Aurox Clarity Laser Free Confocal (Aurox LFC)

Author: Mathias Pasche-Drews, Field Applications Specialist, Teledyne Photometrics

Author: Dan Croucher, Applications Team Manager, Teledyne Photometrics

Introduction

One of the most popular confocal microscopy techniques is spinning disk confocal microscopy; a high-speed, highsensitivity technique that is reasonably simple to implement. Conventional spinning disk confocal microscopy uses a dual disk strategy which focuses excitation light through microlenses on the first disk into the pinholes of the second disk to increase acquisition speed and the amount of light reaching the sample. Emission light then passes back through the second disk and onto the camera.

The Aurox Clarity Laser Free Confocal (Aurox LFC) is a system which combines features of Spinning Disk microscopy and Structured Illumination (SI) to provide an affordable confocal microscope that can be attached to any conventional widefield system.

The LFC effectively captures two images originating from the same plane of focus, one transmitted through the disk and one reflected by the disk. When these images are subtracted from one other (transmitted – reflected), a sectioned image is created which suppresses out-of-focus blur and retains the in-focus image of the sample. The optical sectioning ability of this system is comparable to a point scanning confocal microscope.

The contrast pattern required for SI is imprinted with a reflective mask on the excitation beam and hence onto the sample. The pattern is also contained in the fluorescence emitted by the sample. This means that in-focus structures will see the SI pattern, but those structures above and below focus will disappear rapidly, resulting in an even, widefieldlike illumination of the focal plane.

LFC Principle

The reflective mask on the excitation beam is the essential part of the system and has three different patterns, consisting of line-patterns with different ratios and spatial frequencies, required to suit the characteristics of different objective lenses. The emitted light passes the mask which now works as a reciprocal filter and partiallytransmitting mirror. The emission created in the plane-of-focus will mainly arise from regions excited by the pattern and therefore passes the mask (widefield + confocal, WF+C); but emission that originates from out-of-focus locations will hit the reflective surface on the disk (widefield – confocal, WF-C).

The other important part of the system is to make good use of a camera. Both components of the image – WF+C and WF-C – will be imaged on either two individual cameras or split on the sensor of a single camera. LFC, therefore, benefits from CMOS sensors as they are large enough to allow the one-camera solution which makes registration and image processing easier. Both signals can vary quite substantially in intensity so a large dynamic range is very helpful as well.

 

Figure 1
Figure 1: Figure 1: Aurox LFC unit
Figure 2
Figure 2: Light path of the Aurox LFC. The excitation light is sent through the reflective mask which is mounted at an angle to allow the WF-C image to be reflected to the parallel light path indicated by the white arrow. The part of the image containing the WF+C information will pass through the SI pattern.

Additional Information

The Aurox homepage offers plenty of information and resources, helpful to every potential customer. Apart from the basics, the user can build a customized system or get suggested combinations based on applications, such as developmental questions, plant tissue or electrophysiology. One of the biggest advantages of the LFC is the ability to add it to any existing microscope which means that the functionality of having a widefield microscope is retained.

Cameras for the Aurox LFC

Various cameras have been implemented in the Aurox LFC system but the best performance can be achieved with a sensitive camera with high speed and a large field of view.

Spinning disk confocal microscopy has historically been performed with EMCCD cameras which are highly sensitive but have a relatively small field of view and slow speed.

More recently, one of the more attractive options has been back-illuminated sCMOS cameras which have equivalent sensitivity to an EMCCD camera but with the field of view, speed and pixel size advantages of sCMOS technology. This allows sensitivity and resolution to be maximized without trading off speed and field of view.

The range of pixel sizes offered by modern back-illuminated sCMOS cameras can ensure Nyquist sampling with 60× magnification (with a pixel size of 6.5 µm) or Nyquist sampling with 100× magnification (with a pixel size of 11 µm). Modern back-illuminated sCMOS cameras also have the field of view necessary to get the most out of the diagonal field of view of the Aurox LFC.

Putting all of these specifications together ensures the fastest, most physiologically relevant data acquisition with the highest throughput.

References

http://www.aurox.co.uk/