Live-cell imaging challenges put at ease

Live-cell imaging is crucial when studying the mechanisms and dynamics of biological processes, whether it is at low or high resolution. Therefore, it is essential to choose the most suitable microscopy approach to maintain the correct environment for living cells, obtain meaningful data and high-quality images. For these reasons, performing a successful live-cell imaging experiment may become challenging but this is where advanced microscopy technology aids. As mentioned, the main concerns in live-cell imaging are maintaining the biological sample alive under the correct environmental conditions, setting the correct time-lapse sampling rate and avoiding bleaching of the fluorescent markers over time. In addition, excitation light can bring damage to the cellular compartments causing impairment of the sample physiology and in some cases leading to sample death; therefore, the laser power and exposure time used are very important values to pay attention to. Making sure that these phenomena don’t occur is crucial as these can alter the conclusions drawn from experiments. The choice of non-toxic, bright and light-stable fluorophores is the first step towards successful imaging together with the setup of the correct acquisition settings. However, even if these expedients are observed, things can go wrong if fluorescence excitation and emission efficiency is not optimal and light power reaches the sample for a long period of time.

X-Light V3 

Spinning disk confocal microscopy has many advantages for live-cell imaging over conventional laser-scanning confocal microscopy, especially when equipped with cutting-edge technology and high-quality hardware. CrestOptics X-Light V3 possesses the right hardware setup to be fast and gentle on your sample providing high image quality using low light levels. The optimized optical path (Figure 1) and the optimal disk pattern (Figure 2) make live-cell imaging possible minimizing the chances of photobleaching significantly. The optimized optical path together with an efficient mechanism of fluorescence detection allows the rejection of reflected light. The detection signal is maximized thanks to the use of cameras with a sensitivity up to 95% of quantum efficiency compared to the 45% of a PMT in a point scanning confocal. Moreover, proper matching between the illumination and the camera detection area reduces the generation of phototoxic products beyond the field of view (FOV) compared to widefield fluorescence imaging. This means that low laser power and exposure times can be used to achieve high confocal throughput and speed

Figure 1: Schematic diagram of the X-Light V3 spinning disk confocal. Laser light enters the V3 via a multi-mode fiber, passes through the microlenses array and the excitation filter wheel; collimated light is then focused through the pinholes of the disk to the sample. Emission light coming from the sample goes through a dichroic mirror, emission filters and the selected emission frequency is detected by the camera. 

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Figure 2:  A) High throughput, B) Standard, C) Deep penetration disks. 

Furthermore, our disks can be customized to different pinhole sizes and distancing satisfying the imaging requirements and minimizing the chances of photobleaching (Table 1). For more information on how to match the disk pattern to your application, visit our webpage Technology. The various layouts of the disk are designed to provide the best possible balance between pinhole cross-talk and 3D sectioning capability for your fluorescently marked samples.

In addition, the high rotation speed of our spinning disk (15000 RPM) allows an acquisition speed of 1400 fps, and together with the use of fast and high sensitivity sCMOS cameras, it is possible to capture fast and dynamic biological processes. Another advantageous factor is the size of the FOV. A FOV of 25 mm reduces the number of acquired images needed to collect enough data, lowering again the chances of bleaching. Moreover, thanks to the microlenses technology, a uniform collimated beam gives more than 90% homogeneous illumination over the entire FOV. Lastly, two irises located along the excitation and emission light path assist with maintaining the sample intact and using low excitation light. 

In fact, the sample is exposed to only a small fraction of the excitation light thanks to the presence of an illumination iris positioned before the spinning disk, and therefore its characteristics remain preserved. 

Disk

Application

High throughput

Ultra-fast imaging for thin samples

Standard

Live imaging, cell monolayers or tissues

Deep penetration

Thick and scattering tissues (over 100 um)

Custom 

Customer-specific application

Table 1: X-Light V3 spinning disk layout options. Custom disks also include the choice of a double pattern on the same disk.

The live-cell imaging capabilities of the X-Light V3 are demonstrated in our application notes. With the X-Light V3, it is possible to perform fast live imaging on a large FOV to then focus on a single cell to follow lysosomes dynamics at high resolution (Live-cell details and dynamics with structured illumination microscopy). A time-lapse of IFT-NG cells followed particles moving up and down along ciliary microtubules of green algae at the high speed acquisition rate of 187 fps (Up&down along cilia in green algae).

We performed 3D stacks and time-lapse imaging to accurately study protein localization and fluorescence stability over time, demonstrating that it is possible to visualize endogenously expressed fluorescent proteins preserving the health of cells and avoiding fluorescence bleaching (A dream come true: long-term yeast imaging by confocal microscopy). We were capable of performing a long-term live-cell experiment on Ewing’s sarcoma A637 cells grown on a 3D matrix. The apoptotic effect was followed in a time-lapse experiment of 72 h on a 530 um thick sample (Live Imaging of Anti-Cancer Therapy Effect in VITVO®). 

DeepSIM

Dynamic events of small structures need to be resolved with super-resolution to really appreciate and understand true biological events. A reliable system able to provide both super-resolution avoiding artifacts and high imaging speed plays an important role. DeepSIM, as an add-on module to X-Light V3 (Figure 3A), has the ability to image fast, with adequate depth penetration and without the use of special probes. The DeepSIM preserves all the X-Light V3 functionalities unaltered, from the light source to the camera. This means that all the benefits of performing a successful live-cell imaging experiment are maintained, adding the super-resolution capability with a temporal resolution greater than 10fps (1024×1024 px FOV). Only a simple click is necessary to remove the spinning disk of the X-light V3 from the light path and switch to the super-resolution modality (Figure 3B). 

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Figure 3: A) Schematic diagram of the X-Light V3+DeepSIM add-on module in super-resolution modality. Laser light enters from the V3 via a multi-mode fiber, passes through the microlenses array and the excitation filter wheel; the spinning disk is removed from the light path. Collimated light enters the DeepSIM, it’s focused through the pinholes of the mask and the lattice pattern is projected to the sample via a motorized galvo. Emission light coming from the sample goes through a dichroic mirror and emission filters of the V3, and the selected emission frequency is detected by the camera. B) Schematic diagram of the X-Light V3+DeepSIM add-on in spinning disk confocal modality. Laser light enters the V3 via a multi-mode fiber, passes through the microlenses array and the excitation filter wheel; collimated light is then focused through the pinholes of the disk, which is reinserted into the light path. Excitation light then enters the DeepSIM bypassing the mask and galvo. Excitation light then reaches the sample and emission light is sent to the camera via the same emission path as that of the V3 stand-alone.

The DeepSIM is a lattice multi-point structured illumination system that provides an XY resolution of ~100 nm and optical sectioning with Z resolution of ~300 nm (100X, 1.45 NA). We offer three multi-point mask patterns to satisfy a wide range of applications (see table below).

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Figure 4: A) High throughput, B) Standard, C) Deep penetration masks.

Mask

Application

High throughput

Live imaging, thin samples and thick samples (up to ~50 um)

Standard

Live imaging, thin samples and thick samples (up to ~100 um)

Deep penetration

Dense and homogeneous structures, deep Imaging (over 100 um)

Table 2: DeepSIM lattice mask layout options.

The main strength of the DeepSIM for live-cell imaging is provided by the lattice SIM technology. Lattice SIM overcomes the limitations of classic SIM by using masks with a multi-point pattern rather than grid lines, and the pattern is shifted only laterally, without rotations. In this manner, light and time are efficiently used, enabling fast and gentle 3D super-resolution imaging of live samples. In addition, the multi-point pattern offers higher contrast making image reconstruction more robust. Moreover, the DeepSIM utilizes the X-Light V3 excitation and emission path that maximizes illumination minimizing the changes of photobleaching. 

Refer to our latest DeepSIM application note “Live-cells details and dynamics with structured illumination microscopy” to appreciate the strengths of the CrestOptics DeepSIM for live-cell imaging. We focused on HeLa cells lysosomes and tracked their fast movement for 30 seconds of continuous recording and performed long-term imaging of HeLa cells division for 12 hours and 15 minutes (15 minutes delay).   

CrestOptics X-Light V3 and X-Light V3 coupled with DeepSIM are the right solutions for performing high and super-resolution imaging of living samples thanks to the exploitation of the latest technology advantages.  Another benefit of these systems is the multi-line laser compatibility to allow the use of a wide range of fluorophores. You can choose to image in the visible spectrum and in NIR up to 750 nm. This brings a substantial benefit to live-cell imaging experiments as markers in the far-red and NIR spectrum can be used, lowering the chances of phototoxicity and allowing for multi-marker options avoiding channel bleedthrough. Moreover, the X-Light V3 hosts two camera ports to perform simultaneous imaging in both modalities bringing even more flexibility to your experiments. Lastly, the design of our systems allows the use of microscope environmental chambers to completely encase and maintain your sample in a controlled environment. Being compatible with the most common microscope base brands, the use of stage top incubators is also possible for those samples that require a more controlled environment. 

Visit our product page for more details on the CrestOptics X-LightV3 and DeepSIM. Contact us to book a demo and see their great capabilities with your own eyes.

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