Unlocking the potential of CICERO spinning disk system: exploring diverse applications

At CrestOptics, we aim to make our products accessible to all researchers enabling a variety of applications, and to create products that are as efficient as user-friendly. In this Application Note, we demonstrate the great versatility and strengths of the new CICERO system, a complete widefield (WF) and spinning disk confocal (CF) solution designed to be integrated into any imaging setup, due to its accessibility, compactness, and flexibility. In particular, we will demonstrate its key features, namely its use on both inverted and upright microscopy configurations, its wide spectral range, the high resolution and sectioning power, and its large field of view (FOV).

Flexibility is the key word: CICERO spinning disk system is adaptable on both inverted and upright microscopes

CICERO has been designed to be integrated into any imaging setup transforming it into a reliable confocal system. CICERO fits both upright and inverted microscope frames with a camera port, ensuring maximum flexibility of configuration (Figure 1).

Figure 1: Schematization of microscopy setups. Left: Upright microscope body equipped with CICERO and camera. Right: Inverted microscope body equipped with CICERO and camera.

Inverted and upright microscopes have the same working principle, the difference is in the placement of the objective to the specimen. Each of the two types of microscopes has its advantages and disadvantages, so the researcher must be familiar with the characteristics of the sample to be examined to choose the right microscope. One example where an inverted microscope is more suitable is the imaging of living cells, as the need for stage-top incubators arises and access is required from above. In addition, the objective does not touch the specimen, allowing sterility to be maintained during imaging and reducing the probability of collision between the objective and the sample.

However, some types of experiments necessarily require the use of an upright microscope. The medium in which the sample is included is a limiting factor that determines the instrument to be used for imaging, thus making flexibility an essential feature to answer today’s scientific questions. The use of an upright microscope is often used in histology, histopathology, and electrophysiology experiments. In the latter case, in fact, vital samples, such as cells or slices, are submerged in aqueous solutions designed to provide the necessary nutrients to keep the sample viable during recordings. For this very reason, it is necessary to use water immersion objectives. In addition, the use of a patch pipette, usually mounted on an arm moved via a micromanipulator, inevitably leads to the use of an upright microscope. To date, the combination of electrophysiology and confocal microscopy is widely used when, in addition to describing the electrophysiological profile of a particular cell, it is also necessary to study its morphology. For instance, during patch clamp experiments, recording pipettes are often filled with compounds such as biocytin, associated with fluorescent markers, to be able to reconstruct the morphology of the cell whose currents were recorded.  The thickness of samples used for electrophysiology often exceeds 100 µm, it is therefore necessary to use a system with high axial resolution and high confocality.

As shown in Figure 1, CICERO fits upright and inverted microscopes, providing great flexibility and adaptability. We used CICERO mounted on an upright microscope to obtain images of glial fibrillary acidic protein (GFAP)-positive astrocytes from acute brain slices, namely brain slices kept viable in an artificial cerebrospinal fluid solution. Figure 2 shows a large image of GFAP-positive astrocytes obtained with a 40x water immersion objective in an uptight configuration. This kind of image is useful to have a preliminary screening of astrocyte distribution in the brain area to be studied.

Figure 2: 4×4 large image of GFAP-positive astrocytes. This image was acquired with CFI Apo Lambda S 40XC WI objective (1.25 NA, 0.2-0.16 WD).

Changes in astrocyte morphology, known as a hallmark of reactive astrogliosis, are a common pathological feature in many neurological disorders. Furthermore, the astrocyte morphology is found to be highly dynamic and activity-dependent, making the combination of electrophysiology and confocal microscopy an extremely useful tool. Figure 3A shows a maximum intensity projection of about 30 µm of astrocytes acquired with a 60x water objective in both WF and CF mode and it is clearly visible that, even in acute slices, usually about 200 µm thick, CICERO is able to maintain its great sectioning power. From these images, we managed to isolate a single astrocyte and study its morphology with a 3D reconstruction, shown in Figure 3B.

Figure 3: GFAP-positive astrocytes from mouse brain section. 3A: Comparison of WF and CF spinning disk maximum intensity projections. 3B: Comparison of WF and CF spinning disk 3D projections of single astrocytes isolated from figure 3A. This image was acquired with a CFI Plan Apo VC 60XC WI objective (1.2 NA, 0.31-0.28 WD).

CICERO’s broad range of spectral capabilities, ranging from DAPI to CY7, enables maximum flexibility in staining samples.

CICERO spinning disk system can be used with both laser and LED illumination allowing to cover both entry-level and challenging applications. When used with laser light sources, it allows the use of a wide spectral range, from 390 to 750 nm excitation and 430 to 850 nm emission. The ability to use such a wide range of wavelengths opens the door to the simultaneous use of multiple markers, thus allowing an insightful study of biological samples and enabling both entry-level and challenging applications.

What has just been described is nicely illustrated in the multichannel acquisition shown in Figure 4. NIH/3T3 fibroblast cells are stained with multiple fluorescent markers ranging from DAPI to CY7. The possibility of using several wavelengths allows the researcher to observe at the same time a high number of markers, thus reducing the number of images to be obtained. In addition, near-infrared wavelengths are characterized by good tissue penetration and low autofluorescence, therefore simplifying the image acquisition process.

Figure 4: maximum intensity projection of NIH-3T3 fibroblast cells. Blue: DAPI; green: actin; cyan: β-tubulin; white: heat-shock protein 70 (HSP70); red: fibrillarin. This image was acquired with CFI Plan Apochromat Lambda D 60X oil objective (1.42 NA, 0.15 WD).

All-in-one solution for high-quality WF and CF imaging

CICERO is a complete widefield (WF) and spinning disk confocal (CF) solution. In the context of entry-level applications, CICERO is a well-performing and accessible solution for fluorescence imaging. In WF mode, you can obtain informative data from applications such as cell monolayers and thin tissue sections, as shown in Figure 5 where we show a WF acquisition of retinal ganglion cell monolayer.

Figure 5: Retinal ganglion cell monolayer maximum intensity projections from 11 µm Z-stack obtained in WF mode. Brn3a: white; Map2: cyan. This image was acquired with CFI Plan Apochromat Lambda D 60X oil objective (1.42 NA, 0.15 WD).

Currently, advances in life science research are strictly bound to high-resolution 3D imaging. The study of complex organs requires imaging of very thick tissues, where cells interact to form complex structures. To this end, the use of systems with great optical sectioning power and high axial resolution is essential.

With the slide of an up/down switch, with CICERO we can switch from WF to CF mode to easily evolve into high-quality 3D imaging. We can image pyramidal neurons from a 250 µm thick brain slice sample: Figure 6A shows hippocampal pyramidal neurons acquired in WF or CF modality and, as clearly shown by the 3D reconstructions shown in Figure 6B, with CICERO it is possible to depict neuronal 3D organization in the tissue.  In addition, the 3D reconstructions clearly demonstrate the axial resolution gain obtained by switching to CF mode.

Despite its small footprint, the CICERO confocal solution, like all our spinning disk products, provides the highest image acquisition speed, enabling also fast live imaging acquisitions.

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Figure 6: Hippocampal pyramidal neurons from mouse brain. 6A: Comparison of WF and CF spinning disk maximum intensity projections from 70 µm Z-stack. 6B: Comparison of WF and CF spinning disk 3D projections. These images were acquired with CFI Plan Apochromat Lambda D 60X oil objective (1.42 NA, 0.15 WD).

Due to its large FOV, CICERO offers a minimal scanning process and can capture large samples in a single frame

Modern science often requires the study of complex tissues and structures, thus demanding large amounts of images and data. CICERO‘s FOV of up to 22 mm allows imaging of large structures, with a single image or by stitching a few images together, thus greatly increasing the data throughput.

Figure 7 illustrates several spheroids acquired with a single image at 20x magnification. In particular, the image shown is a maximum intensity projection of about 40 µm of spheroids in which pericentrin and nuclei are respectively stained in orange and blue. Such an image allows the study of morphology, nuclei distribution, and pericentrin expression in numerous spheroids with a single frame.

Figure 7: Maximum intensity projection of spheroids imaged with a CFI Plan Apochromat Lambda D 20x air objective (0.8 NA, 0.8 WD). Blue: DAPI; orange: pericentrin.

As shown by the examples in Figures 8A-D, CICERO ‘s FOV width also allows imaging of large structures and complex tissues with single frames or through the stitching of a few frames, strongly accelerating data collection. In all the examples shown, the uniform illumination provided by our system enables us to obtain images free of “vignetting”, the reduction in brightness at the periphery compared to the centre, an aberration that is common when stitching together several images.

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Figure 8: Examples of large structures images obtained thanks to CICERO large FOV. A: Ipomoea root section, 1 single FOV; B: Cerebral organoid stained for DAPI (blue) and TBRI + neurons (red), 2×2 large image; C: Rat brain stained for DAPI (blue), MAP2 (green), CALBINDIN (orange), MBP (red), 6×6 large image; D: mouse kidney, whole section stitching. These images were acquired with CFI Plan Apochromat Lambda D 20x air objective (0.8 NA, 0.8 WD).

Conclusion

In this Application Note, we aimed to provide an overview of the main features of CICERO through different applications. CICERO spinning disk system is characterized by its great flexibility that allows to use it on both inverted and upright microscopes, by its wide spectral range, large FOV, and by the ability to be used with both LED and laser illumination sources. Moreover, CICERO offers a seamless user experience; any new user can include high-quality WF and CF images in their daily work.

CICERO is an extremely flexible yet high-performing product, enabling new exciting possibilities for basic and applied research.

Acknowledgments

Retinal ganglion cell monolayer displayed in Figure 5 and the cerebral organoid shown in Figure 8B  derive from the laboratory of Prof. Silvia Di Angelantonio, Center for Life Nano-&Science, Rome. (CLN2S – Sapienza Università di Roma – Istituto Italiano di Tecnologia). We thank Dr. Lorenza Mautone and Chiara Dantoni for kindly providing this sample. 

3D spheroids shown in Figure 7 were provided by Dr. Giulia Fianco and Dr. Giulia Guarguaglini from the Institute of Molecular Biology and Pathology, Consiglio Nazionale delle Ricerche, Rome.

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