Microscopy Lecture Notes PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Microscopy: types and principles  Learning outcomes  Recognize the principles and uses of different types of microscopes  Identify the parts of compound light microscope and unde...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Microscopy: types and principles  Learning outcomes  Recognize the principles and uses of different types of microscopes  Identify the parts of compound light microscope and understand their function  Find out the total magnification (objective and ocular lenses)  Understand and follow the rules of microscope use  Focus the compound light microscope at low power and high power magnifications  Define: Field of view (at low and high power), Longitudinal & cross sections and Resolution Quizlet definition: Quizlet definition: https://quizlet.com/527647934/biotechniques_microscopy-flash-cards/  Types of microscopes  Biological objects can be very small to see ❑ Light microscope them with the naked eye. Accordingly scientists ▪ Compound light microscope developed microscopes to view them. ▪ Binocular dissecting microscope (stereomicroscope)  Microscopes come in many different types. ▪ Phase contrast microscope ❑ Fluorescent Microscope ▪ Confocal microscope ❑ Electron microscope ▪ Transmission electron microscope (TEM) ▪ Scanning electron microscope (SEM)  Light microscope  The simplest form of light microscope consists of a single glass lens mounted in a metal frame – a magnifying glass.  The specimen requires very little preparation.  Focusing of the region of interest is achieved by moving the lens and the specimen relative to one another.  The source of light is usually the Sun or ambient indoor light.  The detector is the human eye.  The recording device is a hand drawing.  Compound light microscope  All modern light microscopes are made up of more than one glass lens in combination. The major components are the condenser lens, the objective lens and the eyepiece lens, and, such instruments are therefore called compound microscopes. 1. Light from a lamp is focused at the specimen by a glass condenser lens. 2. The specimen is mounted on a glass slide with a coverslip placed on top. 3. The image is magnified with an objective lens (glass lens). 4. It is projected onto a detector with the eyepiece lens. 5. The detector can be the eye or a digital camera. The parts of the Microscope and their Function Compound light microscope  Lenses and their magnifications OBJECTIVE LENS POWER OF POWER OF TOTAL OCULAR LENS OBJECTIVE LENS MAGNIFICATION SCANING POWER 10X 4X 40 LOW POWER 10X 10X 100 HIGH POWER 10X 40X 400 OIL IMMERSION 10X 100X 1000 Scanning Lower High Oil Total magnification = ocular x objective power power immersion objective objective objective Field of view  Field of view is the area of the slide that you see when you look through a microscope's eyepiece. It is a circle. HP Field of view (40 x) LP Field of view (10x)  Microscope immersion oil  Typically you don’t need the immersion oil with a lower magnification lens. You will need immersion oil when using a higher magnification lens. Microscope immersion oil is a transparent oil that has a unique  Be sure that the lens is optical character and viscosity for use an oil lens. in microscopy, especially in higher magnification.  Stereomicroscope  It is used for the observation of the surfaces of large specimens, when 3D information is required, for micromanipulation and dissections.  Example: routine observation of whole organisms, for example for screening through vials of fruit flies or dissecting a specimen.  A wide range of objectives and eyepieces are available for different applications.  An external light source at different angles serves to add contrast or shadow relief to the images. A research-grade stereomicroscope. The light source is from the side, which can give a shadow effect to the specimen; in this example a vial of fruit flies. The large objective lens above the specimen can be rotated to zoom the image.  Phase contrast microscope  It is used to enhance the contrast of images of transparent and colorless specimens, e.g. viewing unstained cells growing in tissue culture.  It enables visualization of cells and cell components that would be difficult to see using an ordinary light microscope.  The method images differences in the refractive index of cellular structures. ▪ Light that passes through thicker parts of the cell is held up relative to the light that passes through thinner parts of the cytoplasm.  It requires a specialized phase condenser and phase objective lenses.  Fluorescent microscope  Fluorescence microscopy is currently the most widely used contrast technique since it  gives the ability to achieve highly specific labelling of cellular compartments.  gives superior signal to noise ratios, typically white (fluorescent) on a black (non-fluorescent) background.  The most commonly used fluorescence technique is called epifluorescence light microscopy, where ‘epi’ simply means ‘from above’. Here the light source comes from above the sample, and the objective lens acts as both the condenser and the objective lens.  Fluorescent microscope  The light source is the UV into the red wavelengths: ▪ High-pressure mercury or xenon vapour lamp ▪ Lasers ▪ LED sources  This specific wavelength of light is used to excite a fluorescent molecule or fluorophore in the A fluorophore is an organic specimen. molecule with the ability to absorb light at a particular  Light of longer wavelength from the excitation of the fluorophore wavelength and then emit it is then imaged. This is achieved in the fluorescence microscope at a higher wavelength using combinations of filters that are specific for the excitation and emission characteristics of the fluorophore of interest.  Fluorescent proteins & stains  Red fluorescent protein (RFP)  Green fluorescent protein (GFP)  DAPI (4′,6-diamidino-2- phenylindole) is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA Fluorescent image of mobile skin cells (fibroblasts) Immunofluorescence  Confocal microscopes Laser scanning confocal microscopes (LSCM)  Optical sections are produced in the laser scanning confocal microscope by scanning the specimen point by point with a laser beam focused in the specimen, and using a spatial filter, usually a pinhole (or a slit), to remove unwanted fluorescence from above and below the focal plane of interest.  The power of the confocal approach lies in the ability to image structures at discrete levels within an intact biological specimen.  Electron microscope  Uses a beam of electrons that is magnified and focused on the object by means of electron magnetics.  Can view much smaller objects compared to light microscopes, with far greater detail. ▪ Transmission electron microscope (TEM) - analogous to the compound light microscope ▪ Scanning electron microscope (SEM) - analogous to dissecting light microscope  Extensive specimen preparation is required for EM analysis, and for this reason there can be issues of interpreting the images because of artifacts from specimen preparation. Transmission electron microscope Scanning electron microscope Microscope use  Rules for microscope use  Keep both eyes open while using the microscope and do not touch the eyepiece with your eye lashes.  The lowest power objective (scanning) should be in the position both at the beginning and end of the microscope use.  Do not clean lenses with regular paper tissue/wipes.  Do not tilt the microscope when viewing.  Do not remove parts of the microscope.  To locate small objects in slide, first find them with the naked eye or under low power.  Focusing the microscope 1. Always begin focusing with the scanning power objective 4x. 2. With coarse adjustment knob lower the stage. 3. Place a slide on the stage and stabilize it with a clip. 4. While looking through the eye piece with both eyes, slowly raise the stage using the coarse adjustment knob until the object comes into view. 5. Use the fine adjustment knob to sharpen the focus if necessary.  Focusing the microscope Compound light microscopes are parfocal, meaning once an object is In focus with the low power field, it should be almost in focus with the higher power. 1. Always find the object under the low power field before viewing it with the high power field. 2. Make sure the object is centered in the middle field. 3. Move the objective lens to the high power objective. You should hear a “click” sound (note: Parfocal microscope objectives will not hit normal slides when changing the objective if the lowest objective was initially used to focus). 4. If any adjustment is needed, use the fine adjustment knob only. Field of view  Field of view is the area of the slide that you see when you look through a microscope's eyepiece. It is a circle. HP Field of view (40 x) LP Field of view (10x) Low-power field diameter (LPD)  Use the 10X (low power) objective.  Use a clear ruler to measure the field diameter in mm.  Convert from mm to micrometer.  This will be your LPD (always expressed in micrometer).  You can calculate the size of a single cell which is equal to: Diameter of the field of view Number of cells that fit LPF = 2 mm (measure by ruler) = 2000 micrometer 1 cell = 2000/ 5 = 400 micrometers e.g. 2 mm diameter  Longitudinal section vs cross section Longitudinal section: cut through the long axis of an organ Transverse section (cross section): a cut along a horizontal plane, dividing the body or organ into superior and inferior parts  Resolution  The resolution achieved by a lens is a measure of its ability to distinguish between two objects in the specimen.  The shorter the wavelengths of illuminating light the higher the resolving power of the microscope.  The limit of resolution for a microscope that uses visible light is about 300nm with a dry lens (in air) and 200nm with an oil immersion lens.  By using ultraviolet light (UV) as a light source the resolution can be improved to 100 nm because of the shorter wavelength of the light (200–300nm). References  The Different Types of Microscopes – A Comprehensive Guide. https://www.microscopeclub.com/types-of-microscopes/  How to Use Microscope Immersion Oil to Get Higher Resolution Images. https://rsscience.com/how-to-use-microscope-immersion-oil-to-get-higher-resolution- images/