Cast an eye beyond the red: NIR fluorescence in neuronal model systems

At the Center for Life Nano Science (CLNS@Sapienza – Italian Institute of Technology) in Rome, Prof. Silvia Di Angelantonio is carrying out studies on major human neurodegenerative diseases, such as Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic Lateral Sclerosis, to identify biomarkers which might further drug development in a medical area requiring therapeutic progress. Di Angelantonio’s group exploits different human model systems, from post mortem retina tissues to in vitro neuronal cultures, for investigating the molecular basis of such neurodegenerative processes.

More specifically, here we show post mortem human retina (Part 1) and human iPSC-derived cortical neuron (Part 2) samples, both stained for DAPI and Tubulin (secondary antibody: Alexa 750). We used CrestOptics X-Light V2 spinning disk confocal coupled with Celesta laser (Lumencor) having spectral linewidth from 405 nm to 749 nm. Although most fluorophores operate in visible or ultraviolet parts of the spectrum, near-infrared (NIR) fluorescence is a light wavelength of 650–950 nm very promising for detection and imaging. NIR dyes, such as Alexa 750 fluorophore, allow researchers to broaden the scenario of fluorescent markers within the same sample and represent a valid additional channel to the usual set of DAPI/GFP/RFP/Cy5. Moreover, due to its optical features, NIR light penetrates to a much greater depth and is generally preferred for in vivo fluorescence and in-depth imaging in thick specimens, because of its good tissue penetration and low autofluorescence.

Part 1: Post mortem human retina tissue

We report acquisitions of a retina tissue (5 um section thickness) obtained with Prime 95B camera (11 um pixel size; Photometrics) to highlight the cellular organization in three primary layers: the outer photoreceptive layer (i) and the bipolar cell layer (ii) identified by nuclei staining, and the inner ganglion cell layer (iii) identified by nuclei and tubulin stainings (Figure 1). In addition to that, we show detailed portions of the retina in 2D (as single Z plane and Maximum Intensity Projection (MIP) of a Z stack) and 3D volume rendering (Figure 2). We compare raw spinning disk acquisition with post-processing elaboration (i.e. Advanced Denoising (AdDen) tool provided in NIS Element Software, NIKON) pointing out how the smoothing process improves the subcellular structure surface, especially appreciable for nuclei (see the Intensity Profile plot for quantification).

Figure 1: Two different retina areas; 20x air objective; DNA (cyan) and Tubulin-750 (red).

Figure 2: Comparison between Raw spinning disk image and images processed with Advanced Denoising (AdDen) tool; 60x oil objective.  

Raw

AdDen

One Z plane

MIP

Volume View  

Intensity Profile

Raw

AdDen

Part 2: Cortical neurons from human induced-pluripotent stem cells

Cortical neurons have been generated through overexpression of a master gene capable to induce the differentiation of pluripotent stem cells into excitatory cortical neurons, and collected after 15 days in culture. Here we show a large view acquisition of neurons (Figure 3) and a region of interest (Figure 4), both obtained with BSI camera (6.5 um pixel size; Photometrics). By Nyquist sampling theorem, it can be inferred that 60x objective magnification proper matches with camera pixel size of about ~ 6.0 um. Due to this, we run deconvolution processing to investigate differential quality improvement on neuron images. We performed both Advanced Denoising (AdDen) and Deconvolution algorithms (Dec; provided by NIS Element Software, NIKON), alone or in combinations and we highlight that it is worth trying different post-processing procedures to identify which one gives major benefits (e.g. smoothing edges, resolution gain) to specific subcellular structures according to experimental needs (see the Intensity Profile plot for quantification).

Figure 3: Large view of cortical neurons; 20x air objective; Tubulin-750 (red) and Map2-488 (white).

Figure 4: Comparison between Raw spinning disk image and images processed with Advanced Denoising (AdDen) and Deconvolution (Dec) algorithms; DNA (cyan), Tubulin-750 (red) and Map2-488 (white); 60x oil objective.

Raw

AdDen

Dec

One Z plane

MIP

Volume View

Intensity Profile

Raw
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