Fluorescence Microscopy Overview
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Fluorescence Microscopy Overview

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Questions and Answers

What is primarily analyzed in fluorescence microscopy?

  • Fluorescence emitted from a sample (correct)
  • The thermal properties of a sample
  • The refractive index of a sample
  • The change in pressure of a sample

    • Which type of microscope provides better resolution in fluorescence imaging by using optical sectioning?

  • Dark field microscope
  • Epifluorescence microscope
  • Confocal microscope (correct)
  • Simple light microscope

    • What type of light typically excites the electrons in fluorescence microscopy?

  • Visible light
  • Infrared light
  • Ultraviolet light (correct)
  • Microwave radiation

    • Which of the following is NOT a feature of epifluorescence microscopy?

    <p>Utilizes reflection to visualize samples</p> Signup and view all the answers

    What does fluorescence microscopy use to generate an image?

    <p>Fluorescence from excited molecules</p> Signup and view all the answers

    What type of light sources are commonly used in fluorescence microscopy?

    <p>Xenon and mercury lamps</p> Signup and view all the answers

    Which application of fluorescence is not commonly associated with cytogenetic analysis?

    <p>Chemical sensors</p> Signup and view all the answers

    What advantage does Fluorescence in situ Hybridization (FISH) have over routine chromosome analysis?

    <p>Higher specificity and sensitivity</p> Signup and view all the answers

    What imaging system is mentioned as part of cytogenetics work in the lab?

    <p>Zeiss AxioImager Z2</p> Signup and view all the answers

    Which feature is associated with imaging systems in fluorescence microscopy?

    <p>Image acquisition and processing</p> Signup and view all the answers

    What is one of the primary uses of fluorescence in situ hybridization (FISH)?

    <p>Identifying chromosomal abnormalities</p> Signup and view all the answers

    Which of the following techniques involves multicolor fluorochrome banding?

    <p>Color Karyotyping</p> Signup and view all the answers

    What impact does FISH have on the turnaround time of patient results?

    <p>Shortens turnaround time</p> Signup and view all the answers

    What is the primary difference between DIRECT FISH and INDIRECT FISH techniques?

    <p>DIRECT FISH features a reporter plus probe.</p> Signup and view all the answers

    In the context of FISH detection, what does Tyramide signal amplification (T5A) enable?

    <p>Increased sensitivity in mRNA detection.</p> Signup and view all the answers

    Which of the following types of FISH probes is specifically designed to label repetitive sequences?

    <p>Chromosome painting probe</p> Signup and view all the answers

    What is the purpose of denaturation in the FISH process?

    <p>To prepare DNA for hybridization.</p> Signup and view all the answers

    What type of FISH probe is designed to target the centromere of chromosomes?

    <p>Centromeric probe</p> Signup and view all the answers

    What visual pattern does dual color dual fusion fluorescent in situ hybridization (FISH) typically produce?

    <p>Dual separate signals indicating translocations.</p> Signup and view all the answers

    How does the use of Horseradish peroxidase contribute to the Tyramide signal amplification process in mRNA detection?

    <p>It catalyzes the formation of Tyramide radicals.</p> Signup and view all the answers

    What does the term 'break apart' refer to in break apart FISH probes?

    <p>Separation of hybridized probe signals.</p> Signup and view all the answers

    What is the primary function of the BRCA1 protein?

    <p>To maintain genomic stability</p> Signup and view all the answers

    What percentage of breast cancer cases in the United States is associated with abnormal BRCA1 or BRCA2 genes?

    <p>5-10%</p> Signup and view all the answers

    Which test measures the presence of protein receptors or gene copy number for HER2 status?

    <p>Immunohistochemistry (IHC)</p> Signup and view all the answers

    What does FISH specifically involve when analyzing genetic material?

    <p>Hybridizing a fluorescence probe to a target</p> Signup and view all the answers

    What is the location of the BRCA1 gene on the chromosome?

    <p>17q11–21</p> Signup and view all the answers

    How does an abnormal BRCA1 or BRCA2 gene affect women's risk of breast cancer?

    <p>It increases the risk to about 60%</p> Signup and view all the answers

    What color indicates the visualization of BRCA1 probes in the two-color FISH technique?

    <p>Green</p> Signup and view all the answers

    What type of protein is HER2/neu classified as?

    <p>Growth-promoting protein</p> Signup and view all the answers

    What is the percentage of interphase cells analyzed that were found positive for BCR-ABL gene fusion in the first analysis?

    <p>60.8%</p> Signup and view all the answers

    Which translocation is associated with Acute Promyelocytic Leukemia (APL)?

    <p>t(15;17)(q24;q21)</p> Signup and view all the answers

    What is the primary purpose of the FISH technique mentioned in the content?

    <p>To determine XX/XY cell ratio in bone marrow transplants</p> Signup and view all the answers

    Which medication is noted as a molecularly targeted drug for BCR-ABL positive CML patients?

    <p>Imatinib Mesylate (Glivec)</p> Signup and view all the answers

    Which chromosomes are involved in the translocation associated with Acute Promyelocytic Leukemia?

    <p>Chromosome 15 and 17</p> Signup and view all the answers

    What is the expected FISH signal pattern in a normal male cell when hybridized with the CEP X/Y DNA Probe?

    <p>One orange and one green signal</p> Signup and view all the answers

    Which of the following genes is located on Chromosome 17 and is associated with susceptibility to breast cancer?

    <p>BRCA1</p> Signup and view all the answers

    Which method is NOT mentioned as an FDA-approved technology for breast cancer screening?

    <p>Genomic profiling</p> Signup and view all the answers

    Study Notes

    Fluorescence Microscopy

    • Analyzes fluorescence from a sample
    • A beam of light (e.g., UV) excites electrons in molecules of certain compounds, causing them to emit light, often visible light

    Epifluorescence Microscope

    • A type of fluorescence microscope where light passes through the sample in a straight angle, maximizing illumination
    • Uses high-energy light (e.g., xenon, mercury lamp), but now LED is used
    • Uses fluorescence and phosphorescence

    Applications of Fluorescence

    • Mineralogy and gemology
    • Chemical sensors (fluorescence spectroscopy)
    • Fluorescent labeling dyes
    • Biological detectors
    • Fluorescent lamps

    Applications in Cytogenetics

    • Routine karyotyping/cytogenetic analysis
    • Color karyotyping
    • Spectral karyotyping (SKY)
    • Multicolor fluorochrome banding (mBand)
    • Automatic telomere measurement

    Fluorescence in situ Hybridization (FISH)

    • A powerful technique used to detect chromosomal abnormalities/mutations
    • Introduced in the late 1980s
    • Addressed the limitations of routine chromosome analysis
    • Refined the level of cytogenetic screening
    • High specificity and sensitivity
    • Shortened turnaround time (TAT) of patient results

    Types of FISH Detection

    • Direct FISH: Reporter + Probe
    • Indirect FISH: Antibody + Reporter + Probe

    Fluorescent in situ Hybridization (FISH) Protocol

    • Sample DNA is denatured.
    • Probe DNA is denatured and hybridized with the sample DNA.
    • Either a direct or indirect label is used.
    • The sample is washed and detected.

    Different Types of FISH Probes

    • Chromosome painting: Targets whole chromosomes
    • Centromeric probe: Targets the centromere region
    • Telomeric probe: Targets the telomere region
    • Gene-specific probe: Targets a specific gene

    FISH in Hematological Malignancies

    • FISH can be used to identify translocations, deletions, and amplifications in hematological malignancies.
    • Example: In Chronic Myelogenous Leukemia (CML), FISH can detect the BCR-ABL fusion gene caused by the t(9;22) translocation.
    • Imatinib Mesylate (Gleevec) is a molecularly targeted chemo drug for BCR-ABL positive CML patients.
    • Example: In Acute Promyelocytic Leukemia (APL), FISH can detect the PML/RARα gene fusion caused by the t(15;17) translocation.
    • Trans Retinoic Acid is a molecularly targeted drug for patients positive for t(15;17)(q24;q21) or PML/RARα gene fusion.

    FISH in Bone Marrow Transplantation

    • Example: FISH can be used to determine XX/XY cell ratio in sex-mismatched bone marrow transplants using CEP X SpectrumOrange/CEP Y SpectrumGreen DNA Probe Kit:
      • In a normal male cell, the expected pattern is one orange, one green (1O1G)
      • In a normal female cell, the expected pattern is two orange (2G) signals.

    FISH in Solid Tumors

    • Example: FISH can be used to detect gene mutations in solid tumors, such as breast cancer and other malignancies.

    BRCA1 and BRCA2 Genes

    • BRCA1 and BRCA2 are tumor suppressor genes located on chromosomes 13 and 17, respectively.
    • Mutations in these genes increase the risk of breast, ovarian, and other cancers.
    • Women with abnormal BRCA1 or BRCA2 genes have about a 60% risk of being diagnosed with breast cancer during their lifetimes (compared to 12-13% for women overall).

    HER2/neu gene

    • HER2/neu (often just HER2) is a growth-promoting protein located on chromosome 17, near the BRCA1 gene locus.
    • HER2 gene amplification is associated with aggressive breast cancer.
    • Testing for HER2 status can enable the most appropriate therapy decision.
    • Tests should measure either gene copy number or the presence of protein receptors.

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    FISH Techniques and Applications PDF

    Description

    Explore the fundamentals of fluorescence microscopy, including epifluorescence techniques and various applications. Learn about its significance in fields such as cytogenetics, mineralogy, and biological detection methods. This quiz will test your understanding of how fluorescence aids in scientific discoveries.

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