Cost-Effective Live Cell Structured Illumination Microscopy with Video-Rate Imaging

Abstract

Optical nanoscopy is rapidly gaining momentum in the life sciences. Current instruments are, however, often large and expensive, and there is a substantial delay between raw data collection and super-resolved image display. Here, we describe the implementation of a compact, cost-effective, high-speed, structured illumination microscope (SIM), which allows for video-rate superresolved image reconstructions at imaging rates up to 60 Hz. The instrument is based on a digital micromirror device (DMD) and a global-shutter camera, which enables faster pattern cycles and higher duty cycles than commonly used liquid crystal-based spatial light modulators. In order to utilize a DMD for creating illumination patterns by the coherent superposition of laser beams, we carefully studied its blazed grating effect Through both simulation and experimental determination of system parameters, we identified and optimized its alignment for optimal SIM pattern contrast. Raw image data are collected using inexpensive industry-grade CMOS cameras, while a parallel-computing platform allowed us to reconstruct and visualize living cells in real time. We demonstrate the performance of this system by imaging submicron-sized fluorescent beads diffusing in an aqueous solution, resolving bead−bead interactions in real time. We show that the system is sensitive enough to image intracellular vesicles labeled with fluorescent proteins in fixed cells. We also image dynamic fluctuations of the endoplasmic reticulum (ER), as well as the movement of mitochondria in living osteosarcoma cells, where the cellular organelles are labeled with live cell fluorescent stains.

Cost-effective high-speed, three-dimensional live-cell imaging of HIV-1 transfer at the T cell virological synapse

“…The system resembles an open-frame wide-field fluorescence microscopy setup that is specifically optimized to permit fast 3D image acquisition. Such compact microscope systems can now be realized by utilizing uncooled industry-grade cameras (IDS μEye UI-3060CP-M, IDS, Germany), which have been demonstrated to achieve single molecule fluorescence detection sensitivity, 14 , 15 , 24 , 25 while substantially reducing the overall cost of the system. The microscope itself consists of two stacked aluminum optics breadboards, which further decreases the overall footprint of the system so that it can be easily shipped (see Figure 1 A).…”

“… 36 , 37 We have recently made a number of developments toward speeding up the SR-SIM imaging process, while still allowing for instant display of the super-resolved image data, as well as toward the development of more compact and cost effective realizations of SR-SIM. 25 , 37 In SR-SIM, a low single to double digit number of raw images is acquired, while the sample is illuminated with a diffraction-limited (stripe-like) interference pattern. Computational image reconstruction then results in roughly a doubling of the spatial resolution in x, y, and z.…”

An LED-Based structured illumination microscope using a digital micromirror device and GPU accelerated image reconstruction

“…A common and cost-effective alternative for illumination pattern generation is the digital micromirror device (DMD) technology. Laser sources have been frequently used with DMDs for generation of high-contrast illumination patterns in SIM setups [16,17]. Due to its periodic surface, the DMD creates a blazed grating effect and generates multiple diffractive orders.…”

Miniaturised structured illumination microscopy using two 3-axis MEMS micromirrors

“…However, the mechanical manipulation process generates a limit on the achievable temporal resolutions [13,15] from the system. The second method uses spatial light modulators (SLM) or digital micromirror devices (DMD) to create tailored spatial excitation patterns in the back-aperture of a microscope objective, thereby generating the required interference patterns in the sample [8,15,[17][18][19].…”

“…The DMD approach to carrying out SIM however has some limitations, For instance, the “on” and “off” positions of the individual mirror pixels create a sawtooth-like surface which requires the DMD to be treated as a blazed grating. To compensate for this blazed grating effect, the illumination must arrive on the DMD at a wavelength-dependent incident angle to achieve the greatest excitation grating efficiency by equalizing the intensity between the diffraction orders which, if not carried out will be detrimental to the grating contrast obtained [18,19,21]. Additionally, the ensuing requirement to build the microscope around this incident wavelength defined blazed angle means that multi-colour imaging is difficult to implement.…”