VisiTech International’ VT-iSIM Imaging Beyond all Limits

Introduction to VT-iSIM The optical resolution of a confocal microscope to a point source emitter is the product of the illumination and detection PSF’s as per equation 1. Equation 1: 𝑃𝑆𝐹 𝑐𝑜𝑛𝑓 = 𝑃𝑆𝐹 𝑖𝑙𝑙 ∙ 𝑃𝑆𝐹 𝑑𝑒𝑡 The detection PSF of a confocal microscope is equal to the emission PSF convolved with the pin hole, we can therefore expand equation 1 as shown below in equation 2. Equation 2: 𝑃𝑆𝐹 𝑐𝑜𝑛𝑓 = 𝑃𝑆𝐹 𝑒𝑥𝑐 ∙ (𝑃𝑆𝐹 𝑒𝑚 ⨂𝑃𝐻(𝑑)) So as per equation 2 you can see that by setting the pin hole to be infinitely small we should get the best resolution as the effective PSF would just be the product of the excitation and emission PSF’s. In such a hypothetical case the resolution enhancement is √2. However this is in-practical as an infinitely small pin hole would prevent any light reaching the detector. In Practice a pin hole size >1AU is used thus offering improved sectioning ability and axial resolution but limited or no improvements in lateral resolution. 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓

Introduction to VT-iSIM 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓 X 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓 X X/2 If we consider displacing the detection PH by a distance X (in regard to the optical axis), then as the 𝑃𝑆𝐹 𝑒𝑓𝑓 is a product of the 𝑃𝑆𝐹 𝑖𝑙𝑙 and 𝑃𝑆𝐹 𝑑𝑒𝑡 , it would be shifted but narrower. As the overlap decreases with increased displacement the width of 𝑃𝑆𝐹 𝑒𝑓𝑓 decreases, and if an emitter is imaged through the displaced PH the likelihood that is that it will be more precisely localised increases. Therefore as the displacement increases higher frequencies become more pronounced and their proportion rises. The highest probability of the emitters location is within the narrow overlap between illumination and detection PSF’ and hence it can be localised with more precision. However, simply summing multiple 𝑃𝑆𝐹 𝑒𝑓𝑓 at different displacements would give you a blurred image, you must first shift each 𝑃𝑆𝐹 𝑒𝑓𝑓 before summing. Since a PH displaced by X collects an image displaced by X/2 you can shift the signal back to where it belongs. Thus in turn, summing all the signals from all the back shifted PH positions which yields a Gaussian function with a width reduced by a factor of √2. 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓 X 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓 X

Introduction to VT-iSIM This process has been traditionally called “pixel reassignment” and is usually done via post imaging computation. However, with VT-iSIM this is done in real time without any computation, how? It’s quite simple, since a PH displaced by X collects an image displaced by X/2, shrink the image of each PH by a factor of 2 towards the centre of the PH. In VT-iSIM this correction is implemented by using a u-lens to “shrink” the pin holed image by a factor of 2 before they reach the detection camera; no interpolation is required, due to the analogue nature of reality. A super resolution image is therefore generated in real time on the detector with enhanced spatial resolution of √2. In addition the significant increase in high frequency content, as detailed previously, enables simple deconvolution to further enhance spatial resolution a full factor of 2 compared to wide field microscopy. Details of how this technique has been implemented in VT-iSIM is shown on the next set of slides. 0.5x Mag 𝑃𝑆𝐹 𝑖𝑙𝑙 𝑃𝑆𝐹 𝑑𝑒𝑡 𝑃𝑆𝐹 𝑒𝑓𝑓 X X/2 Galvo Scanner Fibre Input Sample Scan Lens Mirror 0.5x FL 1x FL

VT-iSIM Optical Layout Illumination u-Lens Array Mirror Fibre Input Beam Expanding Optics Galvo Scanner Scan Lens Sample 1x FL Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror Mirror

VT-iSIM Optical Layout Illumination Pin Hole Array Mirror Fibre Input Beam Expanding Optics Galvo Scanner Scan Lens Sample Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror

VT-iSIM Optical Layout 2-D Array Scanning Mirror Fibre Input Beam Expanding Optics Galvo Scanner Scan Lens Sample Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror

VT-iSIM Optical Layout Emission Pin Hole Array Mirror Fibre Input Beam Expanding Optics Mirror Mirror Emission u-lens Array Emission Filter Galvo Scanner Scan Lens Scan Lens Sample Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror Mirror

VT-iSIM Optical Layout Emission u-Lens Array Mirror Fibre Input Beam Expanding Optics Mirror Mirror Emission u-lens Array Emission Filter Galvo Scanner Scan Lens Scan Lens Sample 0.5x FL 1x FL Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror Mirror

VT-iSIM Optical Layout Camera Detection Mirror Fibre Input Beam Expanding Optics Mirror Mirror Emission u-lens Array Emission Filter Galvo Scanner Camera Scan Lens Scan Lens Sample Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Mirror Mirror Mirror Mirror

VT-iSIM Optical Layout Additional Features Mirror Fibre Input Beam Expanding Optics Mirror VisiTech’ VT-LMM Laser Engine available with choice of 405, 445 ,488 ,514, 532, 561, 642nm Lasers Optional In/Out u-lens array Mirror Emission u-lens Array Can be used with any research camera* Scan speeds up to 1,000Hz (Full Frame) Emission Filter Galvo Scanner Camera Scan Lens Scan Lens Sample Optional 6-position regular and high speed emission filter wheel Can be used with any research microscope Optional variable Pin Hole Plate (10-64um) Optional adjustable u-lens array Variable Pin Hole Plate Dichroic Mirror Illumination u-lens Array Bright field by-pass and FRAP add-ons are also available Mirror Mirror Optional 3-position automated dichroic changer Mirror Mirror * Note pixel size of 6.5um or lower is recommended for spatial sampling.

VT-iSIM Specifications Spatial Resolution: Up to 125nm Laterally and 350nm Axially* Temporal Resolution: Scan Speed up to 1000fps, full frame With Hamamatsu sCMOS camera, achievable capture rates are: 200fps @ 1024x1024, 400fps @ 1024x512, 800fps @ 1024x256 Pin Holes: Selectable from 10-64um Dichroic Changer: Automated 3-Position Dichroic Changer Emission Filter Changer: Regular 6-Position Emission Filter Changer or high speed (<50mS) 6-Position Filter Changer available Excitation: Up to six solid state lasers selectable from within the visible range Illumination intensity and laser line selection controlled via software FRAP: Fully integrated FRAP add-on available and utilises existing lasers BF by-pass: BF by-pass mode available enabling WF imaging onto same camera Sync: Perfect camera sync comes as standard Camera Specification: For accurate sampling camera must have pixel size <6.5um Camera connection is via regular c-mount Microscope Specification: For quoted resolution numbers high NA high magnification lens must be used, i.e. 100x 1.45NA Microscope connection is via regular c-mount Software: System supplied with VisiTech International VoxCell Scan Acquisition software but can also be supplied with MM and NIS Elements * Spatial resolution quoted for fully integrated system and 100x 1.45NA Lens, see VTi for more options

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