We will cover the following topics:<\/strong><\/p>\n\n\n\n Written by Karen McGeachy<\/p>\n\n\n\n 10-15 minutes Read<\/p>\n<\/div>\n\n\n\n Due to the diffraction of light, traditional light microscopy is unable to resolve structural details less than ~200 nanometres in the lateral dimension and ~500 nm in the axial dimension. For over a century, this was considered a fundamental, unbreakable rule.<\/p>\n\n\n\n After decades of gradual improvements, revolutionary change has arrived with the advent of superresolution imaging. Now it\u2019s possible to see molecular structures as small as 10-20 nm in two dimensions. But this technology lacks axial resolution, failing to capture depth information.<\/p>\n\n\n\n With Double Helix Optics\u2019 Light Engineering technology, it is now possible to capture 3D images with nanoscale precision. By modifying the point spread function (PSF) of the optical system, users of the SPINDLE\u00ae and SPINDLE2 imaging modules are able to capture details as small as 10-15 nm in the lateral dimension and 15-25 nm in the axial dimension. This has sweeping implications for advancing scientific research, drug discovery, and industrial inspection.<\/p>\n\n\n\n PSF, as the name suggests, is the response of an imaging system to a point source. This function describes the \u2018spread\u2019 due to the nature of the optical system. The final image is the convolution of the object with the PSF [Fig 1].<\/p>\n\n\n\n An optical system with resolution performance at the instrument\u2019s theoretical limit is said to be diffraction-limited or Abbe Limit, which is proportional to the wavelength of light and the Numerical Aperture (NA). The NA can be thought of as the ability of a lens to collect light at oblique angles (n sin\u03b8) and can be as high as 1.4 \u2013 1.6. A nice rule of thumb is to half the wavelength, so around 200nm for visible light.<\/p>\n\n\n\n Resolution in microscopy has many different definitions but John William Strutt, 3rd Baron Rayleigh expanded on the work of George Airy and invented the \u2018Rayleigh Criterion\u2019 in 1896, which defines the diffracted limit as when the two point sources are distinguishable from each other.<\/p>\n\n\n\n As mentioned in the introduction, super resolution techniques (such as single molecule localisation microscopy) enable users to beat this criteria by temporally separating the two peaks and reporting on the peaks location, where the localisation precision is dependent on the photon flux.<\/p>\n\n\n\n Double Helix Optic\u2019s Light Engineering technology alters the response system of the PSF of a microscope to capture information that is out of focus with the standard Airy disk of the typical microscope. The image below [Fig.2] demonstrates the ability of the double helix PSF to stay in focus above and below the focal plane while the standard Airy disk and astigmatic PSF go out of focus.<\/p>\n\n\n\n In conjunction with the SPINDLE or SPINDLE\u00b2 modules, a phase mask inserted into the pupil plane modifies the PSF from the Airy disk of the microscope. With a library of e-PSFs available, performance is optimised by selection of the most appropriate phase mask.<\/p>\n\n\n\n The graphic below creates two lobes which rotate round a point, the location of the emitter is based on the centre point of the lobes and the axial position is based on the angle the two lobes make [Fig 3].<\/p>\n\n\n\n Depending on the objective in use, the double helix PSF offers up to 6 times the depth of a conventional microscope system<\/strong>. Additional masks offer up to 30x depth of a conventional microscope. This depth is achieved without the need for scanning the Z stage.<\/p>\n\n\n\n Double Helix Optics\u2019 award-winning SPINDLE family enables easy capture, analysis and tracking of multicolour 3D images down to the single molecule and single particle level as well as extended depth whole cell imaging.<\/p>\n\n\n\n Designed as modular upgrades compatible with most scientific microscopes, the SPINDLE product family seamlessly extend the capabilities to enable 3D superresolution and extended depth of field imaging and tracking.<\/p>\n\n\n\n The SPINDLE and SPINDLE2 modules can be added at the camera port of Sa microscope [Fig 4].<\/p>\n<\/div>\n\n\n\n Science is an ongoing quest to bring new insight. To date scientists using optical microscopy have been limited in their ability to see structures in 3D and with precision below the diffraction limit.<\/p>\n\n\n\n With the SPINDLE and SPINDLE2 modules researchers now have the ability to easily capture and analyse 3D images of cellular structures and intra and inter cellular activity down to the single molecule level<\/strong>.<\/p>\n\n\n\n Optimised to ensure the best performance from the library of phase masks to engineer the PSF [Fig 5], the SPINDLE and SPINDLE\u00b2 add 3D capabilities to your system matched to the precision -depth requirements of your experiment.<\/p>\n\n\n\n Features of the SPINDLE2<\/strong> – Multichannel imaging on a single camera<\/strong> <\/p>\n\n\n\n Like the SPINDLE, the SPINDLE\u00b2 attaches between your microscope and camera. Light from the microscope is split into two paths [Fig 6] by:<\/p>\n\n\n\n and then combined back onto the camera as side by side images.<\/p>\n\n\n\n Each channel can be PSF engineered or left in bypass mode. There are several advantages of imaging on a single camera as opposed to two cameras, an important one being the cost factor.<\/p>\n\n\n\n However, there is also the consideration that there is no need for synchronization between the cameras which can often be complex.<\/p>\n\n\n\n Simultaneous multi-colour imaging<\/strong><\/p>\n\n\n\n The SPINDLE\u00b2 allows for simultaneous multicolour imaging for simultaneous study of two tagged species. Utilizing your phase masks of choice for each channel you can track and analyze colocalization, utilize one channel for fiducial and the second channel for imaging, and track fiducials to merge multiple captures to extend depth range even further. With the multi-colour phase mask option, you can image up to 4 colours simultaneously<\/strong>.<\/p>\n\n\n\n Depending on the phase mask of choice users can realize increased depth range of up to 30x clear aperture, without the need to scan the Z stage. This capture of more data in a single image not only saves significant time performing experiments but also reduces phototoxicity of sample.<\/p>\n\n\n\n Multi modal<\/strong><\/p>\n\n\n\n With the SPINDLE2 users can chose between multiple imaging modalities including 3D particle tracking, 3D Single Molecule Localization Microscopy (SMLM), FRET, SOFI, widefield and more. For example, you can use chose to use a phase mask in one channel and polariser or even a second focus point lens in the second channel.<\/p>\n\n\n\n 3D and non 3D comparisons<\/strong><\/p>\n\n\n\n Another advantage of the SPINDLE\u00b2 is the ability to easily switch between two channels, single channel, multi-focus or by-pass mode for non-3D experiments. This is particular useful if the user would like to compare between 2D and 3D simultaneously<\/strong>.<\/p>\n\n\n\n The data obtained using the SPINDLE\u00ae and SPINDLE\u00b2 can be analysed using Double Helix Optics\u2019 3DTRAX\u00ae software [Fig 7] designed as an easy to use ImageJ\/FIJI plugin. You would capture the data using the same tools that you are familiar with on your microscope and then import them to 3DTRAX for processing and analysis.<\/p>\n\n\n\n Key features of the software:<\/p>\n\n\n\n 3D Single Molecule Localisation Microscopy<\/strong><\/p>\n\n\n\n Single Molecule Localisation Microscopy (SMLM) are a family of techniques, including STORM, PALM and DNA PAINT, which offer the highest resolution methods.<\/p>\n\n\n\n This family of methods tags biological samples by attaching synthetic dyes to target molecules. These dyes are able to enter a blinking state so that only a small number of them are \u201con\u201d during an image frame.<\/p>\n\n\n\n By collecting many frames, often thousands, you can reliably sample the underlying structure and identify the position of the single-molecule to a sub-diffraction localisation.<\/p>\n\n\n\n Adding the Double Helix Optics\u2019 SPINDLE\u00ae enables the recovery of up to 10x more depth information than in a 2D SMLM experiment<\/strong>.<\/p>\n\n\n\n In [Fig 8], you can see sharp, colour-encoded depth throughout the sample in contrast to 2D imaging, where much of this information is out of focus and lost, hiding the structure of the sample.<\/p>\n\n\n\n RHS shows 2D super-resolution image and LHS shows the same sample but with the SPINDLE\u00ae for 3D imaging.<\/p>\n<\/div>\n<\/div>\n\n\n\n SPINDLE\u00b2 expands on this by offering the same 3D information but with multi-colour capabilities [Fig 9].<\/p>\n\n\n\n The 3D nature of the reconstruction is particularly beneficial for these complex three dimensional networks.<\/p>\n\n\n\n Fig 8 & 9 – Images courtesy of Double Helix Optics.<\/p>\n<\/div>\n\n\n\n <\/p>\n<\/div>\n<\/div>\n\n\n\n Particle tracking<\/strong><\/p>\n\n\n\n Conventional 2D tracking requires axial stitching to extend the imaging volume and not lose particles to defocus. Utilising the SPINDLE\u00ae and SPINDLE\u00b2 in conjunction with Double Helix Optics\u2019 3DTRAX software, as the particle moves in the axial position the data is recorded in the angle that the lobes make. So now we can track these particles in 3D with remarkably high precision, enabling a much better reconstruction of the particle\u2019s overall trajectory [Fig 10].<\/p>\n\n\n\n For a more in depth look at how the SPINDLE and SPINDLE\u00b2 can be used for 3D particle tracking with examples such as liquidsolid surface diffusion, understanding cell signaling and diffusion and co-localisation, please see the Double Helix Optics Webinar – http:\/\/www.doublehelixoptics.com\/2020\/06\/02\/webinar-dholight-engineering-for-3d-single-molecule-tracking\/<\/a><\/p>\n<\/div>\n<\/div>\n\n\n\n Light sheet – 3D single-molecule super-resolution microscopy with a tilted light sheet<\/strong><\/p>\n\n\n\n Light Sheet microscopy is a growing field. Unlike traditional illumination methods a light sheet illuminates a thin section of the sample that is orthogonal to the detection path. The light sheet reduces background, increases signal-to-noise ratio and reduces photobleaching of the sample.<\/p>\n\n\n\n However conventional light sheet methods do not allow imaging with high-NA objectives close to the coverslip. In this application from the Moerner Lab at Stanford University, the team combined the powerful methods of light sheet and high-precision 3D SMLM, creating a technique known as \u201cTILT3D\u201d.<\/p>\n\n\n\n Combining light sheet with the Double Helix and Tetrapod PSFs they were able to image a complete HeLa cell with super-resolution precision<\/strong>.<\/p>\n<\/div>\n\n\n\n To do this they matched the thickness of the light sheet with the depth of focus of the Double Helix PSF. They then used a long-range Tetrapod PSF to track fiducials on the coverslip of the imaging chamber. By tracking the fiducials while imaging separate slices of the sample with the Double Helix PSF they were able to image and reconstruct structures throughout whole mammalian cells<\/strong>.<\/p>\n\n\n\n Gustavsson, A., Petrov, P.N., Lee, M.Y. et al. 3D single-molecule super-resolution microscopy with a tilted light sheet. Nat Commun 9, 123 (2018). https:\/\/doi.org\/10.1038\/s41467-017-02563-4<\/a><\/p>\n\n\n\n Multiphoton – Two-Photon PSF-engineering image scanning microscopy<\/strong><\/p>\n\n\n\n This application from the Piestun Lab at the University of Colorado and used a two-photon fluorescence image scanning microscope (ISM) in conjunction with engineered excitation and detection PSFs enabling 3D imaging in a single 2D scan.<\/p>\n\n\n\n Excitation was from a holographic multisport array of focused femtosecond pulses and the Single Helix PSF. This enabled volumetric imaging of biological samples over a depth of field spanning more than 1500nm and with an axial resolution of better than 400nm<\/p>\n\n\n\n Omer Tzang, Dan Feldkhun, Anurag Agrawal, Alexander Jesacher, and Rafael Piestun, “Two-photon PSF engineered image scanning microscopy,” Opt. Lett. 44, 895-898 (2019)<\/p>\n<\/div>\n\n\n\n Beyond life sciences<\/strong><\/p>\n\n\n\n Double Helix Optics Light Engineering technology extends beyond super-resolution microscopy and extended depth of field imaging for life sciences to applications including industrial inspection and machine vision. The principle of engineering the PSF can be applied to any imaging system by scaling the size of the phase mask to the imaging system. When applied to the field of machine vision for example, the approach offers several advantages over existing methods such as stereo-vision, structured illumination, time of flight, and light field:<\/p>\n\n\n\n For more information on how the technology can be used in machine vision please see the following article – https:\/\/www.photonics.com\/Articles\/Optical_Advancements_Enable_HighPrecision_3D\/a64430<\/a><\/p>\n\n\n\n For in depth look at applications see Double Helix Optics\u2019 page which includes a list of publications and specific application notes – http:\/\/www.doublehelixoptics.com\/applications\/<\/a><\/p>\n\n\n\n
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\n\n\n\n1. Introduction<\/h3>\n\n\n\n
\n\n\n\n2. Background on Point Spread Functions<\/h3>\n\n\n\n
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\n\n\n\n3. How does the Double Helix Optics\u2019 Light Engineering\u2122 technology work?<\/h3>\n\n\n\n
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\n\n\n\n4. The SPINDLE\u00ae Product Family \u2013 Key Benefits<\/h3>\n\n\n\n
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\n\n\n\n5. Software<\/h3>\n\n\n\n
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\n\n\n\n6. Applications<\/h3>\n\n\n\n
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