Topic 2 Flashcards
Visualization of Cells
To visualize cells
• Different types of
microscopes are used
Samples may be processed
• Stained or dyed
• Fixed (dead) (fixatives ensure everything stays in place) or alive
• Material may be sliced (specimen needs to be thin in order for light to pass through it), dissociated (enzymes are detergents)
‘Challenges’
• What do the colors tell you about a microscopic image?
• What is the scale/size of a microscopic image?
- scale bars are crucial and colours can tell you if you have different structures and/or cells
- Identify if one or many cells are being viewed
- Labeling with stains/dyes/probes can help to distinguish cellular features
Limit of Resolution
• Microscopes have unique magnifying powers and different limits of resolution
• Limit of resolution:
- How far apart adjacent objects can be in order for them to be seen as separate entities
• Resolving power
- Human eye: mm range
- Light microscope: 200-350 nm
- Electron microscope: 2nm
• Image clarity and details depend on the microscope used and specimen preparation
• A microscope’s resolution is affected by
- The illumination wavelength (most important one)
- The refractive index of the material between the specimen and the objective lens
- Quality of the lenses
- (Other features)
Principles of microscopy
Light microscope
• Bright light is focused onto a specimen by lenses
• Another set of lenses focus the image for the eye
• Compound microscope: Two lenses whose total magnifying power is the product of the magnification of each lens
• Specimen is thin to allow light to pass through
• The illumination source (light) travels in waves of a specific length
• How a specimen changes (perturbs/disturbs) the wavelength of illumination results in the specific image seen
- A light microscope’s limit of resolution is based on the wavelength of visible light
• Light is a form of electromagnetic radiation
• Light microscopy usually utilizes energy ranges within the visible spectrum
• Different optical components reveal varied features of the same living cell
- Unstained cell
— Brightfield
— Phase contrast
— Interference
— Fluorescence
Electromagnetic Spectrum
Range of different forms of electromagnetic radiation
• Photons travel in a continuous wave
• Vary in energy, wavelength, and frequency
• Observed specimens disrupt illumination wave form
- long wavelengths good for seeing big objects
- small wavelengths good for seeing small objects
— small object will not disturb a long wavelength, therefore will not be able to see it
Electron Microscopy
• Light microscopy is limited in resolution due to the wavelength of visible light
• Electrons travel with shorter wavelength→ are focused through electromagnets
- Light magnification : 1000-1500X
- Electron magnification: 20,000-100,000X
Types of electron microscopy
• TEM: Transmission EM
- Electrons travel through thin sectioned samples (100 nm)
• SEM: Scanning EM
- Sputter coat with electron dense material
- Beam of electrons bounces off sample to yield 3D image
• TEM has a higher magnification compared to SEM
Manipulation of cells
• Tissues are too big and thick for light microscopy
• Specific cellular features may be revealed by staining procedures
• Common process:
- Fixation
- Sectioning
- Staining
- These steps kill the cellular material
Sample preparation for microscopy
• Fixation and Permeabilization:
- Detergents may be used to create holes in membranes (done before staining most of the time. For dyes sometimes)
- Acids/aldehydes cross- link proteins in place
• Sectioning:
- Sample is dehydrated (allows for image to be sharp and clear and not messy/mushy)
- Placed in wax/paraffin
- Frozen, and sliced
• Staining
- Used for examination of tissues in light and electron microscopy
Labeling Cells
• Specific macromolecules and structures in cells can be examined using microscopy
• Stains, dyes, and probes are used to increase contrast, to visualize specific components, and to somehow ‘mark’ or ‘identify’ cellular parts
- Cellular features or organelles
- Specific proteins
Cell Staining
• Vital dyes/stains penetrate cells and their color is visualized using brightfield light microscopy (doesn’t kill cell)
• Dyes may interact generally or specifically with cell structures
- Example: H and E Staining. Mouse intestine stained with hematoxylin and eosin
• Eosin: stains DNA purple
- Nuclei are visualized
• Hematoxylin: stains proteins pink
- Cytoplasm is visualized
Cancer staging and cell staining
• Melanoma tumor: human skin (H and E staining)
• Malignant cells underlay normal epithelium: cells seen with dark nuclei and brown melanotic inclusions
• A pathology report is a document that contains the diagnosis determined by examining cells and tissues under a microscope.
• Frozen sections of a tissue sample are done when an immediate answer about a sample is needed.
• The pathology report is usually created after a biopsy or surgery.
• The pathology report includes information about the patient, a description of how cells look under the microscope, and a diagnosis.
• NCI is sponsoring clinical trials that are designed to improve the accuracy and specificity of cancer diagnoses.
Labeling cells with fluorescent probes
• Fluorescence microscopy is a form of light microscopy
• Fluorescent markers are used to visualize certain cell structures or specific molecules
• DAPI is a blue fluorescent dye that binds to DNA
- Cellular nuclei are visualized
Principles of Fluorescence
• Fluorescent molecules absorb light at one wavelength and emit light at a longer wavelength with lower energy
• Atoms within the fluorescent dyes emit light as electrons return to ground state
Fluorescent Molecules
• List of fluorescent dyes, markers, and proteins
• Absorption and emission spectra for each is shown
- Fluorescent microscopes must illuminate sample at the correct wavelength for the fluorescent dye being used
Fluorescence Microscope Optics
Fluorescence microscopy concept
• Shine visible light of all wavelengths onto the specimen
- Use a filter that allows a specific wavelength through, appropriate for the fluorescent molecule used (Eg. Blue)
• Specimen is illuminated and fluorescent molecule absorbs and then emits light
- Emitted light is longer wavelength and lower energy (Eg. Green)
— Additional filter removes the excitation wavelength, so only the emitted light is seen
Markers detect specific cell parts or molecules
• Eosin: stains DNA purple
- Nuclei are visualized
• Hematoxylin: stains proteins pink
- Cytoplasm is visualized
• DAPI is a blue fluorescent dye that binds to DNA
- Cellular nuclei are visualized
• How are SPECIFIC proteins identified using microscopy?
Antibody use in cell biology
• Antibody: proteins made by cells in the immune system
• They attach to specific substances (antigens) and target them for destruction
• Value to researchers:
- Extremely specific against antigen
- Can be created in billions of forms
- Have many applications in research and medicine
• Antibodies combined with fluorescent molecules are typically used to label specific proteins of interest in cells
• Here, a green fluorescent molecule called FITC is labeling a membrane protein called ‘Cadherin’ , but through the use of antibodies
- Unlabeled primary antibody specific for ‘cadherin’ was added to cells
• A FITC labeled secondary antibody was then added
- This fluorescently labeled secondary antibody bound to the primary antibody
Antibody use in microscopy
• Goal: To visualize the expression or localization of Receptor Protein A on cells
• Fluorescent molecule is chemically conjugated to an antibody specific for Receptor Protein A
• These antibodies are added to a dish of cells
• Fluorescence microscopy detects expression of Protein A
• Would this approach also work to detect Protein C?
Antibody use: direct vs. indirect
• Often, a combination of antibodies is used in detection • Primary antibody binds to the antigen
• Secondary antibody is fluorescently labeled and binds to the primary antibody
• What is the advantage and disadvantage of using two antibodies?
I wish to know if a specific protein is found in the nuclei of cells.
Describe how you could investigate this question using microscopy.
• Use a marker that binds to DNA to highlight the location of the nucleus
- E.G. DAPI
• Use a marker (labeled antibody) against your protein of interest
- See if your protein appears in the same location as where the DAPI stain appears
• Image results:
- The expression of the Cadherin protein is seen by the green staining
- The relative location of the Cadherin protein is seen in relation to the nucleus
Using multiple fluorescent molecules
• Double labeling
- Provides information about the localization of structures or
proteins in relation to each other
• Colocalization of proteins can be seen by merging images and observing a ‘yellow’ color in this experiment
- Images are taken separately and then ‘merged’ electronically
Fluorescent Proteins
• Fluorescent proteins occur naturally in some organisms
• Green fluorescent protein (GFP) can be isolated from Aequoria victoria (jellyfish)
• DNA sequence encoding Green Fluorescent Protein can be inserted into organisms
- Exogenously added DNA is transcribed into mRNA and is then translated into green fluorescent protein
• How is this useful when studying cells?
Creation of Fusion Proteins
• DNA sequence for GFP is added to the DNA (gene) encoding the protein of interest
- Modified DNA is transfected (inserted) into cells
• Protein created is a ‘fusion’ containing the amino acids for GFP and for the protein of interest
• Advantages and disadvantages of this process?
Using GFP tagged proteins and Studying cellular functions using GFP
• Living cells express a fusion protein that consists of GFP and the protein of interest
- Allows you to follow expression of your protein in live cells in real time
• GFP fused to a protein that is found on the ends of microtubules
What is the difference between using fluorescent antibodies and fluorescent proteins in microscopy?
• Fluorescent antibodies are proteins conjugated to a chemical
- They specifically attach to a protein of interest and are detected through the fluorescence that is emitted
• GFP is a protein that has natural fluorescence
- A protein of interest can become permanently fluorescent by fusing the DNA of GFP to the DNA of the protein of interest
• This molecule is added to cells, where the resulting expressed fusion protein is a combination of the protein of interest and GFP
- Doesn’t require the use of labeled antibodies to detect it
Summary of cell labeling
• Vital dyes: visualized with brightfield light microscopy
• Fluorescent molecules: visualized with fluorescent light
microscopy
- Dye may directly bind a cellular structure
- Dyes are often attached to antibodies that recognize specific proteins
- Cells stained with vital dyes or fluorescent dyes are frequently fixed and permeabilized (dead)
• Fluorescent proteins, such as GFP, can be used to examine proteins in live cells, over time
