A complete workflow solution for large-scale 3D fluorescence microscopy

A complete workflow solution for large-scale 3D fluorescence microscopy

See the big picture and dive deep into the details

High-resolution, 3D light sheet microscopy of large samples opens up new dimensions in the world of biomedical microscopy.  

Complex 3D structures, such as vasculatures or neuronal networks, can span from micrometers to centimeters. For this reason, they escape truly comprehensive analysis by current standard 2D and 3D microscopy techniques involving tissue sectioning. 

Miltenyi Biotec offers a complete workflow solution that allows you to analyze your intact sample in full and dive deep into the details. 

 

3D light sheet imaging workflow

Acquiring meaningful 3D microscopy data from large samples such as 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.

Miltenyi Biotec’s product portfolio dedicated to large-scale 3D imaging offers validated and ready-to-use protocols and reagents that provide you with the optimal performance when using our microscopes. Streamline your workflow – no matter if you are a microscopy expert or just about to start your 3D imaging project. 
 

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, human tumor samples, 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.

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 a mouse brain hemisphere within only 6 hours (left: before clearing; right: 6 hours after treatment).

Step 3: Large-scale 3D fluorescence imaging

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. 

 

Applications for 3D light sheet imaging

Immuno-oncology

Miltenyi Biotec’s 3D imaging workflow offers many applications for immuno-oncology research, including 3D imaging of infiltrating lymphocytes in entire tumors. It also enables users to localize CAR T cells in a whole organism, and it can provide detailed insight into the tumor microenvironment through high-resolution imaging.

3D fly-through animation of a pancreatic carcinoma allows identification of tumor cells, CAR T cells, and vasculature

The video shows a 3D rendered “flight” through a pancreatic carcinoma. GFP-expressing tumor cells are shown in green, infiltrating CAR T cells in purple, and the vasculature in orange. Imaging was performed using UltraMicroscope technology.  

Neuroscience

For neuroscientists, UltraMicroscope light sheet imaging systems offer the option to generate 3D images of whole brains in unprecedented detail, facilitating studies on the pathology of Alzheimer’s and Parkinson’s diseases in mouse models, for example. 3D rendering allows the detailed analysis of complex structures within the brain, such as the vasculature. 

3D rendering of a whole mouse brain shows subcellular details of the vasculature

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

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