A 3D imaging workflow you must see to believe

The 3D imaging workflow from Miltenyi Biotec offers neuroscientists the ability to generate 3D images of entire biological systems with unprecedented detail, advancing studies in pathology of Alzheimer's and Parkinson's diseases in mouse models. Where once imaging analysis was restricted due to standardized microscopy techniques, Miltenyi Biotec's high-resolution rendering and easy-to-use staining, clearing and imaging protocols are a complete solution that must be seen to be believed.

 

3D light sheet imaging workflow

Acquiring meaningful 3D microscopy data from large samples such as whole brain, hemispheres or brain organoids, whole rodent organs, or even human organs is a three-step process: 

  1. The sample is stained with fluorochrome-conjugated antibodies. Thorough epitope labeling within the entire 3D structure and high signal-to-noise ratios are key for reliable analysis.
  2. The tissue needs to be rendered transparent to allow excitation of fluorochromes deep inside the sample.
  3. The signals are acquired with a microscope that can host large samples and has suitable optics with long working distance to generate both overview and high-resolution images of your large samples.
 

Step 1: Staining with 3D-immunofluorescence antibodies

Our 3D-immunofluorescence (IF) antibodies are specifically validated for whole-mount staining of large samples, such as mouse brain hemispheres, or cerebral brain organoids. Directly conjugated to bright and photostable Vio® Dyes, our REAfinity™ Recombinant Antibodies will accelerate and facilitate your research through shorter experiment times and optimal signal-to-background ratios. For maximum reliability, ultimately producing more conclusive results, all our 3D-IF antibodies are functionally validated with the MACS® Clearing Kit.

Take a look below at a few highlighted 3D-IF antibodies for neuro-imaging.

ProductCloneProduct Number
Calretinin Antibody, anti-mouse/rat, Vio R667, REAfinityREA1157130-131-150
MBP Antibody, anti-human/mouse, Vio R667, REAfinityREA1154130-131-152
NeuN Antibody, anti-human/mouse/rat, Vio R667, REAfinityREA1131130-131-153
β-Tubulin 3 Antibody, anti-human/mouse, Vio B515, REAfinityREA1152130-131-158
β-Tubulin 3 Antibody, anti-human/mouse, Vio R667, REAfinityREA1152130-131-156
Tyrosine Hydroxylase Antibody, anti-human/mouse/rat, Vio R667, REAfinityREA1159130-131-157

Selected markers for the analysis of neural cells. All antibodies were validated for tissue clearing with the MACS Clearing Kit and 3D imaging with the Ultramicroscope Blaze.

Validated antibodies provide conclusive 3D imaging data

The video shows a human cerebral organoid, whole-mount stained with Ki-67 Antibody, anti-human/mouse, Vio R667, REA183 (yellow); SOX2 Antibody, anti-human, Vio B515, REA320 (magenta); and β-Tubulin 3 Antibody, anti-human/mouse, Vio G570, REA1152 (cyan). The sample was processed and optically cleared using the MACS Clearing Kit and imaged with an UltraMicroscope II light sheet instrument. 

 

Step 2: Tissue clearing

To enable clear and distinct imaging of structures deep inside large tissues, light scattering of opaque tissue has to be minimized by tissue clearing. Starting from scratch, establishing a new clearing protocol, can be time consuming and labor intensive.  

Our ready-to-use, non-toxic MACS Clearing Kit effectively renders your samples transparent for optimal 3D imaging results. Easy-to-follow tissue staining and clearing protocols guide you through a hassle-free process step by step. 

Effective clearing of a mouse brain hemisphere within only 6 hours

Hassle-free and effective tissue clearing

The MACS Clearing Kit enables effective clearing of, for example, a human tumor sample or adult mouse brain within only 6 hours.

 

Step 3: Large-scale 3D fluorescence imaging

The UltraMicroscope II and UltraMicrosope Blaze

After staining and clearing, the samples are immersed into the non-toxic MACS Imaging Solution, which matches the optimal refractive index for optimal light sheet imaging with the UltraMicroscope II and UltraMicrosope Blaze™.  

Full automation of the UltraMicroscope Blaze Instrument enables overnight imaging of multiple samples in a single imaging experiment, without manual intervention or monitoring. The batch measurement mode ensures that 3D data will be available for in-depth analysis the next morning. Our pioneering UltraMicroscope technology combined with cutting-edge light sheet optics ensures excellent data quality from macro to subcellular imaging. 

UltraMicroscope Blaze workflow solution

Visualize the complete workflow solution for automated 3D light sheet imaging.

3D rendering of a whole mouse brain shows subcellular vascular detail

Watch the video on 3D rendering of a whole mouse brain vasculature obtained with the UltraMicroscope Blaze Light Sheet Imaging System using the 4x MI Plan objective lens.

Interested in cellular analysis of neural cells?

Check out our complete workflow solutions for cellular analysis providing you seamless support for brain tissue dissociation to cell separation, cultivation and flow cytometric analysis.

Curious for details on 3D light sheet microscopy?

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Our worldwide sales and technical support scientists are happy to help you with any question you have on our neuroscience portfolio and workflows.

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