Microscopy in Oncology Research: Visualizing Tumor Microenvironments

Microscopes have evolved significantly since their first discovery in the 17th century by an English scientist, Robert Hooke. The current high-powered microscopes have enabled visualization of individual cells.

Single-molecule imaging helps track the movement of molecules in a cell.1 The advancements in microscopy have played an important role in cancer research. This article focuses on how different types of microscopy help study tumor microenvironments. 

Image Credit: Gorodenkoff/Shutterstock.com​​​​​​​Image Credit: Gorodenkoff/Shutterstock.com

Microscopy in Oncology Research

Cancer is associated with abnormal growth of cells in an organ, and it can invade nearby tissues through the lymphatic system and blood. This disease accounts for a significant number of deaths worldwide. Over the years, researchers have identified many biological, environmental, and lifestyle factors that increase cancer risk.2 

The recent high-powered microscopes enable scientists to study tumor microenvironments in greater detail.3 Recent methods associated with binding fluorescent probes to protein and DNA in living cells enable long-term observation of living cells. This type of microscopy enabled the visualization of cellular processes in real-time. It also helps determine the timescale of protein integration in cells.4

Real-time imaging of cellular processes also entails visualization of chromosome translocation in living cells. Anomalies in chromosome formation are inherently associated with cancer biology. 

Microscopy has opened up new avenues to learn about the workings of cells. This has helped researchers to understand the events that drive the transformation of healthy cells into malignant cells. Automation in microscopy has saved a significant amount of time, which was required to prepare biological samples for observation.5

Implementing robotics and automated imaging has enabled the processing of numerous samples simultaneously. Specific images of the samples can be collected and processed using computers linked with advanced microscopy-based software. This method helps study protein behavior and identify genes responsible for cancer development.

Types of Microscopy Used in Oncology Research

Early cancer diagnosis is correlated with survival rate. A thorough examination of tissue samples is the basic diagnostic technique for cancer diagnosis. Microscopies, such as hyperspectral imaging, phase-contrast imaging, and optical coherence tomography are used to detect malignant tumors. Some of the common microscopies used in oncology research are discussed below:

 Confocal Microscopy 

Confocal microscopy enables in vivo imaging with ‘en face’ orientation, i.e., the image is parallelly oriented to the surface of the tissue. This type of microscope is commonly used to detect oral cancer.

Some key challenges of the hand-held miniature version of confocal microscopy are the lack of high-resolution imaging, limited fast frame rates, and improper image depth, which is essential for diagnosis.6

Confocal fluorescence microscopy (CFM) is another non-invasive imaging technique that enables in vivo imaging of untreated biological tissues at cellular-level resolution.

In contrast to the hand-held miniaturized version, CFM provides high-resolution three-dimensional imaging by inhibiting any out-of-the-focus plane using a pinhole in front of the detector. The different aperture sizes of the pinhole can be used for selective focal plane imaging of the sample.7 

CFM has been used to detect colorectal cancer during colonoscopy. Two key disadvantages of CFM are loss of the fluorescence signal and intense photobleaching. Reflectance confocal microscopy (RCM) is used to detect oral squamous cell carcinoma.8

Second-Harmonic Generation Microscopy

Second-harmonic generation (SHG) microscopy is based on the interaction of multiple photons with biological samples. This is a non-linear technique that offers low photon toxicity and provides 3D imaging of organs and tissues.

Collagen, present in the extracellular matrix (ECM), undergoes specific morphological changes per cancer stage. These changes can be observed via SHG microscopy. Furthermore, the metastatic stage of cancer can be detected through the SHG signal ratio from fibrillar collagen.9

Photoacoustic Microscopy And Tomography

This type of microscopy has been used in both in vitro and in vivo imaging to visualize tumor growth over the past decade. This technique is based on the optical excitation and acoustic detection modality.

This is a label-free technique for tissue analysis using endogenous chromophores, such as cytochromes in mitochondria, hemoglobin in red blood cells, and melanin in melanosomes.10 

In this microscopic method, blood oxygen saturation can be measured based on the spectral differences between deoxyhemoglobin and oxyhemoglobin. These measurements are compared with other tumor hypoxia imaging methods, such as blood oxygen level-dependent magnetic resonance imaging.

Atomic Force Microscopy

Atomic force microscopy (AFM) indicated the formation of needle-like structures on the surface of leukocytes in leukemia patients.

This microscopic technique also revealed a higher prevalence of cell surface roughness than normal white blood cells. Compared to optical microscopes, the resolution of AFM is higher and can highlight the differences in cells’ ultrastructure. The membrane surface of different types of tumors can be visualized through AFM.11 

Images of cell surface roughness obtained via AFM indicate different stages of human breast cancer tissues. AFM also differentiates the cell membrane morphology between tumor and normal cells. 

Laser scanning microscopy

It is important to remove cancerous tissues with great precision and minimum incisions. Laser scanning oncology is based on the combination of laser-scanning microscopes and fluorescent tumor markers.

This microscopy is used in the operation theatre to assess whether all tissues surrounding the area of the tumor were removed during the surgical procedure.

Multiphoton Microscopy (MPM)

MPM is a detection technique combining two-photon excited fluorescence (TPEF), and second harmonic generation (SHG). The major advantages of this microscopy are real-time, in vivo, and high-resolution imaging.

Two MPM-based strategies, namely, multiphoton tomography (MP) and multiphoton tomography with fluorescence lifetime imaging (MPT-FILM) have been developed to study unlabeled fresh samples.12

Super-resolution microscopy

This robust and high-speed imaging microscopy is used to visualize higher-order chromatin structure on pathological tissue. This technique images significant fragmentation in DNA folding in the early stage of carcinogenesis.13

Future of Microscopy in Oncology Research

In the future, researchers must find a way to simultaneously optimize imaging speed, spatial resolution, photodamage, and signal-to-noise ratio. The current microscopies largely fail to cope with the unpredictability of biological events.

The majority of microscopic techniques cannot image biomolecules that cannot be easily labeled. There is a need to develop both hardware and software solutions to combat the aforementioned challenges. 

In the future, developing “smart” microscopes with well-expanded multiplexed molecular labeling technologies will play a crucial role in shaping imaging science.14 


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  2. Seyfried TN, Shelton LM. Cancer as a metabolic disease. Nutr Metab (Lond). 2010;7:7. https://nutritionandmetabolism.biomedcentral.com/articles/10.1186/1743-7075-7-7
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  8. Levine A, Markowitz O. Introduction to reflectance confocal microscopy and its use in clinical practice. JAAD Case Rep. 2018;4(10):1014-1023.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232695/
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  12. Treacy PJ, Khosla A, Kyprianou N, et al. Value of multiphoton microscopy in uro-oncology: a narrative review. Transl Androl Urol. 2023;12(3):508-518. https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-018-0428-0
  13. Liu Y, Xu J. High-resolution microscopy for imaging cancer pathobiology. Curr Pathobiol Rep. 2019;7(3):85-96. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500261/
  14. Balasubramanian, H., Hobson, C.M., Chew, TL. et al. Imagining the future of optical microscopy: everything, everywhere, all at once. Commun Biol 6, 1096 (2023). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500261/

Further Reading

Last Updated: Jun 19, 2024

Dr. Priyom Bose

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.


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