Transcript Theory of instant SIM - VisiTech International

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:
𝑃𝑆𝐹𝑐𝑜𝑛𝑓 = 𝑃𝑆𝐹𝑖𝑙𝑙 ∙ 𝑃𝑆𝐹𝑑𝑒𝑡
• 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:
𝑃𝑆𝐹𝑐𝑜𝑛𝑓 = 𝑃𝑆𝐹𝑒𝑥𝑐 ∙ (𝑃𝑆𝐹𝑒𝑚 ⨂𝑃𝐻(𝑑))
• So as per equation 2 you can see that by setting the pin hole to be infinitely small we
𝑃𝑆𝐹𝑖𝑙𝑙
should get the best resolution as the effective PSF would just be the product of the
excitation and emission PSF’s.
𝑃𝑆𝐹𝑑𝑒𝑡
𝑃𝑆𝐹𝑒𝑓𝑓
• 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.
X
Introduction to VT-iSIM
𝑃𝑆𝐹𝑖𝑙𝑙
X displacing the
• If we consider
detection PH by a distance X (in regard to the optical
𝑃𝑆𝐹
axis), then as the 𝑃𝑆𝐹𝑒𝑓𝑓 is
a𝑑𝑒𝑡product of the 𝑃𝑆𝐹𝑖𝑙𝑙 and 𝑃𝑆𝐹𝑑𝑒𝑡 , it would be shifted
X/2
𝑃𝑆𝐹𝑒𝑓𝑓
but narrower.
X
• As the overlap decreases with increased displacement the width of
𝑃𝑆𝐹𝑒𝑓𝑓 decreases, and if an
is imaged through the displaced PH the
𝑃𝑆𝐹emitter
𝑖𝑙𝑙
likelihood that is that it will
be
𝑃𝑆𝐹
𝑑𝑒𝑡 more precisely localised increases.
X more
• Therefore as the displacement
𝑃𝑆𝐹𝑒𝑓𝑓 increases higher frequencies become
𝑃𝑆𝐹𝑖𝑙𝑙
pronounced and their proportion rises.
𝑃𝑆𝐹𝑑𝑒𝑡
• The highest probability of the emitters location is within the narrow overlap between
𝑃𝑆𝐹
illumination and detection PSF’ and hence it can be localised with more precision. 𝑒𝑓𝑓
𝑖𝑙𝑙
• However, simply summing multiple 𝑃𝑆𝐹𝑒𝑓𝑓 at different displacements would give𝑃𝑆𝐹
you
𝑃𝑆𝐹𝑑𝑒𝑡
a blurred image, you must first shift each 𝑃𝑆𝐹𝑒𝑓𝑓 before summing.
𝑃𝑆𝐹𝑒𝑓𝑓
• 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
• 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
X 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
0.5x
X/2due to the analogue nature of reality.
required,
Mag
• A super resolution image is therefore generated in real time on the detector
with
Fibre
Input
enhanced spatial resolution
of √2.
𝑃𝑆𝐹𝑖𝑙𝑙
• In addition the significant 𝑃𝑆𝐹
increase
in high frequency content, as detailed previously,
𝑑𝑒𝑡
enables simple deconvolution
to further enhance spatial resolution a full factor of 2
𝑃𝑆𝐹𝑒𝑓𝑓
compared to wide field microscopy.
• Details of how this technique has been implemented in VT-iSIM is shown on the next
set of slides.
Galvo
Scan Lens
Scanner 0.5x FL
1x FL
Sample
VT-iSIM Optical Layout
Illumination u-Lens Array
Fibre
Input
Beam
Expanding
Optics
Galvo
Scanner
1x FL
Illumination
u-lens Array
Scan Lens
Variable Pin
Hole Plate
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
Illumination Pin Hole Array
Fibre
Input
Beam
Expanding
Optics
Galvo
Scanner
Scan Lens
Variable Pin
Hole Plate
Illumination
u-lens Array
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
2-D Array Scanning
Fibre
Input
Beam
Expanding
Optics
Galvo
Scanner
Scan Lens
Variable Pin
Hole Plate
Illumination
u-lens Array
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
Emission Pin Hole Array
Fibre
Input
Beam
Expanding
Optics
Emission
u-lens Array
Emission
Filter
Scan Lens
Galvo
Scanner
Scan Lens
Variable Pin
Hole Plate
Illumination
u-lens Array
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
Emission u-Lens Array
Fibre
Input
Beam
Expanding
Optics
Emission
u-lens Array
Emission
Filter
Scan Lens
Galvo
Scan Lens
Scanner 0.5x FL
1x FL
Variable Pin
Hole Plate
Illumination
u-lens Array
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
Camera Detection
Fibre
Input
Beam
Expanding
Optics
Emission
u-lens Array
Camera
Emission
Filter
Scan Lens
Galvo
Scanner
Scan Lens
Variable Pin
Hole Plate
Illumination
u-lens Array
Dichroic
Mirror
Sample
VT-iSIM Optical Layout
Additional Features
Fibre
Input
Beam
Expanding
Optics
Emission
u-lens Array
Can be used with any
research camera*
Emission
Filter
Camera
Optional
adjustable
u-lens array
Illumination
u-lens Array
VisiTech’ VT-LMM Laser Engine
available with choice of 405, 445
,488 ,514, 532, 561, 642nm Lasers
Optional
In/Out u-lens
array
Optional 6-position
regular and high speed
emission filter wheel
Scan speeds up to
1,000Hz (Full Frame)
Scan Lens
Galvo
Scanner
Scan Lens
Sample
Can be used with any
research microscope
Optional variable Pin
Hole Plate (10-64um)
Variable Pin
Hole Plate
Dichroic
Mirror
Bright field by-pass
and FRAP add-ons
are also available
Optional 3-position
automated dichroic
changer
* Note pixel size of 6.5um or lower is
recommended for spatial sampling.
VT-iSIM Specifications
•
•
Spatial Resolution:
Temporal Resolution:
•
•
•
Pin Holes:
Dichroic Changer:
Emission Filter Changer:
•
Excitation:
•
•
•
•
FRAP:
BF by-pass:
Sync:
Camera Specification:
•
Microscope Specification:
•
Software:
Up to 125nm Laterally and 350nm Axially*
Scan Speed up to 1000fps, full frame
With Hamamatsu sCMOS camera, achievable capture rates are:
200fps @ 1024x1024, 400fps @ 1024x512, 800fps @ 1024x256
Selectable from 10-64um
Automated 3-Position Dichroic Changer
Regular 6-Position Emission Filter Changer or high speed (<50mS)
6-Position Filter Changer available
Up to six solid state lasers selectable from within the visible range
Illumination intensity and laser line selection controlled via software
Fully integrated FRAP add-on available and utilises existing lasers
BF by-pass mode available enabling WF imaging onto same camera
Perfect camera sync comes as standard
For accurate sampling camera must have pixel size <6.5um
Camera connection is via regular c-mount
For quoted resolution numbers high NA high magnification lens must be
used, i.e. 100x 1.45NA
Microscope connection is via regular c-mount
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
Thank You!