Light microscopy is essential for clinical diagnosis and studying cancer's progression. However, traditional light-field microscopes have limitations when it comes to visualising tissue abnormalities and nuclear morphology. Fortunately, new imaging techniques have emerged in recent years, enhancing both basic and clinical research in pathobiology. One of these techniques is epifluorescence microscopy, which has found extensive use in life sciences research labs and can be an invaluable tool when properly optimised.
Basics of epifluorescence microscopy
Epifluorescence microscopy (also known as Fluorescent Widefield Microscopy) is a type of fluorescence microscopy in which both the excitation and emission light travel through the same objective lens. The sample is illuminated with a high-intensity light source, and the resulting fluorescent light is separated from the surrounding radiation with filters, allowing the observer to see only the fluorescing material.
When it comes to fluorescence and epifluorescence microscopy, the key distinction lies in the path of the excitation and emission light. In fluorescence microscopy, these light paths are separate. On the other hand, in epifluorescence microscopy, both the excitation and emission light pass through the same objective lens. This setup is known as epifluorescence microscopy because "epi" means "same" in Greek. Epifluorescence microscopy proves particularly useful when imaging thick samples that are over 10µm deep. The illumination beam can penetrate the entire depth of these samples, allowing for effortless observation.
| Excitation filter | This filter only allows light of specific wavelengths to pass through, which is used to excite the fluorophores in the specimen |
| Objective lens | This lens is used to focus the excitation light onto the specimen and to collect the fluorescence emitted by the specimen |
| Dichroic lens | This lens reflects the excitation light towards the specimen and allows the fluorescence emitted by the specimen to pass through to the detector |
| Emission filter | This filter only allows light of specific wavelengths emitted by the fluorophores to pass through to the detector |
| Tube lens | This lens is used to focus the fluorescence emitted by the specimen onto the detector |
| Image | The image produced by epi-fluorescence microscopy shows the fluorescence of specific fluorophores in the specimen |
Biomarkers and Specific Markers
In epifluorescence microscopy, biomarkers play a crucial role in identifying specific cells or tissues, including cancer cells or other pathological conditions. These biomarkers are molecules that help to illuminate the sample under examination. The most commonly utilised markers are fluorescent proteins, like green fluorescent protein (GFP), and fluorescent dyes, such as fluorescein and rhodamine. When exposed to light of a particular wavelength, these markers re-emit light at a longer wavelength, which can then be detected under the microscope.
The markers used in epifluorescence microscopy vary depending on the type of sample under analysis. In cancer diagnosis, specific markers are employed to identify cancer cells. One example is HER2, which is found to be overexpressed in certain types of breast cancer. Fluorescently labelled antibodies that bind to HER2 are used to visualize the protein. The antibodies are conjugated to a fluorophore and can be excited by light with a wavelength of 488 nm. Another marker is EGFR, which is overexpressed in certain types of lung cancer and can be excited by light with a wavelength of 405 nm. These markers serve as valuable tools for guiding localised cancer therapy and monitoring treatment response.
Epi-fluorescence Microscopy in Cancer Diagnosis
Epi-fluorescence microscopy is also used in various ways in cancer diagnosis, including:
- Detection of cancer stem cells: One way to improve cancer diagnosis and treatment is by using epifluorescence microscopy to detect and isolate cancer stem cells. This technique relies on specific biomarkers expressed by these cells, which allows for the development of targeted therapies that specifically address cancer stem cells. By identifying and targeting these cells, we can enhance the effectiveness of cancer treatment strategies.
- Tumour microenvironment analysis: Epifluorescence microscopy has been used to study the tumour microenvironment, specifically examining how cancer cells interact with stromal cells. This analysis holds promise for identifying potential targets for cancer treatment and enhancing the effectiveness of current therapies.
- Cellular imaging and drug response assessment: epifluorescence microscopy has been used to study how cancer cells respond to anti-cancer drugs, including evaluating drug resistance and toxicity. By utilising this method, experts can optimise the selection and dosage of drugs in cancer therapy.
- Detection of circulating tumour cells: One way to monitor cancer progression and evaluate the treatment response is by using Epifluorescence microscopy to detect circulating tumour cells in patients' blood. This technique allows for an examination of disease status and can be a valuable tool in cancer management.
Use of Epifluorescence Microscopy in Cancer Therapy
Epifluorescence microscopy is used in various ways in cancer therapy, including:
- Photodynamic therapy (PDT): Epifluorescence microscopy is used to track the accumulation and photobleaching of the photosensitiser. This provides real-time monitoring of the procedure, enabling evaluation of the depth at which the photosensitiser localises in cases involving topical administration or intravenous injection. Real-time monitoring of PDT helps optimise treatment and minimise harm to healthy tissue.
- Fluorescence-guided surgery: Epifluorescence microscopy lets surgeons precisely define tumour location and margins during surgery, resulting in more complete resections. By avoiding unnecessary damage to normal tissue, this approach improves safety, reduces operative time, and minimises the need for additional surgeries.
(Source: https://opg.optica.org/boe/fulltext.cfm?uri=boe-6-12-5063&id=333112)
- Drug delivery and nanoparticle tracking: Epifluorescence microscopy has proven helpful in tracking drug delivery and nanoparticle movement during cancer therapy. This technique aids in the optimisation of drug administration, ultimately enhancing the effectiveness of cancer treatment.
- Cellular imaging and drug response assessment: epifluorescence microscopy can be used to evaluate how cancer cells react to anti-cancer drugs, including examining drug resistance and toxicity. This valuable tool aids in optimising the selection and dosage of anti-cancer medications for effective cancer therapy.
(Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712660/pdf/JBO-022-116010.pdf)
New Biomarkers and Techniques for Epifluorescence Microscopy in Cancer Therapy and Diagnosis
Researchers are constantly developing new methods to enhance the effectiveness of epifluorescence microscopy in diagnosing and treating cancer. One promising approach involves using quantum dots as fluorescent probes. Quantum dots are tiny semiconductor nanocrystals that emit light when stimulated by a light source. They offer several advantages over traditional fluorescent dyes, including brighter and more stable fluorescence, as well as a wider range of excitation wavelengths. Another innovative technique is super-resolution microscopy, which enables the visualisation of cellular structures with resolution beyond the limitations imposed by the diffraction of light. This advanced imaging method allows for a detailed examination of cellular organisation and dynamics, providing valuable insights into the mechanisms underlying cancer development and progression.
In addition to traditional methods, researchers are also working on developing new biomarkers that can enhance the detection of cancer cells and pathological conditions. One such promising avenue is the study of exosomes, which are small vesicles released by cells and contain important biomolecules like proteins and nucleic acids. Studies have shown that exosomes play a significant role in cancer progression and metastasis, making them potential biomarkers for cancer diagnosis and prognosis. Other potential biomarkers being investigated for their role in cancer diagnosis and therapy include microRNAs, circulating tumour cells, and cancer stem cells.
(Source: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0253222#sec011)
Combining Epifluorescence Microscopy with Other Imaging Techniques to Improve Cancer Therapy and Diagnosis
Combining epifluorescence microscopy with other imaging techniques can enhance cancer therapy and diagnosis. One such combination is epi-fluorescent light microscopy and confocal microscopy. By utilising a pinhole to eliminate the out-of-focus light, the resolution and contrast of images improve significantly. This powerful technique allows for the examination of the three-dimensional structure of cells and tissues, enabling a better understanding of the mechanisms underlying cancer at both the cellular and molecular levels.
In addition to its standalone applications, pi-fluorescent light microscopy can be combined with other imaging techniques, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), to enhance cancer diagnosis and treatment. While MRI allows the visualisation of tissue anatomy, PET provides insights into tissue metabolism. By combining epifluorescence light microscopy with MRI and PET, researchers gain a comprehensive understanding of tissue structure and function, enabling more accurate cancer diagnosis and tailored treatment approaches.
One example of advanced imaging techniques in cancer research is the use of epifluorescence microscopy combined with MRI. By visualising the distribution of fluorescently labelled nanoparticles in tumours, researchers can enhance drug delivery and improve cancer treatment effectiveness. Similarly, by combining epifluorescence microscopy with PET, scientists can observe how radiolabeled tracers are absorbed in tumours, enabling them to monitor disease progression and evaluate response to treatment. These innovative approaches offer valuable insights for optimising cancer therapy.
Choosing the right microscope setup for Epifluorescence Microscopy
As a researcher, there are several tips that can help you optimise your use of Epi-fluorescent microscopy. Firstly, it is important to critically assess every step of your imaging experiment, from design to execution to communication to data management, for bias, rigour, and reproducibility. Secondly, when selecting a research microscope, consider the type of specimen you want to explore, as well as the characteristics of the microscope that are essential for your experiment. For example, if you are working with living cells, an inverted microscope with a motorized objective revolver and an automatic focus adjustment may be necessary. Thirdly, it is important to select fluorophores that are spaced as far apart as possible to reduce bleed-through. Fourthly, try the gentlest excitation light intensity and check if you can observe the signal in a realistic exposure time. Finally, it is important to use the right microscopy tools and techniques to obtain high-quality images without affecting cell viability.
With the above said, a good Epi-fluorescent microscope should have the following features:
- LED Light Source: A fluorescent microscope should use a discreet LED light source that is controllable from the stand and is recommended for use on multi-colour fluorescent samples. Single channel illumination is preferable, as it decreases autofluorescence. Look for stable and homogenous illumination with very low consumable cost.
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High-resolution sCMOS camera: A high-resolution sCMOS camera can also enable researchers to observe cellular processes and dynamics in real time. This can be especially valuable for studying complex biological structures or identifying particular cells or tissues, including cancerous cells and other pathological conditions. By observing real-time cellular processes and dynamics, researchers can gain a more comprehensive understanding of the specimen and pinpoint potential targets for cancer treatment.
For example, the Moticam series microscope cameras have ultra high-resolution options such as the Moticam S20 for 20-megapixel capture resolution, or the Pro S5 Plus that has a global shutter and better scanning FPS. Different cameras provide different strengths that can be used for various applications.
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Motorized stage: The motorized stage feature enables researchers to scan larger areas of the sample, providing a more comprehensive understanding of the sample under examination.
The PA53 FS6 Scan series has a 100mm x 100mm high-precision motorized stage with the smallest step size of 10nm (repeatability better than 200nm). This feature allows for relocation even with a 100x objective, making it possible to mark individual features on a Whole Slide Scan (WSI).
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Fluorescence filter cubes: Depending on application, a user can choose to use a multi-band cube for convenience, or dedicated cubes for each channel for more accurate color representation.
The PA53 BIO FS6 models come with a 6-position encoded fluorescence turret that can handle multi-colour staining in combination with a channel-merge software plug-in. The standard configuration of PA53 BIO FS6 comes with DAPI, FITC, and TRITC filter cubes, but many more can be added.
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| PA53 BIO FS6 | PA53 BIO Scan | PA53 FS6 Scan |
Conclusion
Epifluorescence microscopy has revolutionised the field of cancer diagnosis and therapy. This powerful tool allows scientists to visualise cellular events in real-time, down to the molecular level. By doing so, it helps researchers uncover the intricate mechanisms of underlying cancer development. Epifluorescence microscopy finds numerous applications in cancer diagnosis and therapy, such as identifying cancer stem cells, guiding localised treatment approaches, and monitoring treatment response. Furthermore, when combined with other imaging techniques, epifluorescence microscopy provides complementary information about tissue structure and function. This comprehensive approach enhances both cancer diagnosis and treatment strategies. Scientists are continuously developing new biomarkers and refining the techniques to further optimise the use of epifluorescence microscopy in treating cancer patients. These advancements not only contribute to a deeper understanding of cancer but also improve patient outcomes significantly.
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