. It is observed that the circle fits the lens profile with $>99\%$ approximation (Appendix A.2).

Fig. 7

Radius of curvature and focal length characterization of PDMS lenses fabricated using (a–c) 1 mm and (d–f) 5-mm aperture diameters. (a) and (d) The side-view images of the lenses. Plots (b, e) and (c, f) show the radius of curvature and focal length obtained for different guide points, respectively. The error bars represent the deviations across three lenses. A total of 24 lenses were fabricated to study the reproducibility of the fabrication technique.

The plots of radius of curvature versus guide point are shown in Figs. 7(b) and 7(e) for 1- and 5-mm lens diameters, respectively. The experimental radius of curvature is estimated to be the radius of the circle that fits the lens profile in the side-view image. The theoretical radius of curvature is geometrically computed as in Eq. (3), where $a$ is the radius of $\mathbf{A}$ and $k$ is an integer ranging from 1 to 5 corresponding to the guide points $C_1$ to $C_5$, respectively,

It is noticed from Fig. 7(b) that the experimental radius of curvature does not change significantly with the position of the guide point beyond $C_3$. The plot also shows that lenses are fabricated with high reproducibility (up to 8% error) for higher lens curvatures $C_3$, $C_4$, and $C_5$. Similarly, the plot for radius of curvature versus guide point for 5-mm-diameter lens [Fig. 7(e)] shows that larger lenses of higher curvatures ($C_3$) can be fabricated with even higher reproducibility (0.8% error).

In both the plots, the lower curvatures of the lenses ($C_1$ and $C_2$) show larger deviation from theoretical estimates. This can be majorly attributed to (1) the tilt angle of the imaging system and (2) visual error. The visual error is induced during fabrication in (a) choosing $P_1$, $P_2$, and $P_3$, and (b) passing the meniscus through the desired guide point. The percentage error is calculated as $100*(\frac{2*\sigma }{\mu })$, where $\mu$ and $\sigma$ are the mean and standard deviation, respectively, for experimental radius of curvature for a given guide point across three lenses.

Figures 7(c) and 7(f) show the front focal length obtained for different guide points for 1- and 5-mm aperture diameters, respectively. The setup used for the focal length measurement is described in Appendix A.3. The theoretical values are computed using simplified Lens Maker’s formula as in Eq. (4), where $R$ is the radius of curvature of the lens and $n$ is the refractive index of PDMS.

The propagation of error from the measured radius of curvature is attributed to be the major factor for the variation in the measured focal length values. The plot shows $<4\%$ deviation in measured and theoretical focal lengths for curvatures $C_2$ and $C_3$. The percentage deviation is calculated as $100*\frac{|\text{Measured}-\text{Theoretical}|}{\text{Theoretical}}$.

3.3.

Development of a Smartphone Microscope (${\mathbf{I}}_{\mathbf{\text{smart}}}$)

We developed a smartphone microscope (${\mathbf{I}}_{\mathbf{\text{smart}}}$) by placing a single fabricated PDMS lens of high optical power externally to a smartphone [Fig. 8(a)]. The lens is fabricated using 1-mm aperture $\mathbf{A}$ diameter and guide point $C_3$ (focal length, $f=1.2\mathrm{mm}$). A 0.6-mm-diameter microaperture is self-assembled on the lens using the capillary encapsulation technique as described in Fig. 4. The presence of a microaperture prevents the peripheral rays from getting collected by the PDMS lens. Figure 8(b) shows the actual image of the portable 3-D printed prototype of ${\mathbf{I}}_{\mathbf{\text{smart}}}$. The smartphone is placed on a fixed platform. A vertically movable translational stage with resolution of 0.01 mm is used for sample movement. A slip-on module is designed to house the self-assembly of a PDMS lens and a microaperture [Figs. 8(c) and 8(d)]. The intensity of illumination is varied using a potentiometer. The working distance of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is measured to be 2.11 mm.

Fig. 8

Smartphone microscope (${\mathbf{I}}_{\mathbf{\text{smart}}}$). (a) Schematic representation showing the various components. (b) Portable 3-D printed prototype. (c) and (d) The actual and schematic diagram of the slip-on module, respectively. The slip-on module houses the self-assembly of a PDMS lens and a microaperture.

The performance of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is evaluated in comparison with a conventional microscope as shown in Fig. 9. Figures 9(a) and 9(b) show an image of a positive USAF 1951 resolution target acquired using ${\mathbf{I}}_{\mathbf{\text{smart}}}$ and the conventional microscope, respectively. Figure 9(c) shows the normalized line intensity of Element 4 Group 8 from ${\mathbf{I}}_{\mathbf{\text{smart}}}$ and the conventional microscope for comparison. The plot shows that line pairs of $1.38\mu \mathrm{m}$ spacing can be separated by ${\mathbf{I}}_{\mathbf{\text{smart}}}$. The fringe visibility ($V$) is computed as⁸

Fig. 9

Performance metrics of ${\mathbf{I}}_{\mathbf{\text{smart}}}$. Images of a positive USAF 1951 resolution test target (Group 8) acquired by (a) ${\mathbf{I}}_{\mathbf{\text{smart}}}$ and (b) a conventional microscope ($50\times$ objective). (c) Comparison of the normalized line intensity across Element 4 for images acquired from ${\mathbf{I}}_{\mathbf{\text{smart}}}$ and microscope. The resolution of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is estimated as $1.4\mu \mathrm{m}$. (d) Plot showing the minimum intensity computed across line AA′ for images below and above the best focused plane. The distances are normalized with respect to the best focused plane. The depth of field is computed as the distance within which the change in the minimum intensity is $<20\%$ of its value at the best focused plane. (e) Line AA′ across a gold pattern on quartz wafer. (f) Image of a hemocytometer glass slide acquired using ${\mathbf{I}}_{\mathbf{\text{smart}}}$. The field of view of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is estimated as $\sim 200\text{-}\mu \mathrm{m}$ diameter.

The depth of field of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is estimated as $45\mu \mathrm{m}$ using the axial intensity profile of a gold pattern on quartz.²⁶ The image of the gold pattern is shown in Fig. 9(e). The minimum intensity is obtained across line AA′ for planes below and above the best focused plane. Figure 9(d) shows the normalized minimum intensity for each plane versus distance of the plane from the best focused plane. The depth of field is computed as the distance within which the change in the minimum intensity is $<20\%$ of its value at the best focused plane. The field of view of the system is estimated as $\sim 200\text{-}\mu \mathrm{m}$ diameter by imaging a standard hemocytometer glass slide [Fig. 9(f)].⁷

3.4.

Biological Sample Imaging

In this section, the potential of ${\mathbf{I}}_{\mathbf{\text{smart}}}$ to image biological samples is demonstrated and compared with a conventional microscope (Fig. 10). Image quality functions are computed in MATLAB R2015a using images of a thyroid tissue sample acquired using a conventional microscope as the reference image [Fig. 10(a)], and ${\mathbf{I}}_{\mathbf{\text{smart}}}$ as the test image [Fig. 10(b)]. The peak signal-to-noise ratio (PSNR) and structural similarity index (SSIM) are computed as 19.04 dB and 0.87, respectively. The value of 20 dB of PSNR is considered as recognizable with respect to the reference image.²⁷ SSIM ranges between 0 and 1, where 1 is for high similarity between the test and reference images.²⁸ The values of the image quality metrics show that the images acquired by ${\mathbf{I}}_{\mathbf{\text{smart}}}$ have similarity to the images from a conventional microscope.

Fig. 10

Biological sample imaging. The images of thyroid gland tissue sample are acquired by (a) monochrome CCD camera mounted on a conventional microscope, and (b) ${\mathbf{I}}_{\mathbf{\text{smart}}}$ (image converted to grayscale). The images malaria (P. falciparum) infected human blood smear stained with field’s stain are acquired using (c) smartphone placed at the eye-piece of the conventional microscope, and (d) ${\mathbf{I}}_{\mathbf{\text{smart}}}$. The conventional microscope was fitted with a $50\times$ objective for both the samples. The infected cells are highlighted by dotted rectangles. (e) The spots (highlighted by dotted rectangles) computed using the color filter algorithm show the presence of malarial infected cells in the sample.

Figure 10(c) shows the malaria (P. falciparum) infected cells in a stained human blood smear (Field’s stain) which can also be seen in the image acquired by ${\mathbf{I}}_{\mathbf{\text{smart}}}$ [Fig. 10(d)]. Further, the image acquired by ${\mathbf{I}}_{\mathbf{\text{smart}}}$ is subjected to an image processing algorithm (Appendix A.4) to locate the infected cells in a given image. A color filter algorithm is designed to indicate the malaria infected cells with black spots. As can be seen from Fig. 10(e), the algorithm is able to identify the infected cells clearly. Thus, we have demonstrated ${\mathbf{I}}_{\mathbf{\text{smart}}}$ to be a potential affordable microscope capable of acquiring images of pathological samples.