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Light microscopy ZMB
Nicolas Schilling
Created on October 1, 2024
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Transcript
Widefield
Confocal
spinning disk
Multiphoton
lightsheet
Versatility
XY resolution
Z resolution
Thick sample compatibility
Live-cell friendliness
Image Fidelity
Multicolor
Temporal resolution
Provides high temporal resolution due to rapid simultaneous multi-point scanning, ideal for capturing fast cellular dynamics (Nakano, 2002; Graf et al., 2005).
Temporal Resolution
Moderate temporal resolution due to point-by-point scanning speed limitations.
Temporal Resolution
High fidelity in thick specimens due to minimal background noise and reduced scattering outside the focal plane (Denk et al., 1990; Helmchen and Denk, 2005).
Image Fidelity
Moderate image fidelity due to the presence of out-of-focus light, which reduces contrast and clarity in thicker samples (Diaspro et al., 2005).
Image Fidelity
Multiphoton Microscopy
Multiphoton microscopy utilizes near-infrared laser light to excite fluorophores through the simultaneous absorption of two or more photons, which occurs only at the focal point. This method restricts excitation to the focal plane, minimizing photobleaching and phototoxicity outside the imaging plane. It is particularly well-suited for deep tissue imaging in live animals and thick specimens (Denk et al., 1990; Helmchen and Denk, 2005).
Confocal laser scanning microscopy
Confocal microscopy uses a laser to illuminate a single point within the specimen and employs a pinhole in front of the detector to block out-of-focus light, enhancing optical sectioning and contrast. By scanning the laser across the sample in a raster pattern, confocal microscopy can generate high-resolution images of thin optical sections, allowing for three-dimensional reconstruction of the specimen. This technique is widely used in biological and medical research for imaging fixed and live samples, particularly where high resolution and optical sectioning are required (Pawley, 2006; Lichtman and Conchello, 2005).
Poor axial resolution because it captures all emitted light, including out-of-focus fluorescence, leading to significant loss of contrast and detail in the Z-axis (Diaspro et al., 2005). Deconvolution techniques can improve Z-resolution to some extent.
Z Resolution
Similar axial resolution to point-scanning confocal but in very thick samples, pinhole crosstalk (emission overspill between adjacent pinholes) can degrade axial resolution by allowing out-of-focus light to pass through, diminishing image clarity (Nakano, 2002; Graf et al., 2005).
Z Resolution
Limited lateral resolution due to the inclusion of out-of-focus light, which can cause blurring and reduce image clarity, especially in thicker samples (Diaspro et al., 2005).
XY Resolution
Provides good lateral resolution (~200 nm) due to the elimination of out-of-focus light using a pinhole aperture, though it is still limited by the diffraction of light (Lichtman and Conchello, 2005; Pawley, 2006).
XY Resolution
Effective for multicolor imaging with reduced phototoxicity, suitable for observing multiple fluorophores in live specimens (Huisken and Stainier, 2009; Power and Huisken, 2017).
Multicolor
Superior for live-cell imaging, enabling long-term observations with minimal phototoxicity and bleaching due to selective plane illumination (Huisken and Stainier, 2009; Power and Huisken, 2017).
Live-Cell Friendliness
Produces high-fidelity images with good contrast by rejecting out-of-focus light, though photobleaching can reduce signal over time (Pawley, 2006; Lichtman and Conchello, 2005).
Image Fidelity
High image fidelity with minimized photodamage and effective optical sectioning, making it ideal for 3D imaging of large samples (Huisken and Stainier, 2009).
Image Fidelity
Provides excellent axial resolution for thick tissues, thanks to localized excitation at the focal point and reduced out-of-focus fluorescence (Denk et al., 1990; Helmchen and Denk, 2005).
Z Resolution
Confocal Spinning Disk Microscopy
Spinning disk confocal microscopy uses a disk with multiple pinholes that rotate rapidly, allowing simultaneous multi-point illumination and detection. This design enables faster imaging with lower phototoxicity and photobleaching compared to traditional point-scanning confocal microscopy. Spinning disk confocal is particularly useful for live-cell imaging and capturing dynamic processes in real-time (Nakano, 2002; Graf et al., 2005).
Less versatile than other forms of microscopy due to specific requirements for fluorophores and longer excitation wavelengths. However, it excels in deep tissue imaging and live-animal studies (Denk et al., 1990).
Versatility
Better suited for thicker samples than point-scanning confocal due to reduced photobleaching and faster acquisition speeds(Nakano, 2002). However, in very thick samples, pinhole crosstalk (emission overspill between adjacent pinholes) can degrade axial resolution by allowing out-of-focus light to pass through, diminishing image clarity
Thick Sample Compatibility
Lower lateral resolution than confocal microscopy due to the use of longer excitation wavelengths, which decreases resolution but allows deeper tissue penetration (Zipfel et al., 2003; Helmchen and Denk, 2005).
XY Resolution
Similar versatility to point-scanning confocal but with enhanced speed and reduced phototoxicity, making it better suited for dynamic live-cell studies (Nakano, 2002).
Versatility
The resolution is primarily determined by the thickness of the light sheet and the NA of the detection objective (often water immersion objevtives of NA 1.0. Lateral (XY) resolution ranges around 200–300 nm.
XY Resolution
Confocal microscopy is suitable for live-cell imaging when laser power and pixel dwell time are carefully controlled to minimize phototoxicity (Pawley, 2006).
Live-Cell Friendliness
Highly versatile for imaging a wide range of samples, particularly thin or monolayer specimens. It is commonly used in cell biology, histology, and high-throughput screening (Diaspro et al., 2005).
Versatility
Superior temporal resolution due to fast plane illumination and orthogonal detection, ideal for dynamic live-cell imaging and real-time 3D reconstructions (Huisken and Stainier, 2009; Power and Huisken, 2017).
Temporal Resolution
Suitable for imaging moderately thick specimens, but performance declines due to photobleaching and reduced penetration depth (Lichtman and Conchello, 2005).
Thick Sample Compatibility
Widefield Microscopy
Widefield microscopy involves illuminating the entire specimen and capturing fluorescence from all focal planes. This technique provides rapid imaging and is particularly suited for thin specimens and high-throughput applications. However, it suffers from lower resolution and contrast for thick specimens due to significant out-of-focus light (Diaspro et al., 2005).
Capable of multicolor imaging but limited by the range of available excitation wavelengths and the need for specific fluorophores (Zipfel et al., 2003).
Multicolor
Suitable for live-cell imaging in thick tissues, though requires careful control of laser power to avoid photodamage (Helmchen and Denk, 2005).
Live-Cell Friendliness
Highly suitable for live-cell imaging, particularly for thin specimens, due to its rapid imaging capabilities and low phototoxicity (Diaspro et al., 2005).
Live-Cell Friendliness
Supports multicolor imaging with minimal spectral overlap, making it ideal for complex imaging studies (Pawley, 2006).
Multicolor
Highly suitable for live-cell imaging due to rapid image acquisition and lower phototoxicity (Graf et al., 2005).
Live-Cell Friendliness
Offers improved axial resolution over widefield microscopy by optically sectioning, but resolution is limited by the size of the pinhole and diffraction effects (Pawley, 2006; Lichtman and Conchello, 2005).
Z Resolution
Highly compatible with thick samples due to its ability to penetrate deep into tissues with minimal scattering and photodamage, making it ideal for in vivo imaging (Denk et al., 1990).
Thick Sample Compatibility
Confocal microscopy is highly versatile and can be adapted for a wide range of biological imaging applications, including live-cell imaging, fixed tissue imaging, and various advanced techniques like fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP).
Versatility
Offers moderate temporal resolution due to the time required for point-by-point scanning (Pawley, 2006).
Temporal Resolution
Source Zeiss
Light-Sheet Microscopy
Light-Sheet Fluorescence Microscopy (LSFM) uses a thin sheet of laser light to illuminate a single optical plane of the specimen while an orthogonal detection system captures fluorescence from the illuminated plane. This method allows fast, high-resolution imaging of large, live specimens with minimal phototoxicity, making it ideal for developmental biology and neuroscience applications (Huisken and Stainier, 2009; Power and Huisken, 2017).
Effective for multicolor imaging but can suffer from cross-talk between channels, especially in thicker samples (Diaspro et al., 2005).
Multicolor
Provides excellent temporal resolution because it captures the entire field of view in a single exposure, making it ideal for fast imaging applications (Diaspro et al., 2005).
Temporal Resolution
Comparable to point-scanning confocal, but potential slight blurring can occur due to light scatter from the disk (Nakano, 2002; Graf et al., 2005).
XY Resolution
The resolution is primarily determined by the thickness of the light sheet and the NA of the detection objective (often water immersion objevtives of NA 1.0. Axial (Z) resolution typically reaches 300–500 nm.
Z Resolution
Highly compatible with thick samples due to the thin light sheet that selectively illuminates one plane at a time, minimizing photobleaching and photodamage (Huisken and Stainier, 2009).
Thick Sample Compatibility
Versatile, allowing rapid 3D imaging of large samples such as embryos or entire organs. It is highly suitable for live imaging and developmental studies (Huisken and Stainier, 2009).
Versatility
Not suitable for thick specimens due to the substantial accumulation of out-of-focus light, which severely degrades image quality (Diaspro et al., 2005).
Thick Sample Compatibility
Excellent for multicolor imaging with appropriate filter sets, minimizing spectral crosstalk (Nakano, 2002).
Multicolor
High image fidelity, though slightly lower than single-point confocal due to potential light scatter from the disk (Nakano, 2002).