Despite rapid technological advances in molecular diagnostics, optical microscopy remains the gold standard in pathology, cancer diagnosis, and disease monitoring. Considering that the optical principles of light microscopy and histological preparation methods have seen little change over decades or even centuries, the development of new microscopy and histological preparation methods seems to be long overdue.
Limitations of standard optical microscopy in pathology
In standard optical microscopy, light is transmitted through a specimen. Therefore, biopsied tissues need to be sectioned into thin slices, typically 4 to 6 microns in thickness, for the pathologists to be able to obtain high-resolution and high-contrast images that can provide meaningful histological information.1,2 However, obtaining such thin sections without impacting tissue architecture and integrity can be challenging.
When using optical microscopy to examine tissue specimens, tissues need to go through multiple processing steps before sectioning, making traditional histopathological analysis laborious. Typical pathology protocols involve tissue fixation, dehydration, paraffin embedding, sectioning, mounting onto glass slides, staining, and coverslipping. This multi-step process is often hard to standardize and significant variability can be introduced due to potential differences in tissue preparation and staining methods.3 Such variations may complicate pathological interpretation and diagnosis and thereby compromise patient care.
Furthermore, standard tissue preparation methods for optical microscopy are time-consuming and may delay diagnosis.4 Such delays in diagnosis and treatment may significantly affect treatment outcomes, especially in patients with aggressive, fast-growing tumors.
Microscopy with UV surface excitation
Microscopy with UV surface excitation (MUSE) has emerged as a promising microscopy method that offers rapid disease diagnosis by bypassing the need for time-consuming and cumbersome traditional tissue preparation procedures. This new technology was first described in 2015 by Stavros Demos (currently at the University of Rochester) and researchers at the University of California, Davis, in an article published in Proceedings of SPIE.5 This slide-free histological examination technology has been made available commercially by MUSE Microscopy Inc., a company founded in 2015.
MUSE lays its foundation on the fact that deep-UV light (~200–280 nm) has a penetration depth of approximately 10 microns, which is not much greater than the thickness of standard histology slides.6 As the penetration depth of deep-UV light is considerably smaller than that of visible light, this new microscopy method bypasses the requirement for tissue sectioning without compromising image quality and resolution.5
“MUSE eliminates any need for conventional tissue processing with formalin fixation, paraffin embedding or thin sectioning,” said Richard Levenson, professor at UC Davis, a co-author of the manuscript, and co-founder and CEO of MUSE Microscopy Inc.7
In MUSE, fresh tissue is placed on UV-transparent glass connected to an XYZ-stage. A light-emitting diode (LED) is used to emit UV light (~280 nm), which obliquely illuminates tissues briefly stained with fluorescent dyes. The emitted visible light is directed by a conventional microscope lens to a standard color camera.5
Advantages of MUSE over routine histological evaluation using optical microscopy
The most important advantage of histological evaluation using MUSE instead of standard optical microscopy is the fact that MUSE overcomes the need for laborious and time-consuming traditional histopathology techniques. In MUSE, tissues do not need to be fixed, embedded, or sectioned, considerably reducing the time to diagnosis, as well as the requirement for microtomes and toxic solvents.6
In a study comparing the diagnostic value of MUSE and traditional H&E slides, Qorbani et al.8 showed that MUSE identified most skin structures seen on routine H&E. Notably, the average acquisition time for MUSE images was only 5 minutes, considerably shorter than the time required for traditional histological examination involving tissue preparation and H&E staining.8
Biopsy tissues are generally scarce, and often all of the available material is used for pathological analyses using standard histology methods. As MUSE is a nondestructive imaging technique, unprocessed tissue can subsequently be used for other analyses, such as molecular testing.9 Preliminary findings published in Nature Biomedical Engineering suggest that MUSE imaging has no adverse effects on the results of subsequent molecular analyses, including RNA sequencing and fluorescence in situ hybridization (FISH).10
“It has become increasingly important to submit relevant portion of often tiny tissue samples for DNA and other molecular functional tests,” said Richard Levenson.7 “Making sure that the submitted material actually contains tumor in sufficient quantity is not always easy and sometimes just preparing conventional microscope slices can consume most of or even all of small specimens. MUSE is important because it quickly provides images from fresh tissue without exhausting the sample.”
As the quality of H&E staining is influenced by the staining protocol, the tissue preparation procedures, and the reagents used,9 MUSE offers higher reproducibility than routine histological evaluation methods by bypassing the need for tissue processing. Thus, MUSE has the potential to improve the accuracy of diagnosis.
Furthermore, as MUSE preserves tissue structure, it can provide three-dimensional information that is lost by thin sectioning during traditional histology.10 Hence, MUSE can provide valuable insight into aspects of tissue organization and architecture.
Advantages of MUSE over other slide-free histological evaluation techniques
In addition to MUSE, several other imaging techniques have been developed to evaluate fresh tissue specimens without the need for tissue fixation and sectioning. These techniques employ different methods to collect light from the target layer of the specimen while rejecting the light from the other layers of the tissue and thereby reducing image blurring.
For instance, optical coherence tomography enables nondestructive two- and three-dimensional imaging by employing low-coherence light and collecting light from the desired depth.11 Similarly, multiphoton microscopes can be used to image intact biological tissues by employing two or more photons of different wavelengths, thereby limiting out-of-focus flare.12 Structured illumination and light-sheet microscopy have also emerged as promising approaches to obtain images with high spatial resolution from thick tissue specimens.13
Compared with these imaging techniques, MUSE is based on less complex optical principles. MUSE uses a simpler optical setup, requiring only a LED (~280 nm), an objective, a tube lens, and a color camera.14 As the excitation light illuminates the specimen without passing through the objective first, no dichroic mirrors are required in MUSE. Additionally, no excitation filters are needed to filter out the excitation light because the lens itself blocks UV light.14
Unlike most multichannel fluorescence microscopes, MUSE can generate multicolor fluorescence images without requiring complex hardware as multiple fluorescent dyes in the sample can be excited by the single UV light source. In addition, emission filters are not needed in MUSE, and, thus, standard color cameras can be used to collect the emitted light.14 As image acquisition requires sub-second exposure times, MUSE allows for rapid imaging of large areas and immediate diagnosis.
“It [MUSE] doesn’t require lasers, confocal, multiphoton or optical coherence tomography instrumentation, and the simple technology makes it well suited for deployment wherever biopsies are obtained and evaluated,” said Richard Levenson.7
Limitations and perspectives of MUSE
The recent development of powerful deep-UV LED sources has made MUSE a feasible approach to analyze fresh tissue specimens. Despite the advantages of using MUSE in pathology, several technical limitations need to be addressed. Importantly, the fact that the LED source must be placed adjacent to the tissue specimen permitting only a short working distance for the objective, can make the positioning of the objective challenging. Therefore lenses with relatively long working distances need to be used.14
With a simultaneous decrease in cost and increase in power of LEDs, the requirement for lenses with high numerical aperture and objectives with long working distance is decreasing. However, powerful and affordable LEDs below 270 nm are currently lacking,5 so further development of LEDs emitting light lower than 280 nm may be needed to increase the flexibility and multiplexing ability of MUSE.
References
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8. Qorbani A, Fereidouni F, Levenson R, et al. Microscopy with ultraviolet surface excitation (MUSE): A novel approach to real-time inexpensive slide-free dermatopathology. J Cutan Pathol. 2018;45(7):498-503. doi:10.1111/cup.13255
9. M.D. RML, Harmany Z, Demos SG, Fereidouni F. Slide-free histology via MUSE: UV surface excitation microscopy for imaging unsectioned tissue (Conference Presentation). In: Alfano RR, Demos SG, eds. Optical Biopsy XIV: Toward Real-Time Spectroscopic Imaging and Diagnosis. Vol 9703. SPIE; 2016:100. doi:10.1117/12.2219407
10. Fereidouni F, Harmany ZT, Tian M, et al. Microscopy with ultraviolet surface excitation for rapid slide-free histology. Nat Biomed Eng. 2017;1(12):957-966. doi:10.1038/s41551-017-0165-y
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12. Benninger RKP, Piston DW. Two-photon excitation microscopy for the study of living cells and tissues. Curr Protoc cell Biol. 2013;Chapter 4:Unit 4.11.1-24. doi:10.1002/0471143030.cb0411s59
13. Chatterjee K, Pratiwi FW, Wu FCM, Chen P, Chen B-C. Recent Progress in Light Sheet Microscopy for Biological Applications. Appl Spectrosc. 2018;72(8):1137-1169. doi:10.1177/0003702818778851
14. Huang CZR, Wood RW, Demos SG. Adaptation of microscopy with ultraviolet surface excitation for enhancing STEM and undergraduate education. J Biomed Opt. 2018;23(12):1. doi:10.1117/1.jbo.23.12.121603
