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VisiTech International’ VT-iSIM Imaging Beyond all Limits
• 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:• 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:• 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.
Introduction to VT-iSIM
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
• If we consider displacing the detection PH by a distance X (in regard to the optical axis), then as the is a product of the and , it would be shifted but narrower.
• As the overlap decreases with increased displacement the width of 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 at different displacements would give you a blurred image, you must first shift each 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.
Introduction to VT-iSIM
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
X
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
X
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
X
X/2
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
X
• 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.
Introduction to VT-iSIM
𝑃𝑆𝐹 𝑖𝑙𝑙
𝑃𝑆𝐹 𝑑𝑒𝑡
𝑃𝑆𝐹 𝑒𝑓𝑓
X
X/2
Galvo Scanner
Fibre Input
SampleScan Lens
Mirror
0.5x FL
1x FL
0.5x Mag
VT-iSIM Optical LayoutIllumination u-Lens Array
Beam Expanding Optics
Illumination u-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Fibre Input
SampleScan Lens
Mirror
Mirror
MirrorMirror
Mirror
1x FL
VT-iSIM Optical LayoutIllumination Pin Hole Array
Beam Expanding Optics
Illumination u-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Fibre Input
SampleScan Lens
Mirror
Mirror
MirrorMirror
VT-iSIM Optical Layout2-D Array Scanning
Beam Expanding Optics
Illumination u-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Fibre Input
SampleScan Lens
Mirror
Mirror
MirrorMirror
VT-iSIM Optical LayoutEmission Pin Hole Array
Beam Expanding Optics
Illumination u-lens Array
Emissionu-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Emission Filter
Fibre Input
SampleScan Lens Scan Lens
Mirror
Mirror Mirror
Mirror
MirrorMirror
Mirror
VT-iSIM Optical LayoutEmission u-Lens Array
Beam Expanding Optics
Illumination u-lens Array
Emissionu-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Emission Filter
Fibre Input
SampleScan Lens Scan Lens
Mirror
Mirror Mirror
Mirror
MirrorMirror
Mirror
0.5x FL
1x FL
VT-iSIM Optical LayoutCamera Detection
Beam Expanding Optics
Illumination u-lens Array
Emissionu-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Emission Filter
Fibre Input
SampleCamera Scan Lens Scan Lens
Mirror
Mirror Mirror
Mirror
MirrorMirror
Mirror
VT-iSIM Optical LayoutAdditional Features
Beam Expanding Optics
Illumination u-lens Array
Emissionu-lens Array
Variable Pin Hole Plate
Galvo Scanner
DichroicMirror
Emission Filter
Fibre Input
SampleCamera Scan Lens Scan Lens
Mirror
Mirror Mirror
Mirror
MirrorMirror
Mirror
Optional adjustable u-lens array
Optional 3-position automated dichroic changer
Optional variable Pin Hole Plate (10-64um)
Scan speeds up to 1,000Hz (Full Frame)
VisiTech’ VT-LMM Laser Engine available with choice of 405, 445 ,488 ,514, 532, 561, 642nm Lasers
Optional 6-position regular and high speed emission filter wheel
Optional In/Out u-lens array
Can be used with any research microscope
Can be used with any research camera*
* Note pixel size of 6.5um or lower is recommended for spatial sampling.
Bright field by-pass and FRAP add-ons are also available
• 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 1.45NAMicroscope 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
VT-iSIM Specifications
* Spatial resolution quoted for fully integrated system and 100x 1.45NA Lens, see VTi for more options
Thank You!
