Direct single-molecule detection and super-resolution imaging with a low-cost portable smartphone-based microscope

Between the years 2008-2018, ‘light’ has taken the center stage in the Nobel Prizes. It has won the prestigious award in 2008 (Chemistry) for the discovery of GFP and its application in understanding various biochemical processes, in 2014 (Chemistry) for the ground-breaking work in super-resolution and in 2018 (Physics) for the invention of optical tweezers. Although these techniques are important for all scientists across disciplines, somehow these discoveries fail to reach the undergraduate classrooms because of their high cost and the requirement of specific expertise. This is where our labcoat-wearing-heroes swoop in with their low-cost inventions to make the latest scientific discoveries accessible to all. One such invention is the work by Loretan and colleagues, as part of which they made a small, portable, low-cost smartphone-based microscopic device that can perform Single Molecule Localization Microscopy (SMLM).

Optical super-resolution can be achieved in mainly two ways- 1) Beam scanning, where the laser scans through the sample using a donut shaped beam (STED, MINFLUX) and 2) Camera based SMLM, in which long videos of blinking fluorophores in the sample are recorded (STORM, PALM, PAINT). These microscopic techniques can be commercially purchased at a price that is generally unaffordable for undergraduate colleges or can be built at home which requires a lot of time and expertise. Once set up, the microscopes cannot be moved around easily. However, the small microscope made by Loretan and team has dimensions less than airline carry-on luggage, weighs almost as much as a duty-free bottle of wine and costs less than a one-way trans-Atlantic flight. The authors assembled the microscope, such that the laser excites the fluorophores in the sample in a total internal reflection (TIR) or highly inclined and laminated optical sheet (HiLo) mode. After passing through the sample, an emission filter is used to filter out the unwanted wavelengths from the signal. The emission is collected by the smartphone camera. Then, the data is processed using the raw RGB data from the recorded videos, which allows the user to choose the correct detection channel for the emission photons. The sample stage and the objective holder can be moved using screws to adjust the focus.

Key results

Smartphone-based microscope can detect single molecules– Instead of being specific to just one smartphone, the authors could demonstrate the compatibility of their custom-made microscope with any smartphone equipped with a camera. To test the resolution and efficiency of the microscope, they used standard DNA origami structures labelled with two dyes within a short distance. One of the dyes is suitable for imaging using a high-end super-resolution microscope while the other can be imaged using the new smartphone-based microscope. The authors concluded that the point-spread-function of the detected photons on the smartphone-based microscope is broader than that of the high-end microscope.

Super-resolution benchmark with DNA origami model– As mentioned above, SMLM is a super-resolution technique that requires the detection of fluorescence from single molecules with nanometer resolutions. Since this was achieved in the authors’ previous results, the authors then used DNA-PAINT to image standard DNA origami structures. DNA-PAINT causes the fluorescent molecules to ‘blink’ by constant association and dissociation between two DNA strands (docking strand, immobilized on the sample and imaging strand, floating in the solution). The smartphone-based microscope records long videos as image sequences of the ‘blinking’ in the sample. After processing these image sequences, the resulting images agree well with those taken using a high-end microscope.

Imaging microtubules in a cell– Finally, the authors imaged microtubules in fixed cells using the new device and a high-end microscope. The resolution of the microtubules obtained on the smartphone-based microscope was less than the resolution of the high-end microscope. Even with the lower resolution, the image from the smartphone-based tool was impressively better than the diffraction-limited images.

Why I like this preprint

I like this work due to its simplicity and efficiency (cost, experience and technique). The described imaging tool can be easily used in schools, museums and other education facilities. The components of the smartphone-based microscope are easily available, and the use of the tool requires minimal experience in optics. A brief tutorial and short training session enables anyone to beat the limits set by diffraction. The simple assembly of this device can be used as a teaching tool or opportunity for young students studying microscopy or photonics. Such a microscope-building exercise adds to the classroom teaching experience and could lay the foundation for developing an interest in microscopy. The authors have already established the efficiency and resolution of the new device by imaging in DNA origami models and cellular systems, so this lightweight microscope can be used to conduct field studies at locations where there is no access to high-end microscopes.

 

Questions for the authors

• Once the microscope has been assembled (let’s suppose in an undergrad college), is it possible for each student to get their own smartphone and record super-resolution images without much readjustment to the microscope?
• Does the ‘zoom’ feature on the smartphone camera affect the imaging or the final image quality?

 

doi: https://doi.org/10.1242/prelights.38068

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