On-chip platform for lens-free fluorescent imaging over a large FOV of 2.5 cm × 3.5 cm. Fluorescent excitation is achieved by using side illumination through a rhomboid prism (conveniently a different prism geometry could also be used). A simple LED or a Xenon lamp tuned by a monochromator is used for excitation. Lens-free holographic imaging14 of the same FOV is achieved through vertical incoherent illumination (another LED) which uses the flat top part of the prism. Drawing is not to scale. Dimensions: prism height, p (17 mm); imager active area, w₁ × w₂ (25 × 35 mm); depth of the solution reservoir, k (∼10–100 μm); distance of the vertical source, h (∼5–10 cm); distance of the fluorescent excitation source, f (∼1–2 cm). Not shown here, an index matching gel can also be used to avoid TIR and undesired scattering at the bottom facet of the prism. Note also that to better control the vertical distance between the sample micro-channel and the active region of the sensor, we removed the protective coverglass of the chip. The thin absorption filter shown above acts as a protective layer in this case, isolating the active region of the sensor chip from the micro-channels.

(a) Lens-free fluorescent imaging is illustrated over a FOV of >8 cm² for 10μm fluorescent beads (excitation/emission: 495 nm/505 nm). The raw lens-free image (a) was pumped through an LED (see Fig. 1) and was acquired with an integration time of 1 s, (b) illustrates the digitally deconvolved fluorescent image of the same FOV. The zoomed images (c–e) are cropped from the raw fluorescent image (a), and next to each zoomed image, the result of the deconvolution process is illustrated as a function of the number of iterations (100 vs. 600). In (c) and (d), the letters and the dashed lines within the frames refer to (f) and (h), which illustrate different cross-sections of the raw and the deconvolved fluorescent images, demonstrating ∼5× improvement in fluorescent spot size. Through the iterative deconvolution process two particles that almost completely overlap at the raw lens-free image, see for instance (c–d), can now be separated from each other as shown to the right of (c–d) and in (f, h). Note that the pixel size of the CCD in this experiment is 9 μm, and the resolution of the deconvolved lens-free fluorescent image could be further improved with a smaller pixel.

Lens-free fluorescent imaging (a) and the results of the iterative deconvolution algorithm (b) are demonstrated for Calcein Labeled WBCs. The zoomed images at the bottom are cropped from (a) and (b) to illustrate the success of the deconvolution. (f) and (g) illustrate the cross-sections of fluorescent signatures for the raw lens-free image (blue curve) as well as for 100 (green curve) and 600 (red curve) iterations of deconvolution. The FWHM of the deconvolved fluorescent signature in (f) improved by ∼5× from 191μm to 38μm. (g) illustrates a similar situation for two overlapping fluorescent signatures.

Lens-free holography14 and on-chip fluorescent imaging is demonstrated within regions shown with the dashed squares of Fig. 2(a–b). In (a–c) left column, the raw lens-free fluorescent images are shown. To the right of these images, the results of digital deconvolution are presented. In (ii) on the far right, lens-free holographic imaging results of the same field of view are presented, which show the shadow signatures of all the particles, both fluorescent (F) and non-fluorescent (NF), whereas the other images on the left only show the fluorescent signatures.