Swept-Field Confocal Microscopy

Mathias Pasche-Drews, Field Applications Specialist, Photometrics

Dan Croucher, Applications Team Manager, Photometrics

Introduction

One of the most popular confocal microscopy techniques is spinning disk confocal microscopy; a high-speed, high sensitivity 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. This method does have drawbacks, however, such as high crosstalk, low spatial resolution, limited objective lens selection and the acquisition speed is ultimately limited by the rotational speed of the disks.

Swept-field confocal microscopy overcomes these limitations by using a positionable aperture plate containing a variety of pinhole columns and slit apertures instead of pinholes embedded on a spinning disk. This one-dimensional pinhole/slit array can be swept across the sample by galvanometric and piezo-controlled mirrors to collect confocal information from structures within the plane of focus and reject out-of-focus information at high speed and high resolution with much-reduced crosstalk.

The swept-field microscope is manufactured and distributed by Bruker as the Opterra II swept-field confocal microscope.

The Swept-Field Principle

Bruker has optimized the spacing between individual pinholes (Figure 1) which results in the system showing half or less crosstalk usually observed on a conventional spinning disk system relying on a twodimensional array (i.e. having a second disk with microlenses).

Depending on the selected microscope objective lens, the system can use three differently sized pinhole dimensions to allow the user to match the pinhole size to their imaging requirements resulting in optimal results under each condition.

In slit-scan mode, speed can be vastly increased at the expense of spatial resolution. The system has slits of four different widths to give users the choice to best accommodate their sample.

Figure 1
Figure 1: Opterra II Swept field pinhole pattern and light path

The optical system is highly efficient and is able to capture more photons than spinning disk confocal microscopy with reduced back reflections from incoming light, crosstalk, and noise. The stationary pinhole apertures allow speed to be increased without the distortions typically seen at high frame rates on other scanning confocal systems.

The entire system is software controlled and the user is able to switch between pinholes/slits swiftly without the need to manually change hardware components. Moreover, a bypass mode is available to enable regular phase, DIC, brightfield and epifluorescence on the same system.

Figure 2
Figure 2: Pancreas tumor section. Montage of 2 mm x 1.5 mm area at 60x magnification (from Bruker)

Bruker claims that the Opterra II produces a much improved uniformity in both sample illumination and detected emission, resulting in better images and hence more quantitative data. The roll-off of signal across the FOV is less than 10% on the Opterra II compared to 30% on an optimally adjusted conventional spinning disk system.

Bruker also claims the fastest available 10-position filter-wheel (45 to 110 ms between positions), for fast emission wavelength switching. The light source is a Helios laser launch housing up to five solid-state lasers, optimized by blanking to reduce exposure time.

Cameras for the Opterra II

The Opterra II was designed to work with an EMCCD camera, such as the Photometrics Evolve 512 Delta (Figure 3), due to the fast imaging capabilities and the high sensitivity. The increased sensitivity allows the user to further reduce the excitation light intensity to avoid side effects such as photobleaching and phototoxicity.

With the arrival of the Photometrics Prime 95B back-illuminated scientific CMOS camera, a step forward has potentially been made. The almost perfect, 95% quantum efficient (QE) sensor has equivalent sensitivity to an EMCCD camera but with the much larger field of view (1200x1200 pixels, 18.66 mm diagonal) and higher speed (82 fps, full frame) expected of a CMOS device.

Figure 3
Figure 3: Bruker Opterra II on a Nikon TiE equipped with a Photometrics Evolve 512 Delta

The large, 11x11µm pixels provide additional sensitivity and have a large 80,000 efull well capacity with a low 1.6 eread noise, giving the 95B a very high dynamic range, ideal for performing high contrast imaging. The 11x11µm pixel size also fits perfectly with high magnification objectives, achieving Nyquist sampling without the need for any additional optics with 100x magnification.

The Prime 95B may represent another solution for the Opterra II in addition to the great performance already delivered by the Evolve 512 Delta EMCCD camera.

References

https://www.bruker.com/products/fluorescence-microscopes/opterra-confocal-microscopy/overview.html