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  Principles of Operation


Principles of Operation Figure 1: Basic confocal microscopy set-up


Confocal microscopy - Principles

Confocal microscopy has been invented by Marvin Minsky in 1955 at Harvard University.

In conventional microscopy the whole sample is illuminated and, as a result, the received light at the detector contains not only the light from the focal plane of the microscope objective, but also other planes in the body of the sample.

Minsky used a pair of pinholes, one in front of the source to illuminate the object point by point and the other in front of the detector to receive only the focused light from the focal plane, effectively blocking the out of focus light.

This technique resulted in increased resolution and reduction in blur and significantly improved the observation power of a microscope. Figure 1 shows the schematic diagram for a basic confocal microscopy system.

In this set up, the source light passes through a pinhole and a beam splitter and is focused by a lens on a sample. The reflected light from the focal plane (Shown in grey), and also other planes (shown in blue), travel backwards to the beam splitter and get reflected towards the detector. However, a second pinhole placed at the conjugate focal plane of the objective lens (hence the name confocal) blocks the grey beam which is from an out-of-focus plane and only allows the focal plane beam to pass through. The resolution of the microscope therefore significantly increases. Due to reduction in intensity of the received light, more exposure time will be required. Since the illumination mechanism is point by point the whole image from a local plane can be constructed either if the sample is moved or if optical beam is moved over the sample.

  The Confocal Spinning Disk

Confocal Spinning Disk

Fig. 1



Here two different approaches to confocal microscopy are shown.

This panel shows how the focused light is applied to the specimen by either laser raster scanning (Fig. 1) or through a Nipkow (Fig.2, 2A) disk system such as the X-LIGHT Confocal System.

The sample is illuminated simultaneously with > 2300 discrete points of light over a circular area 15 mm in diameter.
A 1⁄2 inch CCD camera at 60X magnification covers an area 150 µm by 100 µm.

The CrEST X-LIGHTconfocal imager utilizes a Proprietary spinning disk design containing multiple sets of spirally arranged pinholes placed in the image plane of the objective lens.

The column of excitation light is projected through 1000 pinholes to simultaneously scan the entire field six times every millisecond, thereby creating a full image of the focal plane in realtime with no striping effect.

Emitted light is collected and imaged using either a high resolution and high quantum efficiency CCD camera, or a fast sCMOS device.

The variable intensity light from a Laser and/or an LED light source is reflected by a dichroic mirror towards the sample.

The emitted light passes through the dichroic mirror and emission filter before entering the CCD camera.
The Spinning disk can be moved in and out of the light path to produce a confocal or a wide field fluorescence image.

All movable parts including the filter wheels, spinning disk shutters, and mirrors are automated and are controlled via touchpad or 3rd-party software.


Confocal Spinning Disk

Fig. 2A


Confocal Spinning Disk

Fig. 2

  Principles of Operation with VCS ( Video Confocal SuperResolution)


Video-Confocal Super-resolution Microscopy (VCS) is based on narrow-field illumination using scanning patterns and wide-field collection of raw images.

Detection algorithms [1] already developed are able to super-resolve 3D structures in both compact and sparse specimens.

Although other techniques proposed and industrially developed mainly dedicate their efforts in extracting information from a relatively low spatial frequency range, VCM detection methods harness non-linear calculations exploiting the tops more than the belly of the raw signal intensity, collected as a function of illumination and detection positional parameters.

Figure 3 shows the schematic diagram for a basic VCS microscopy system.
In this setup, the light source is focused onto a specified pattern that is optically conjugated to the microscope conjugate focal plane through a relay system. The mask is moved in the optical path with an x-y piezo motor system at each camera acquisition. The emission is collected widefield.
Calculations are performed in parallel during images acquisitions through CUDA technology exploiting Gpu processors.


Spinning Disk with VCS


Fig. 3



Figure 4 shows a typical illumination pattern projected onto the sample


typical illumination pattern projected onto the sample

Fig. 4


[1] - “Improved confocal microscopy methods and devices” patent WO2013144891 (2013)

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