Microscope in Cardiology: A Comprehensive Guide for Physics Students

Summary

Microscopy has become an indispensable tool in the field of cardiology, enabling researchers and clinicians to measure various quantifiable data, including cellular mass, volume, and density. This comprehensive guide delves into the intricacies of two primary approaches used in microscopy for cardiac applications: noninterferometric quantitative phase microscopy (NIQPM) and Hilbert transform differential interference contrast microscopy (HTDIC). Additionally, the use of intravital microscopy to study the beating heart in mice is explored, highlighting the Prospective Sequential Segmented Microscopy (PSSM) technique.

Noninterferometric Quantitative Phase Microscopy (NIQPM)

NIQPM is a powerful technique that allows for the measurement of total cell mass and subcellular density distribution. This method is based on a simplified model of wave propagation, with three underlying assumptions:

1. Low Numerical Aperture (NA) Illumination: The use of low NA illumination ensures that the light scattering and absorption by the specimen are weak, simplifying the wave propagation model.

2. Weak Scattering: The specimen under investigation must exhibit weak scattering properties, which is typically the case for biological samples.

3. Weak Absorption: The specimen must also have weak absorption characteristics, allowing the light to propagate through the sample with minimal attenuation.

The NIQPM technique utilizes a combination of bright field and differential interference contrast (DIC) imagery to extract quantitative information about the specimen. By analyzing the phase shifts and intensity variations in the light passing through the sample, researchers can determine the total cell mass and the distribution of subcellular densities.

NIQPM Theoretical Foundations

The NIQPM method is based on the following theoretical principles:

1. Wave Propagation Model: The wave propagation through the specimen is described by the Helmholtz equation, which governs the behavior of electromagnetic waves in a medium.

2. Phase Shift Calculation: The phase shift experienced by the light as it passes through the specimen is directly proportional to the product of the specimen’s refractive index and thickness.

3. Intensity Variation Analysis: The intensity variations in the bright field and DIC images are used to extract information about the specimen’s refractive index and thickness, which can then be used to calculate the total cell mass and subcellular density distribution.

NIQPM Practical Considerations

While NIQPM offers a powerful approach for quantitative microscopy in cardiology, there are a few practical considerations to keep in mind:

1. Specimen Size Constraints: The NIQPM method is limited to specimens with diameters ranging from 0.2 to 10 micrometers, due to the effects of diffraction.

2. Temporal Constraints: The slow z-stack acquisition time on commercial microscopes currently restricts the investigation of phenomena faster than 1 frame per minute.

3. Illumination Requirements: The NIQPM technique requires low NA illumination, which may limit the achievable spatial resolution and signal-to-noise ratio.

Hilbert Transform Differential Interference Contrast Microscopy (HTDIC)

HTDIC is another approach used in microscopy for cardiac applications, focusing on the acquisition of volumetric information from through-focus DIC imagery under high NA illumination conditions.

HTDIC Principles

The key principles underlying HTDIC are:

1. High NA Illumination: The use of high NA illumination enables enhanced sectioning of the specimen along the optical axis, providing improved depth information.

2. Hilbert Transform Processing: The Hilbert transform is applied to the DIC image stacks, which greatly enhances edge detection algorithms for the localization of specimen borders in three dimensions. This is achieved by separating the gray values of the specimen intensity from those of the background.

HTDIC Advantages and Limitations

The primary advantages of HTDIC include:

1. Volumetric Information: HTDIC allows for the acquisition of three-dimensional information about the specimen, including its size, shape, and spatial distribution.

2. Technological Accessibility: Like NIQPM, HTDIC can be implemented using “off-the-shelf” microscopes, making it a relatively accessible technique.

However, HTDIC also has some limitations:

1. Specimen Size Constraints: The HTDIC method is limited to specimens with diameters up to 20 micrometers, due to the effects of diffraction.

2. Temporal Constraints: Similar to NIQPM, the slow z-stack acquisition time on commercial microscopes restricts the investigation of phenomena faster than 1 frame per minute.

Intravital Microscopy for Cardiac Applications

In addition to NIQPM and HTDIC, intravital microscopy has emerged as a powerful tool for studying the beating heart in mice. This approach allows for the direct observation and measurement of cardiac dynamics in a living organism.

Prospective Sequential Segmented Microscopy (PSSM)

One specific technique used in intravital microscopy for cardiac applications is Prospective Sequential Segmented Microscopy (PSSM). PSSM adopts a prospectively triggered acquisition scheme, which enables precise synchronization of image acquisition with the cardiac cycle.

PSSM Advantages

1. Exact Reproducibility: PSSM provides exact reproducibility in the cardiac cycle, as it is not subject to physiologic variations in heart rate or rhythm.

2. Motion Artifact Reduction: By shifting the phase between image acquisition and the pacemaker signal, and grouping together all ‘segments’ that belong to the same time points of the cardiac cycle, PSSM can obtain motion artifact-free image reconstructions of the heart at all phases of the contractile cycle.

PSSM Technical Details

The PSSM technique involves the following steps:

1. Pacemaker Signal Acquisition: A pacemaker signal is used to trigger the image acquisition, ensuring precise synchronization with the cardiac cycle.

2. Phase Shifting: The phase between the image acquisition and the pacemaker signal is shifted, allowing for the reconstruction of the heart at different time points in the cardiac cycle.

3. Segmentation and Grouping: The acquired images are grouped together based on their corresponding time points in the cardiac cycle, enabling the reconstruction of motion artifact-free images.

By leveraging the PSSM technique, researchers can obtain detailed, high-resolution images of the beating heart in mice, which can provide valuable insights into cardiac function and pathophysiology.

Conclusion

Microscopy has become an indispensable tool in the field of cardiology, enabling researchers and clinicians to measure various quantifiable data, including cellular mass, volume, and density. The two primary approaches discussed in this guide, NIQPM and HTDIC, offer unique capabilities and advantages, while also having their own limitations. Additionally, the use of intravital microscopy, particularly the PSSM technique, has opened up new avenues for studying the beating heart in living organisms.

As physics students, understanding the principles, applications, and limitations of these microscopy techniques in cardiology is crucial for advancing our understanding of cardiac function and developing new diagnostic and therapeutic approaches. By mastering the technical details and practical considerations outlined in this guide, you can become well-equipped to contribute to the exciting field of biomedical imaging and microscopy.

References

1. Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope. (n.d.). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162510/
2. Imaging the beating heart in the mouse using intravital microscopy. (n.d.). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380003/
3. Why bioimage informatics matters. (n.d.). https://doi.org/10.1038/nmeth.2024