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The readership of this journal will appreciate the high cost and complexity that is connected with optical microscopy and imaging flow cytometry equipment. As a direct consequence, despite their gold-standard status in many diagnostic applications, these techniques are not available to most physicians' offices and their use in remote, resource-limited areas of developing countries are unthinkable. The extreme inequality in this “supply and demand” situation has driven the custom design of simple, miniaturized, inexpensive, battery-operated solutions for point-of-care diagnostic purposes as a matter of course. The widespread availability of mobile phones with embedded camera function has played into the hands of this development by providing a globally connected network of telemedicine nodes around which to build remote optical imaging applications.

In combination with the growing availability of powerful light-emitting diodes (LEDs) in virtually every color, even fluorescence applications have become affordable. Microscope adapters and microscopy snap-on modules for mobile phone cameras (1–3) have been reported in the literature and diagnostic fluorescence imaging tests have been performed on diseases that are relevant for developing locations.

Single Lens Miniature Microscopes

Low-tech, however, does not necessarily mean low quality or sophistication. Already the early single lens microscopes from the 17th century were capable of very high magnification and possessed impressive image quality, as was recently demonstrated on an original surviving microscope by Anthoni van Leeuwenhoek (4). The only optical element was a small, high curvature, biconvex bead-like lens with equally short focal length, allowing for high magnifications (up to 118× in an original van Leeuwenhoek microscope). Shrinking the dimensions and complexity of modern multilens objectives, therefore, is the first step in making miniature microscopes. Ironically, the imaging properties of cell phone lenses, which also sport a focal length of only a couple of millimeter—as the distance between the sensor and the lens in the cell phone is equally short—come very close to the original van Leeuwenhoek lenses. In fact, modern replicas of the van Leeuwenhoek microscope (http://museumboerhaave.wordpress.com/2011/07/29/replica-van-leeuwenhoek-microscope-english-version/) are fitted with a single cell phone lens and can reach 275× magnification. The closest cell phone-based approximation of the van Leeuwenhoek microscope uses a 1 mm ball lens, placed in front of the camera lens. The camera lens and the CMOS chip here replace the human lens and retina. The ball lens suffers from image distortion and a small field-of-view, which can be partially corrected for using a multi-focus fusion algorithm that could also assist in unsupported manual focussing on the sample (5).

The imaging of cellular-level processes in the brain of living and freely moving mice has been a major motivation for the construction of miniaturized microscopes (Fig. 1). A fiber-based epifluorescence miniaturized microscope with a single lens objective was developed and used for this purpose (6). However, as with the van Leeuwenhoek microscope, this miniature solution does not include the illumination source (and filters) or the detector, the sun and the eye, corresponding to a mercury arc lamp and an EM-CCD camera in the modern implementation. It only encompasses the primary objective, a millimeter-sized cylindrical GRIN lens (plus focusing and coupling lens) and focusing mechanics (a motor and gear assembly). Using this 1.1 g device, cerebral microcirculation and calcium spiking could be imaged at 3 μm resolution, but the fiber connection still limited the movement of the animal because of its limited bending radius. The same group, therefore, pushed the development of the microscope further to include all of the components in one, self-contained, device with 1.5 μm resolution (7). Besides the advantage of permitting more freedom of movement for the animal, the elimination of the fiber cable improved the optical throughput of this 1.9 g device by fivefold. A small, integrated Complementary Metal Oxide Semiconductor (CMOS) camera, therefore, sufficed, to construct, together with a small LED light source and miniaturized filters, the entire microscope. An untethered miniaturized microscope that includes a small radio transmitter and battery, weighing about 4.5 g and about twice the size, allows for 7 h “field” observations but shows a poor resolution of 20 μm (8). It is likely that further engineering improvements will increase the resolution while still keeping the size and weight within bearable limits. Even though these microscopes were designed for brain imaging, their utility extends to any application where portable, affordable, easily transportable, energy-efficient microscopes can replace their large and expensive counterparts. Besides microscopy-based disease diagnosis in remote locations, other possibilities that are enabled by these specialized “radically minimalist design” optical solutions include imaging inside incubators or other instrumentation and high-throughput screening by the parallelization of tiny microscopes.

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Figure 1. Component integration in miniaturized optical systems.

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Lensless Imaging Systems

The simplification and miniaturization can be taken one step further by eliminating the lens altogether. Lensless microscopy provides a compact and mechanically robust design that avoids alignment issues. It builds on the idea that the pixel array of the sensor chip possesses an intrinsic high resolution because of the micron-scale size of the individual pixels. An object, placed directly on the chip, i.e., at zero working distance, will, therefore, be imaged at the pixel resolution. This has been used also for fluorescence microscopy. Resolution can be increased beyond pixel size by masking the chip with a metal coating on the detector chip with subpixel size apertures. Translating the object along the aperture mask can compensate the loss in continuity of the image that is now observed. This is, in a way, similar to the scanning operation of scanning near-field optical microscopes. A convenient implementation of this idea is to move the object in a microfluidic device. Lines of apertures, arranged at an angle to the direction of movement, scan the object as it is moved through the channel. In combination with the use of an electrokinetic drive that reduces object rotation, a resolution of 0.8 μm was reached (9). In this optofluidic microscope design principle, the aperture array can also be replaced by a Fresnel zone plate array in the coverslip over the microfluidic channel. Resolution is determined by the focus spot size of the Fresnel zone plates (10). The reintroduction of these diffractive elements no longer makes this implementation lensless, but their quasi-two-dimensional nature still aids the miniaturization goal. Fluorescence detection can be easily introduced by the addition of colored films on the cover slide and on the CMOS sensor at the bottom of the microfluidic channel, as was shown in the latter implementation of the optofluidic microscope design.

Lensless imaging can also be achieved by holography, a technique also known from X-ray microscopy. In one application, an arrangement of 23 fiber-coupled LEDs was used to produce an equal number of shifted in-line holograms from which the image amplitude and phase information can be obtained by a pixel super-resolution algorithm (11). This way, submicron resolution was achieved at the full area of the CMOS chip (here 24 mm2), even though the pixel size was 2.2 μm2. The holographic lensless microscope easily compared with a bright-field microscope fitted with a 40× NA = 0.65 objective and was demonstrated to identify the presence of Plasmodium falciparum malaria parasites inside erythrocytes in a standard blood smear, especially in the phase information. These images can be transmitted to centralized analysis centers by the use of a mobile phone—especially when the CMOS chip of a camera phone is used directly—or analyzed on a smart phone that should possess sufficient computational power for the reconstruction of the images from the holograms. The multiple holographic images needed to achieve pixel super-resolution transmission images can also be achieved by translating the objects (at a distance of 5–10 cm) along a large (50–100 μm) aperture in a microfluidic channel (12).

Miniature Imaging Cytometry

A second advanced application that could become emancipated from the domain of the specialized laboratory is fluorescence imaging flow cytometry. A custom-designed optofluidic device is mounted onto the camera objective of a cell phone (13). Doubling as a waveguide for the excitation light that is delivered by two LEDs, the cells flowing through the optofluidic unit are captured by the cell phone camera in video mode. As the excitation light propagates perpendicular to the detection path, a simple absorption filter is sufficient to capture the fluorescently stained cells. In a direct comparison, the results from the cell phone device for fluorescence-based white blood cell counting in whole blood were in very good agreement with a commercial hematology counter and could, thus, be used for rapid and sensitive cell counting analysis of bodily fluids, which is standard clinical practice for a wide range of diseases. It should be technically trivial to include additional morphological parameters or a second fluorescence detection channel, such that advanced clinical and research cytometric analyses become possible on this simple hand-held platform.

Recent advantages in the field of optofluidic design hold the promise to replace the simple channel that was used in the current device, with designs that also support the high throughput that is typically achieved in “large size” flow cytometers. Sheath flow that supports hydrodynamic focusing is possible in optofluidic designs but perhaps the most promising solution is the massive parallelization of the interrogation channel. The insertion at the sample inlet of a 256 parallel straight channel “filter” was shown to produce the ordered flow of cells in an equal amount of parallel tracks (14). Large field-of-view imaging solutions like afforded by the lensless approaches can then be used to interrogate cells flowing at rates up to a million cells per second.

Current microscope and cytometry equipment will not likely be replaced any time soon by the miniaturized versions. In the last years, customers have accepted and appreciated more compact and portable solutions to classical optical equipment. For instance, some commercially available microscopes can no longer be immediately recognized as one or even demand an eyepiece. The scaling-down development that we describe here, however, goes beyond that. This will no doubt come at the cost of functionality or quality, but there are many applications where portable, energy-efficient, robust, and affordable, easy-to-operate and fully networked devices that can be transported in the operator's pocket answers a currently unfulfilled demand. In these niche areas, cell phones become microscopes and large and expensive equipment will fit in the palm of your hand, such that selected and specialized optical applications can become available to all health care providers in a diagnostically democratized world.

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Fred Wouters*, Johannes T. Wessels†, * Laboratory for Molecular and Cellular Systems, Department of Neurophysiology, Centre II: Physiology and Pathophysiology, University of Medicine, Göttingen, Germany, † Central Core Facility Molecular & Optical Live Cell Imaging (MOLCI), University of Medicine, Göttingen, Germany, Department of Nephrology/ Rheumatology, Center for Internal Medicine, University of Medicine, Göttingen, Germany.