Light Microscopy PDF

**Light Microscopy** **What is Light Microscopy?** Definition Uses light to view, discover, and magnify small objects. Light Source Utilizes the visible spectrum of light (white light). White light spans from UV range (\~200 nm) to near-infrared (\~700 nm). **Components of a Brightf...

**Light Microscopy** **What is Light Microscopy?** Definition Uses light to view, discover, and magnify small objects. Light Source Utilizes the visible spectrum of light (white light). White light spans from UV range (\~200 nm) to near-infrared (\~700 nm). **Components of a Brightfield Microscope :** Light Source : Usually a halogen bulb. Focused by a condenser to illuminate the specimen. Specimen : Typically stained tissue or cells for contrast. Collects transmitted light and focuses it to produce the image , First point of magnification (4x to 100x). **Imaging Modalities in Brightfield Microscopy :** Brightfield Imaging : Utilizes white light through a contrasted specimen. Areas of high contrast absorb transmitted light. Applications : Useful for diagnosing disease states from patient samples. Allows for long-term imaging of samples. Limitations Cannot image thick tissue or multiple optical sections. Live cells cannot be counterstained due to dye toxicity. **Alternative Imaging Techniques :** Techniques for Unstained Samples Adjusting light paths enhances contrast without staining. Differential Interference Contrast (DIC) Excellent for unstained samples like cells, nematodes, and bacteria. Requires additional components such as polarizers and a compensator. **Differential Interference Contrast (DIC)** : Polarizer splits light into a single linear beam. Beam traverses the first amass q prism, splitting into two perpendicular beams. Waves alter based on sample thickness and refractive index. Waves recombined in a second prism provide contrast. Positive/negative bias can alter the appearance of optical paths. Often combined with fluorescence microscopy for unstained living samples. **Phase Contrast Microscopy :** Enhances contrast for low contrast or unstained samples. More effective for thinner samples. Requires a condenser annulus and a phase plate. Condenser annulus produces a hollow cone of light. Phase shift occurs due to diffraction by the specimen. Results in bright or less bright regions in the image. **Polarized Light Microscopy :** Requires two polarizers. First polarizer converts light into a linear wave. Birefringent specimens exhibit double refraction. Light refracted into two rays: ordinary and extraordinary waves. Bright or dark regions produced by constructive or destructive interference. **Darkfield Microscopy :** Contrasts bright specimens against a dark background. Requires a disk or stop in the light path. Only oblique rays illuminate the sample. Scattered light enters the objective lens, creating bright images. **Sample Preparation Considerations :** Some techniques require fixation. Fresh frozen tissue can be cut with cryo microtome or vibra tone. Sample thickness is crucial for light transmission. Poor penetration may occur with incorrect thickness. Proper labeling and staining techniques are important. **Image Acquisition Optimization :** Proper alignment of incident light or white light is crucial. Avoids uneven illumination, glare, and shadowed portions (veneering). Maximizes contrast and improves resolution. Each objective change requires matching light settings. Align conjugate planes in the light path for full field illumination. Adjust diaphragms: condenser aperture diaphragm and field diaphragm. **Color Illumination :** Condenser aperture impacts image quality. Open aperture allows excessive light, causing glare. Closed aperture darkens image and introduces refraction artifacts. Correctly collated system balances detail visibility and contrast. **Numerical Aperture (NA) :** NA determines the resolving power of an objective. Takes into account refractive index and cone of light angle (theta). Higher NA captures more light, improving image signal. **Refractive Index :** Measures light ray bending between media. Denser materials like water and glycerin enhance NA. Essential for understanding image resolution. **Resolution in Microscopy :** Ability to differentiate close points in images. Dependent on numerical aperture. Higher resolution images show clearer details upon zooming. **Camera Types and Imaging :** CCD vs. CMOS cameras. CCD: More power, low noise images. CMOS: Less power, faster but noisier images. Cameras convert photons to digital images through pixels. Pixel intensity represented from black to white with gray tones. **Digital Image Quality :** Affected by camera megapixels and bit depth. Higher megapixel cameras yield better quality images. Bit depth determines tone variation. **Magnification :** Process of enlarging object size in microscopy. Total magnification = Objective magnification + Eyepiece magnification. **Lookup Table (LUT):** Represents digital image pixel intensity distribution. Capturing maximum dynamic range is essential for image quality. **Dynamic Range and Lookup Tables :** Importance of lookup tables Helps visualize maximum dynamic range Ensures capturing maximum image information **Over and Under Saturation :** Definition of saturation : Measurement of pixel saturation in images Good images have few over or under saturated pixels Effects of saturation on data Over/under saturated pixels are rejected by the camera Results in substandard analysis **Image Comparison :** Analysis of images with saturation settings Left image has few red/blue pixels (good) Right image has excessive red/blue pixels (bad) **White Balancing:** Purpose of white balancing Removes color cast from light sources or optics Reflects true color of the sample Example of unbalanced vs. balanced images Unbalanced images show color casts Balanced images provide true representation **Exposure Time :** Importance of exposure time Affects over and under saturation of pixels Examples of exposure effects on images Low exposure: dim image, indistinct details Optimized exposure: well-lit, noticeable details High exposure: overexposed, glare, indistinct structures