Background

Smartphone microscopes can be effective tools for a broad range of imaging applications. Particularly, smartphone microscopes have proven to be useful in contexts where access to benchtop microscopes is limited. At present, however, smartphone microscope designs face a tradeoff between cost, imaging performance and functionality. Moreover, the advantages of smartphone-based microscopy are hindered by the need of sometimes complex sample preparation. To address these limitations, in their work Liu et al (1) introduce Pocket Microscopy with Ultraviolet Surface Excitation (MUSE).

Key findings and developments

Overview of Pocket MUSE basic design

This prototype is fabricated from readily available low-cost electronics, and is based on a small optical module that attaches over the rear lens of the smartphone, and enables multichannel fluorescence over a 10x field of view. It consists of only 4 major components: an objective lens, a sample holder, UV LED light sources (powered directly with the smartphone battery via the USB port through a step regulator), and a base plate.

Beyond this, a challenge for portability, is the need to develop versatile sample preparation techniques, without sacrificing the applicability of smartphone microscopes. To address some of the main hindrances, including the need for tissue sectioning, the authors explored the use of ultraviolet illumination, which is strongly absorbed by biological structures but can only penetrate a few microns deep. Without subsurface signals contributing to blur and background, this eliminates the need to prepare flat thin sections for mobile setups. As sub-285 nm UV light is blocked by common optical materials, it is unnecessary to filter out the excitation light with designated filters, making it possible to capture the entire visible range of the emitted light with an RGB camera in a single shot. Another limitation addressed by Pocket MUSE is that compact smartphones have very small working distances. This makes it difficult to fit most conventional sub-285nm light-emitting diodes (LEDs). To overcome this challenge, Pocket MUSE is designed to deliver light using frustrated total internal reflection (TIR) through a UVC transparent optical window, which is also the sample holder pre-aligned at the focus of the smartphone microscope objective. This results in uniform illumination of the full field of view, and eliminates the need for a focusing system. As a further step, the authors improved Pocket MUSE resolution in order to allow visualization of sub-cellular structures.

Pocket MUSE is designed to take quality images while holding the phone in any orientation with one hand, which is convenient for field for applications, where a stable working bench is not always available. Following use, the sample holder can be easily cleaned, either while attached to the device, or detaching from it.

Sample preparation

In addition to the optical configuration, Pocket MUSE is compatible with a series of simple, portable and user-friendly sample preparation strategies that can be directly implemented for various microscopy applications.

Slide-free histology is one of the best established MUSE applications, and therefore this was tested in the Pocket MUSE design here presented by the authors. The authors explored single-dip staining followed by a brief tap-water washing step, and demonstrated high image contrast in various tissue samples. Comparable to resolution achievable by a 10x objective, basic staining for use with Pocket MUSE allows identification of sub-cellular structures such as nuclei, and is therefore already useful for various histology-based applications. Moreover, a color remapping technique could be implemented to mimic the color contrast of conventional H&E staining. Also, conventional immunohistochemistry (IHC) staining can be used for imaging tissues using Pocket MUSE. In terms of fluorescence imaging, overnight staining allowed the use of fluorophore-conjugated antibodies, and signal from multiple fluorophores can be separated by unmixing the RGB channels.

Proof of concept

The authors demonstrated the use of Pocket MUSE for imaging various plants and other environmental samples. They also explored the use of bright-field and hybrid imaging on a blood-smear. Trans-illumination BF microscopy can be used by directing the sample holder towards a bright diffusive surface in the far field, using regular room light or natural light as illumination. This can be used in combination with fluorescent dyes targeting the nucleus to identify white blood cells. The authors also explored the use of Pocket MUSE for mucosal smear imaging, of a sample collected using a cotton swab and dipped in dyes. MUSE fluorescence results showed high contrast between cell bodies nuclei ad background. Interestingly, Pocket MUSE allowed cells to be imaged directly from the cotton fiber matrices, and a large population of cells could be visualized by simply repositioning the swab. Finally, the authors explored the possibility of visualizing bacteria using Pocket MUSE. They found that although individual bacteria are smaller than the resolution limit of Pocket MUSE, they can be visualized if sparsely dispersed in a fluid sample. Moreover, Pocket MUSE could differentiate different populations of microorganisms for example, stained with different dyes.

What I like about this preprint

             I am a big supporter of open science initiatives in all senses. The work presented here makes it possible for many people to access the microscopic world. Moreover, having worked in field settings, I think the possibility of using this setup for screening and diagnostics has great potential, as it could potentially allow the integration of imaging with other tools currently implemented in mobile devices (such as geographical information systems used for epidemiology).

References

1. Liu et al, Pocket MUSE: an affordable, versatile and high performance fluorescence microscope using a smartphone, bioRxiv, 2020

 

 

doi: https://doi.org/10.1242/prelights.26115

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