Lensless fluorescence imaging with height calculation

Abstract

Lensless fluorescence imaging (LFI) is the imaging of fluorescence from cells or microspheres using an image sensor with no external lenses or filters. The simplicity of the hardware makes it well suited to replace fluorescence microscopes and flow cytometers in lab-on-a-chip applications, but the images captured by LFI are highly dependent on the distance between the sample and the sensor. This work demonstrates that not only can samples be accurately detected across a range of sample-sensor separations using LFI, but also that the separation can be accurately estimated based on the shape of fluorescence in the LFI image. First, a theoretical model that accurately predicts LFI images of microspheres is presented. Then, the experimental results are compared to the model and an image processing method for accurately predicting sample-sensor separation from LFI images is presented. Finally, LFI images of microspheres and cells passing through a microfluidic channel are presented.

The Use of Motion Analysis as Particle Biomarkers in Lensless Optofluidic Projection Imaging for Point of Care Urine Analysis

“…Due to the laminar parabolic flow in the channel, the particles exhibit a different flow speed based on their position in the channel. The image size and the flow speed of objects in the images can be used to calculate the actual size and height of the particle 26 . The number of blood cells in urine can be counted without sample preparation, however as there is a much higher www.nature.com/scientificreports www.nature.com/scientificreports/ concentration of blood cells in blood and the blood sample must be diluted if this platform should be extended to hemocytometry.…”

Lensless Imaging and Sensing

“…Lensless fluorescence imaging (57,58) is conceptually similar to shadow imaging, except that (a) the scattering-based partially coherent or coherent signal is now replaced with incoherent fluorescent emission from the sample and (b) the excitation light and captured emission light are of different wavelengths. The experimental setup thus requires an emission filter to be placed between the sample and the sensor to reject the excitation wavelength [unless the sensor is not sensitive to the excitation wavelength (59)]. For many fluorescence applications, even with an emission filter, signal-to-background levels are still a concern, and extra measures can be helpful in reducing leakage of excitation light.…”

Unconventional methods of imaging: computational microscopy and compact implementations

“…In terms of experimental setup, the only additional required component in fluorescence imaging, compared to shadow imaging, is the presence of a filtering mechanism between the sample and image sensor that blocks the excitation light. In order to improve the signal-to-noise performance of these lensfree fluorescent imaging systems, additional strategies may also be used to reduce leakage of excitation light onto the image sensor, including the selection of light source and sensor such that the sensor is insensitive to this light source [145], integrating optical filters directly into the image sensor [146][147][148], delivering the excitation through total internal reflection [93,145,149,150], and structuring the illumination field [151].…”

“…For the most basic lensfree fluorescence on-chip imaging systems, the resolution is intuitively similar to those of the previously discussed shadow imaging systems, where the diffraction of the emitted light over the distance between the sample and sensor is the limiting factor. This diffraction generates a relatively large point spread function (PSF) at the imager chip, whose characteristic size is approximately the same as the sample-sensor distance [145,149]. As this distance is often several hundreds of microns, the resolution of these fluorescent imaging systems is relatively poor compared to traditional microscopy approaches such as objective-based epi-fluorescence.…”