Microscopy, Mitosis, Cells, Tissues Lab 1 Flashcards
What are the parts of Compound Light Microscope?
The main parts of a Compound Light Microscope include:
Eyepiece (Ocular Lens):
The lens at the top of the microscope that you look through, usually with a magnification of 10x or 15x.
Body Tube:
Connects the eyepiece to the objective lenses, ensuring proper alignment of the optical components.
Nosepiece (Revolving Turret):
Holds the objective lenses and rotates to switch between different magnifications.
Objective Lenses:
Typically, there are 3-4 lenses (e.g., 4x, 10x, 40x, 100x oil immersion) with varying magnifications.
Stage:
The flat platform where the slide is placed. It often includes clips or a mechanical stage to hold the slide in place.
Stage Clips:
Secure the slide in position on the stage.
Coarse Adjustment Knob:
Moves the stage up and down quickly to bring the specimen into rough focus.
Fine Adjustment Knob:
Allows for precise focusing by moving the stage in smaller increments.
Diaphragm (Iris):
Controls the amount of light passing through the specimen. It can be adjusted to enhance contrast.
Illuminator (Light Source):
Provides the light needed to view the specimen. Older models use mirrors to reflect ambient light, but modern microscopes often have built-in electric light sources.
Condenser:
Focuses light onto the specimen. It is located below the stage.
Arm:
Supports the body tube and connects it to the base. It is used to carry the microscope.
Base:
The bottom part of the microscope that provides stability and houses the light source (if applicable).
Mechanical Stage Controls:
Knobs that allow you to move the slide left, right, forward, or backward with precision.
What is resolution?
the level of detail, ability to see two neighboring objects as distinct entities
What is limit of resolution?
Smallest distance for two points to be separated and discerned as separate entities
3 Factors that govern resolution?
- Wavelength of the light
- Angular aperture
- Refractive index of the medium
What is field of view?
The area that you can see through the ocular lens and objective
What is the depth of field?
Speciment thickness that is in focus at one focal setting, which decreases as magnification increases.
What is a stereomicroscope?
A stereomicroscope, also known as a dissecting microscope, is a low-magnification optical microscope designed for observing larger, three-dimensional specimens. Unlike a compound microscope, it uses two separate optical paths to provide a three-dimensional view of the sample, making it ideal for detailed surface observations. Each eye receives a slightly different image due to the separate optical paths, which enhances depth perception. Fine dissection work.
What are the different types of light microscopy?
Brightfield Microscopy:
The most common and basic form of light microscopy.
Illuminates the sample with white light, and the specimen appears darker against a bright background.
Best for observing stained or naturally pigmented samples.
Phase-Contrast Microscopy:
Enhances contrast in transparent, unstained specimens by exploiting differences in refractive indices.
Commonly used for observing live cells, organelles, and thin tissue slices.
Fluorescence Microscopy:
Uses high-energy light (UV or blue) to excite fluorescent molecules in the sample, which then emit light at longer wavelengths.
Allows for specific labeling of cellular components and is widely used in molecular biology and medical diagnostics
Confocal Microscopy:
Uses laser light and a pinhole aperture to create sharp, high-resolution images of optical sections, which can be reconstructed into 3D images.
Ideal for detailed analysis of thick specimens or tissues.
Differential Interference Contrast (DIC) Microscopy:
Similar to phase-contrast but uses polarized light to enhance contrast and provide a pseudo-3D appearance.
Suitable for observing live cells and fine structural details.
What are the steps of Histological sample preparation? Explain?
- Fixation:
The tissue is immersed in a fixative (e.g., formalin) to preserve its structure and prevent decomposition.
Fixation stabilizes proteins, prevents enzymatic degradation, and maintains the morphology of cells and tissues.
Common fixatives include formaldehyde for light microscopy and glutaraldehyde for electron microscopy. - Embedding:
The cleared tissue is infiltrated with molten paraffin wax or another embedding medium, such as resin.
The tissue is placed in a mold filled with molten wax, which hardens upon cooling.
Embedding provides support for the tissue and facilitates cutting into thin sections. - Sectioning:
The embedded tissue block is trimmed and sliced into thin sections (typically 3–5 micrometers thick) using a microtome.
Thin sections are essential for light penetration and detailed visualization under a microscope. - Staining:
Staining enhances contrast and highlights specific structures or components within the tissue.
Common stains include hematoxylin and eosin (H&E):
Hematoxylin stains nuclei blue or purple.
Eosin stains cytoplasm and extracellular matrix pink.
Special stains (e.g., PAS, Masson’s trichrome) or immunohistochemistry may be used for specific components or molecules.
What is a Barr Body?
A Barr body is an inactivated X chromosome present in the nuclei of somatic cells of females in mammals. It appears as a dense, darkly staining structure when viewed under a microscope, typically located near the nuclear envelope. The formation of a Barr body is part of a process called X-chromosome inactivation (XCI), which ensures dosage compensation between males (XY) and females (XX) by silencing one of the two X chromosomes in females. Located at the edges of nuclei of female epithelial.
What are the steps of using a microscope
- Setup and Preparation:
Place the microscope on a stable, flat surface.
Ensure the light source (built-in or external) is plugged in and functional.
Clean the lenses (ocular and objective) with lens paper to remove dust or smudges. - Prepare the Specimen Slide:
Place the specimen on a clean glass slide.
Cover it with a coverslip to protect the objective lens and stabilize the sample.
If using a wet mount, add a drop of water or stain before placing the coverslip. - Turn On the Light Source:
Switch on the illuminator or adjust the mirror if using a non-electric microscope.
Adjust the diaphragm to control the amount of light reaching the specimen. - Position the Slide:
Place the slide on the stage and secure it with stage clips or a mechanical stage.
Center the specimen over the light source using the stage controls. - Select the Objective Lens:
Start with the lowest magnification objective lens (e.g., 4x) to locate the specimen.
Rotate the nosepiece to position the objective lens above the slide. - Focus the Microscope:
Use the coarse adjustment knob to raise the stage until the objective lens is close to the slide.
While looking through the eyepiece, slowly lower the stage with the coarse adjustment knob until the specimen comes into focus.
Use the fine adjustment knob for precise focusing. - Adjust the Light and Contrast:
Use the diaphragm to increase or decrease the light intensity for better contrast.
Adjust the condenser if needed to focus the light more sharply on the specimen. - Increase Magnification (if needed):
Once the specimen is in focus under low magnification, switch to a higher magnification objective lens (e.g., 10x, 40x).
Refocus using the fine adjustment knob.
Adjust the light and diaphragm settings to suit the new magnification. - Examine the Specimen:
Observe the specimen through the eyepiece.
Move the slide gently using stage controls to view different areas of the sample. - Finish and Clean Up:
When finished, return the microscope to the lowest magnification.
Remove the slide and clean it if necessary.
Turn off the light source, unplug the microscope, and cover it to prevent dust accumulation.
what are two main mechanisms of fixation are
cross-linking and coagulation. Cross-linking involves covalent bond formation both within proteins and between them, which causes tissue to stiffen and therefore resist degradation. Coagulation is caused by the dehydration of proteins through the use of alcohols or acetone, which deform protein tertiary structure, so that hydrophobic, or water fearing, regions move the protein surface. Coagulative fixation can help embedding media, like paraffin wax, penetrate tissue.
Before fixation, you need to consider a few things:
- Diffusivity of the sample
- Volume and pH of the fixative
