04/15/23

Digital Pathology 3.0: Spatial Pathology

Light-sheet microscopy for slide-free non-destructive pathology 


This piece is an extension to Digital Pathology 2.0, where I discuss 3D histopath solutions via volumetric data. In that exploration, I failed to consider another technique that can be used for 3D imaging: light-sheet microscopy.

What is light-sheet microscopy? 

Light sheet fluorescence microscopy (LSFM) uses a thin beam of light to illuminate a fluorescent sample and then observes the resulting emission from that plane at a perpendicular angle. This selective illumination approach, employed by a light sheet microscope (LSM), prevents the excitation of fluorophores and organic molecules outside the focal volume. This means that it causes minimal damage to surrounding tissue via phototoxicity. The light-sheet microscopy approach achieves optical sectioning (rejection of out-of-focus light) 

Benefits

The benefits of this technique are particularly evident when imaging complex or dynamic three-dimensional (3D) distributions of fluorophores, ranging from entire embryos to organelles. In terms of histopath, it is a powerful technique to study something like distribution of cancer cells. 

In Contrast

Wide-field fluorescence microscopy takes pictures of the whole sample, but the images are blurry and not focused on a specific area. Confocal fluorescence microscopy also takes pictures of the whole sample, but it only focuses on a certain area by using a pinhole. By taking pictures of the sample in different areas, a 3D image can be created. However, because the whole sample is illuminated for each picture, it can cause damage to the sample.

Why

LSFM can create clear images by illuminating only the focal volume, so there is no out-of-focus signal. The sample is moved through the focal volume to create a stack of optical sections, but because the illumination is only on this region, the sample is only irradiated once per 3D image. This means that LSFM causes much less photobleaching and phototoxicity than confocal microscopy. LSFM captures images quickly with cameras for signal detection, and thousands of images can be taken in a few seconds as millions of voxels are acquired in parallel. LSFM is used to create 3D images of specimens of varying sizes, from small to large.

Essentially, LSFM is highly adaptable due to its 

  • fast recording speed 

  • low phototoxicity

In 2014, Nature Methods announced LSFM Method of the Year. Vast technical progress over the past decade has led to the widespread application of LSFM in almost every biological discipline. 

Nature Biomedical Engineering: Open-top light-sheet microscope for clinical pathology. a, An illumination light sheet enters the bottom surface of a tissue sample at an oblique 45° angle (purple). The specimen is placed on a modular glass-plate sample holder, which is inserted into a two-axis translation stage and scanned in a serpentine pattern of volumetric image strips to enable 3D imaging over a large lateral extent. Fluorescence emission (cyan), which is generated along the light sheet, is collected in the orthogonal direction by an objective lens. The fluorescence signal is then transmitted through an emission filter (green) and a dual-channel image splitter (for two-colour imaging) before being imaged onto a high-speed sCMOS camera. b, To provide aberration-free imaging, a solid immersion lens (SIL) and oil layer are used for refractive-index matching of both the illumination and collection beams into and out of the glass plate and tissue sample. c, As the sample is translated in the primary scanning direction, x, oblique 2D light-sheet images with width w and adjustable height h are captured in succession to form a 3D imaging volume. d, In contrast to conventional microscopy methods that have a shallow fixed depth of focus and slow 3D imaging rates, the deep depth of focus and adjustable vertical field of view of the open-top light-sheet microscope makes it optimal for both rapid microscopy of irregular/tilted tissue surfaces, and deep volumetric microscopy of clinical specimens. The imaging speeds (v) shown correspond to acquiring single-channel images with height h. 

What if instead of scanners, what we need is just better and different microscopes? Isn’t there less inertia in replacing a technology rather than inserting a new one into the workflow?

Wouldn’t this increase adoption of digital and computational pathology? 

This paper shows that an open-top light sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. 

They demonstrate its utility for the following applications: 

  1. Wide-area surface microscopy to triage surgical specimens 

  2. Rapid intraoperative assessment of tumour margin surfaces

  3. Volumetric assessment of optically cleared core-needle biopsies 

Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens. 

You don’t need to slice the specimen because you can view it in its entirety in 3D. Digital adoption on the clinical side has been very low due to change management – institutional inertia hinders the adoption of incremental changes in the centuries-old technological framework of histopath – a practice deemed foundational to patient-management decisions. 

Efforts to digitize histology using whole-slide scanners have not made the process of preparing and interpreting histology slides simpler. Instead, they have introduced costly equipment and increased the time taken for pathology workflow, with unclear diagnostic advantages.

Being able to use open-top light-sheet microscopy system that circumvents the need to prepare histology slides through non-destructive imaging of fresh clinical specimens on a glass plate could be revolutionary for digital pathology.

This easy-to-use technology is highly versatile for a diversity of applications in clinical pathology, enabling both rapid surface microscopy and volumetric imaging of clinical specimens

  • streamlining the pathology workflow 

  • guiding surgical oncology 

  • improving the diagnosis and grading of biopsied lesions

These technologies will likely naturally lead to efforts in human-computer interaction (computer-aided diagnosis) + automated image interpretation.

Ultramicroscopy

The paper discusses the technique of ultramicroscopy, which is a type of light-sheet-based microscopy used to achieve high-resolution 3D reconstructions of intact macroscopic specimens. It is closely related to other light-sheet-based microscopy approaches and is useful for imaging samples in the size range of ∼1–15 mm. The article also highlights the differences between ultramicroscopy and light sheet microscopy. Both techniques have their advantages and disadvantages and are used based on the specific needs of the experiment.


Commercialization: Alpenglow Biosciences

Alpenglow Biosciences spun out Lightspeed Microscopy (their previous name / the technology) to commercialize this promising technology with the potential to accelerate drug development and improve clinical diagnostics.  Since then, the platform has claimed to be AI-enabled with cloud-based computing and analysis to produce remarkable 3D spatial analysis of tissue samples at high speed.

They use an open top light-sheet (OTLS) microscope with patented geometry that allows for ease of use by analyzing entire tissue samples or conventional multi-well plates.  This fully automated system facilitates high throughput analysis, while cloud computing and AI-enabled software allows researchers to visualize and quantify complex spatial biology applications in 3D. 


Their justification for why 3D

Most biologic structures are best quantified in 3D: vasculature, neurons, lymphatics, glands, etc.  Alpenglow’s technology provides critical 3D Metrics: volume, total path length, mean diameter, branch count, branch angle, fractal dimension, etc.

Use cases:

  • Neuroscience, enteric nervous system, peripheral nervous system

  • Vascular changes in dementia, tumors, ischemia, neoangiogensis

  • Fibrosis in wide variety of disease including liver, kidney, and lung conditions

Via Alpenglow Biosciences: The original 3D image of this ileocecal sample contains more pixels than stars in the Milky Way. There are more than 840 billion pixels in the 3D scan of this sample encompassing a total volume of 2,350 cubic millimeters.

3D is the best way to understand complex architectures (i.e., microenvironment) in tumors, fibrosis, immune mediated inflammatory disorders, etc.

Use cases:

  • Tumor microenvironment

  • Amyloid & tau protein distribution

  • Immune cell composition / colocalization

Identify rare cells or drug targets, 2D sectioning will often miss these objects.

Use cases:

  • Genetically labeled rare cells

  • Biodistribution of drug at target studies

  • Stem / progenitor cell studies

  • Subclones within PDX models

Some other solutions for drug discovery, translational research & clinical diagnostics includes efficacy and dosage analysis, toxicity and biodistribution analysis, failed asset screening, and predictive capabilities. 

Works Cited

Becker, Klaus, et al. "Ultramicroscopy: light-sheet-based microscopy for imaging centimeter-sized objects with micrometer resolution." Cold Spring Harbor protocols 2013.8 (2013): pdb-top076539.

Glaser, Adam K., et al. "Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens." Nature biomedical engineering 1.7 (2017): 0084.

Sabdyusheva Litschauer, Inna, et al. "3D histopathology of human tumours by fast clearing and ultramicroscopy." Scientific reports 10.1 (2020): 17619. 

Stelzer, Ernst HK, et al. "Light sheet fluorescence microscopy." Nature Reviews Methods Primers 1.1 (2021): 73. 

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