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High-throughput fluorescence microscopy techniques with a sufficiently high spatial and temporal resolution are a necessary tool for the comprehensive 3D visualization of complex systems such as biological cells and organisms.
A newly-developed imaging technique called Coded Light-Sheet Array Microscopy (CLAM) improves upon existing 3D imaging approaches by using a simple yet ingenious optical setup that permits simultaneous illumination of the entire sample volume imaged by the microscope. This will allow scientists to follow long-term dynamic processes with minimal photodamage to the sample.
Some of the main limitations of the existing fluorescence imaging techniques are the induced phototoxicity and photobleaching of the sample. These occur when organic molecules naturally present in the biological samples or fluorophores are introduced to the samples (as fluorescent probes), absorb the excitation light, and become damaged once they react with oxygen or other reactive molecules.
How to Minimize the Damage of Biological Specimens During Long-Term Imaging?
These limitations become particularly relevant when following dynamic biological processes over long periods and with a high spatial and temporal resolution (live specimen imaging), where it is critical to ensure that the effects of the imaging on the sample are minimal and well understood.
Light-sheet fluorescence microscopy (LSFM) overcomes many drawbacks associated with sample photodamage by illuminating the specimen from the side with a thin light sheet of light that overlaps with the focal plane of the detection lens placed orthogonally to the illumination lens.
3D Imaging and Optical Sectioning by Selective Sample Excitation
This arrangement only permits excitement of the relevant part of the sample that is being imaged, unlike the epifluorescence confocal systems that use the same lens to illuminate the whole volume of the sample and collect signal only from the lens' focal plane.
The intrinsic properties of LSFM make the technique well-suited to 3D imaging by scanning the sample (by mechanical or optomechanical means) through the illuminating light sheet. This ensures minimal phototoxic damage or photobleaching outside a very small sample volume close to the focal plane that is excited by the light sheet.
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Sequential Scanning Limits the Throughput
The sample scanning, however, comes at the expense of imaging speed and throughput, since the entire sample volume needs to be sequentially scanned and the scanning speed is limited by the mechanical response of the microscope components.
A research team led by Dr. Kevin Tsia at the Department of Electrical and Electronic Engineering, University of Hong Kong (HKU), has taken LSFM a couple of steps further and developed CLAM - a very high-throughput imaging technique capable of fast 3D visualization of complex and dynamic biological specimens without any scanning mechanism.
Parallel Light-Sheet Array Illumination with Tuneable Density and Intensity
At the core of the new technique is the concept of highly parallelized sample illumination and signal acquisition. CLAM uses a pair of near-parallel mirrors (or an 'infinity mirror') that, after several reflections, convert an incident laser beam into many virtual beams, which are then shaped into a high-density array of 30 to 40 light-sheets, spreading the fluorescence excitation over the entire volume of the specimen.
A distinct feature of CLAM is the ability to control the spatial density and coherence of the light-sheet array by tuning the mirror separation and tilt angle. Importantly, the adjustable degree of coherence of the light-sheets can minimize the imaging artifacts due to speckle generation, especially in highly-scattering biological samples.
Telecommunications Know-How Helps to Keep Track of Each Light-Sheet
To take full advantage of the light-sheet array illumination of the sample, the research team had to devise a method enabling simultaneous detection of the fluorescence signal generated from each individual light-sheet.
The scientists got their inspiration from a frequency modulation technique called Orthogonal Frequency Division Multiplexing (OFDM) which is widely used in telecommunication for sending multiple signals simultaneously. By modulating the light intensity of each individual light-sheet at a particular frequency, by means of a fast-spinning patterned reticle, OFDM tags each light-sheet with a unique temporal 'code'.
This, in turn, 'codes' the fluorescence emission from the individual light-sheets (illuminating 2D cross-sections at different depths within the sample volume) and allows all the data to be multiplexed into a single 2D frame and simultaneously registered on a conventional image sensor.
Multiplexing Increases the Readout Speed and Minimizes Photodamage
The frequency encoding permits the data to be digitally reconstructed into a 3D image of the sample. The multiplexing nature of CLAM significantly improves the technique's sensitivity as the voxels (or volumetric pixels) in the imaging volume are recorded in parallel, hence maximizing the readout time without compromising the imaging rate.
This reduces the required illumination intensity and consequently the photobleaching and photodamage of the sample, which is a critical requirement for the preservation of biological specimens during long-term data acquisition.
As a proof-of-principle demonstration, CLAM was capable of imaging rapidly moving fluorescence microbeads within a microfluidic device (flow rate of 20 μm s-1) at a volumetric imaging rate of up to 13 volumes per second, which is comparable to current state-of-the-art imaging technology.
Fast 3D Imaging of Challenging Biological Specimens
The real strength of CLAM, however, became evident when the technique was applied to a 'real world' problem - imaging of highly-scattering specimens such as thick biological tissues and whole organisms.
Optically cleared mouse kidney and intestine tissues (treated to equalize the refractive index throughout the specimen without altering its anatomical structure) were imaged at a volumetric imaging rate of over 6 volumes per second. This revealed in great detail the finest network of capillaries in the kidneys, known as glomeruli, as well as the intestine blood vasculature.
The research team behind the new technique anticipate a wide range of potential applications of CLAM - its excellent adaptability, high throughput, and low impact in terms of photodamage/phototoxicity enable long-term dynamic imaging of live cells, tissues, and organisms, as well as a rapid examination of a large number of biological samples.
References and Further Reading
Y.-X. Ren et al., (2020) Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array. Light: Science and Applications, 9, 8. Available at: https://doi.org/10.1038/s41377-020-0245-8
P. Loza-Alvarez, (2020) Parallel array with axially coded light-sheet microscope. Light: Science and Applications, 9, 65. Available at: https://doi.org/10.1038/s41377-020-0310-3
E. Reynaud et al., (2015) Guide to light-sheet microscopy for adventurous biologists. Nature Methods, 12, 30 – 34. Available at https://doi.org/10.1038/nmeth.3222
V. Rees (2020) Novel 3D imaging technology could improve fluorescence microscopy, [Online] www.drugtargetreview.com Available at: https://www.drugtargetreview.com/news/60118/novel-3d-imaging-technology-could-improve-fluorescence-microscopy (Accessed on 20 May 2020).