Line-scanning Confocal

Increasingly, scientists are being challenged to study more biologically-relevant model systems, and this often means imaging thick, demanding 3D samples that may also be alive. To extract the required information, imaging these sample types requires careful selection of the most appropriate imaging technology and flexibility of the experiment’s design within that technique. IRIS and EDGE line-scanning confocal microscopy offer a flexible solution capable of delivering high-quality, rapid imaging of even the most complex 3D live samples.


Fig 1. Maximum intensity projection of a Drosophila oocyte, showing the nuclei (cyan, DAPI) and actin fibers (magenta, phalloidin). Image was acquired on the IN Cell Analyzer 6500HS using a 60x 0.7 NA objective in EDGE confocal mode.

A choice of technologies

Confocal microscopy is commonly used to refine optical sectioning and improve the signal-to-noise ratio, particularly when imaging thick specimens or samples that scatter light significantly. Two of the most commonly used methods are laser scanning confocal microscopy (LSCM) and spinning disk confocal microscopy (SDCM). However, line scanning confocal microscopy is an alternative method which combines the strengths of an adjustable aperture used in LSCM with the increased speed of SDCM.

Laser scanning

In LSCM, the sample is illuminated by a single laser beam and the emitted signal is focused onto a detector through a pinhole. The size of the pinhole is variable, allowing the user to adjust how much out-of-focus light is rejected during imaging, effectively changing the optical slice thickness.
Among the main disadvantages of this method is the slow acquisition speed because the sample must be scanned point by point by a single laser beam. Furthermore, LSCM requires high levels of laser light exposure leading to elevated phototoxicity for live cells.

Spinning disk

With SDCM, the sample is illuminated by a laser beam passed through a set of spinning disks with thousands of pinholes. With multiple focused laser beams scanning the sample at once, SDCM achieves faster imaging speeds when compared to LSCM. However, since the pinhole size is fixed on SDCM systems, it is not possible to optimize imaging for various objectives or channels or adjust the level of confocality achieved.

Line scanning

In this method, a focused laser line is swept across a sample in one direction. Since movement occurs only in one direction, line scanning confocal is significantly faster than LCSM. Additionally, since there are no physical pinholes, it is possible to adjust the level of confocality achieved with line scanning confocal microscopy.

Optimizing image quality with IRIS confocal

IRIS confocal is an innovative line scanning imaging module integrated onto the IN Cell Analyzer 6000/6500HS and the DeltaVision™ OMX SR/Flex microscopes. Based on a design presented by Mei et al.(1), it uses the rolling shutter mode on modern sCMOS cameras to create a virtual confocal aperture that enables fast, flexible, and reliable confocal imaging.

The precise optical implementation of IRIS confocal varies between the IN Cell Analyzer and DeltaVision OMX but the basic principles are the same. A Powell lens is used to transform the laser illumination from a spot to a line. Galvanometers are then used to control the illumination line such that it can be swept in the Y direction very precisely to illuminate the sample line by line. On the emission side, the rolling shutter mode on high quantum efficiency (QE) sCMOS cameras is utilized to read out the camera sensor line by line. These two components are then synchronized such that the illumination line and active rows on the camera sensor sweep from top to bottom at the same time (Fig 2).

    A

    B

Fig 2. Light path schematic of IRIS line scanning confocal (A) Diagram showing the synchronized sweep of the laser line and active pixel rows on a sCMOS camera sensor (B).

The width of the illumination line is dependent on the magnification used and is therefore fixed, but the number of rows being read and integrated from the sCMOS camera sensor can easily be varied in rolling shutter mode. When fewer rows are activated at once, the system rejects more out-of-focus light, resulting in an image with a higher level of confocality and a thinner optical section. Conversely, when more rows are activated at once, the system collects more out-of-focus light resulting in an image that appears more similar to a widefield image.In practice, the level of confocality is defined in Airy units (AU). An open aperture would be approximately equivalent to a widefield image, while an aperture set to 1 AU would result in a highly confocal image.


Fig 3. Single Z section of mouse kidney tissue showing membranes (Alexa488-conjugated wheat germ agglutinin) at varying levels of confocality (aperture width). A) Open aperture, B) 3 AU, C) 1 AU. Imaged on DeltaVision OMX Flex with 60x 1.2 NA water immersion objective, scalebar 5 µm.

Additionally, since the level of confocality is controlled by activating greater or fewer rows on the camera sensor, IRIS confocal can easily be optimized for any objective lens or can be used to collect data at varying AU values for each channel within the same experiment.

Increasing contrast with EDGE confocal

IRIS technology provides great speed and flexibility in terms of optical slicing; however, its axial resolution is diffraction-limited. This is because there is no removal of out-of-focus signal along the laser line illumination and signal is blocked only in the direction perpendicular to the laser line.

One of the solutions proposed to compensate for insufficient axial resolution is based on a computational approach. A study by Poher, V. et al(2) has served as an inspiration for EDGE confocal and requires acquisition of three images for each Z section instead of the usual one.

The first image is an in-focus image synchronized with the laser sweep. The other two are taken with the illumination line slightly offset from the active rows on the camera sensor. In the leading scan, the laser line is swept slightly behind of the active rows, while in the lagging scan, the laser line is swept slightly ahead the active rows (Fig 4). These two images measure the out-of-focus light associated with the in-focus image and are subsequently averaged and then subtracted from the in-focus image.



Fig 4. EDGE confocal principle. Three sequential scans are acquired and used to create an EDGE image (shown at top). The first scan captures the in-focus image (A), by synchronizing the active rows on the camera sensor with the laser sweep. In the second (leading) scan (B), the active rows on the camera are positioned slightly ahead of the laser sweep and in the third (lagging) scan (C) the active rows are behind the laser sweep. The images resulting from the leading and lagging scan are used to calculate out-of-focus light which is subsequently subtracted from the in-focus image to create the resultant EGDE image (top).


The resulting images acquired in EDGE confocal mode have a significant improvement in signal-to-noise ratio. This enhancement is especially prominent for tissue sections (see Fig 5.) and cells grown in 3D culture such as spheroids and organoids (see Fig 6.), where out-of-focus light dramatically affects image contrast.


Fig 5. In conventional light microscopy (A), the depth of field imaged with each optical slice can create a significant amount of out-of-focus light that decreases the signal-to-noise ratio in the resultant image. The use of EDGE confocal mode creates a thinner optical section and results in enhanced signal-to-noise (B), revealing more structural detail in the sample. Images were taken on the IN Cell Analyzer 6500HS, using a 20x 0.75 NA air objective.

Benefits of IRIS and EDGE imaging modes

Flexibility

With the variable aperture of IRIS and EDGE imaging modes, confocal images are optimized regardless of objective lens or channel choice. Combine varying aperture sizes or a mix of IRIS and EDGE images in a single experiment to ensure you are collecting exactly the data required to answer your unique biological question.

Higher contrast and background rejection

Use EDGE confocal or low AU imaging to acquire high-quality data from even the most challenging samples such as thick 3D specimens with significant amounts of out-of-focus fluorescence and those used in wash-free assays.


Fig 6. Imaging of spheroids and organoids in conventional microscopy often results in images in which the central region of the sample is significantly blurred, and the object edges are indistinct (A) Removal of the out-of-focus light in EDGE confocal mode allows clearer delineation of individual nuclei (cyan, DAPI) and the edges of the spheroid (B). Maximum intensity projection of 3D dataset was taken on IN Cell Analyzer 6500HS using 20x 0.75 NA air objective.

Increased axial contrast and resolution

EDGE confocal mode improves axial resolution two-fold enabling you to localize structures more accurately in the Z dimension (Fig 7.).


Fig. 7. X/Z section through kidney tissue. Imaging with EGDE confocal mode allows finer detail to be in the membranes (cyan, Alexa Fluor 488 conjugated wheat germ agglutinin) and actin fibers (magenta, Alexa Fluor 568 conjugated phalloidin) in kidney tissue. Imaged on DeltaVison OMX Flex using 60x 1.42 NA oil objective in IRIS (A) and EDGE (B) modes.

Better segmentation/analysis

The higher contrast images acquired using IRIS or EDGE confocal modes will give you more reliable results when downstream segmentation or quantitative analysis is needed (Fig 8.).


Image
Segmented Image
Open apertureIRIS (1 AU)EDGE (1 AU)

Fig 8. Single Y/Z section showing nuclei within a spheroid acquired in open aperture (A), IRIS (B) and EDGE (C) modes. Top row shows image data and bottom row shows overlay with a segmentation mask. In open aperture mode, identifying individual nuclei within the spheroid is very challenging due to the amount of out-of-focus light captured. When IRIS or EDGE mode are used, segmentation quality is greatly improved thus improving downstream analysis. Imaged on the IN Cell Analyzer 6500HS using a 10x 0.45 NA air objective.

References

  1. Mei, E. et al., A line scanning confocal fluorescent microscope using a CMOS rolling shutter as an adjustable aperture. Journal of Microscopy, 247, 269-276 (2012).
  2. Poher, V. et al. Improved sectioning in a slit scanning confocal microscope. Opt. Lett.33(16), 1813–1815 (2008).

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