Histology Microscope Techniques PDF

Microscope PATRICK JUMAR S. BUENAFLOR, RMT, MSMT(C) Microscope in Histology Laboratory  It is an instrument that enlarges the images and allows the visualization of morphological cellular details that are too small to be seen by the unaided eyes.  Two purpose  For Patho...

Microscope PATRICK JUMAR S. BUENAFLOR, RMT, MSMT(C) Microscope in Histology Laboratory  It is an instrument that enlarges the images and allows the visualization of morphological cellular details that are too small to be seen by the unaided eyes.  Two purpose  For Pathologist  For Histotechnologist Objectives of the use of Microscope 1. Magnify the object 2. Resolve the details of the objects 3. Make these details visible Parts of the Microscope  Main framework  Lens system  Illumination system  Main Framework of the Microscope 1. Base  Provides support for the microscope 2. Arm  Supports and holds the magnifying and adjustment system 3. Stage  Flat platform where the slide is placed for examination 4. Sub-stage  Holds the condenser and the diaphragm  Main Framework of the Microscope 5. Mechanical stage  Permits the movement of the stage while holding the slide in place 6. Adjustment knob  Fine adjustment knob  Coarse adjustment knob 7. Mechanical Stage control  Moves the stage in X and Y axis  Lens system of the Microscope 1. Nosepiece  Holds the objectives 2. Objectives  Consist of system of lens that increases or decreases magnification 3. Eyepiece  Lens system nearest to the eye that receives the image from the lens of the objectives 4. Focal Length  Distance between outer lens of the objective and the cover glass of the slide  Lens system of the Microscope Monocular head Binocular head Trinocular head  Illumination system of Microscope 1. Light source 2. Condenser  Receives light rays from the source and forms a cone light that can be focused in the objective 3. Iris diaphragm  Control the amount of light received by the condenser Optical Components Magnification and Calibration Magnification  Process that increase the size of the structure under examination  Achieved by the use of the lens system Total magnification – product of the magnifying power of objectives and eyepiece, with a normal tube length of 160mm Magnification and Calibration Magnification  Process that increase the size of the structure under examination  Achieved by the use of the lens system Total magnification – product of the magnifying power of objectives and eyepiece, with a normal tube length of 160mm  Magnification and Calibration Example: If microscope is fitted with a draw tube, the body length will increase, Objective lens = 40x and the body tube magnification will also increase Eyepiece = 10x Example Total Magnification Objective lens = 40x = 400x Eyepiece = 10x Working Tube Length = 180mm Normal Tube Length = 160 𝑇𝑜𝑡𝑎𝑙 𝑀𝑎𝑔𝑛𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 = 40 𝑥 10 𝑥 1.25 = 450𝑥 Type of Microscopes  Compound Microscope Electron Bright-field Microscope  Microscope  Dark-field Microscope Transmission  Phase-Contrast Microscope (TEM)  Polarizing Microscope Scanning (SEM)  Fluorescent Microscope Bright-Field Microscopy  Used compound microscope  Light is either pass through or reflected off, a specimen  Properties of light are not altered  Viewing a dark and contrasted specimen against bright viewing field  Light is absorbed by stains, pigmentation, or dense areas Dark-field Microscopy  Used to observe unstained and transparent samples  Bright sample against dark background (purely black)  Blocks the central light with a condenser so that only oblique rays hit the object Dark-field Microscopy Phase-Contrast Microscopy  Optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast image  Reveals many cellular structures that are not visible with a simpler bright field microscope Phase-Contrast Microscopy Polarizing Microscopy  Contrast-enhancing technique that improves the quality of the image obtained with birefringent materials  Examine specimens that are visible primarily due to their optically anisotropic character Polarizing Microscopy Fluorescence Microscope  Microscope that uses fluorescence to generate an image  Wavelengths of light are used to cause sample to fluoresce  When a certain compounds are illuminated with high energy light, they emit light of a lower frequency  Uses fluorochrome for staining  Fluoroscein or rhodamine Fluorescence Microscope  Light source used  Xenon arc lamps  Mercury-vapor lamps with excitation filter  Lasers  High-power LED  Used in immunostaining Fluorescence Microscope Electron Microscope  Attains extremely high resolution using an electron beam instead of a beam of light to illuminate the object of study. 1. Transmission electron microscopes  require the preparation of films so thin that they are transparent to a beam of electrons 2. Scanning Electron Microscope  Uses a narrow beam of electrons that scans the surface of a sample and forms a corresponding image from the backscattered electrons or secondary electrons Electron Microscope Electron Microscope Fresh Tissue Examination Fresh Tissue Examination HISTOLOGY HISTOPATHOLOGY The procedures adopted for the preparation of materials for such studies are known as histologic or histopathologic technique. Fresh Tissue Examination  Structural and chemical components of the cells  Natureand amount of the tissue to be evaluated  Needfor an immediate examination of a tissue structure Fresh Tissue Examination  Received in fixative or fresh.  Should Have request form Patient information Clinical history Description of the site of origin  Assigned accession number Surgical Procedures performed to obtain tissue specimen  Fine Needle Aspiration  Core Needle Biopsy  Incisional Biopsy  Excisional Biopsy  Punch Biopsy  Shave Biopsy  Curettings Surgical Procedures performed to obtain tissue specimen  Fine Needle Aspiration  Simplest and least invasive  Uses small needle to remove cells from the area of abnormality  Core Needle Biopsy  Removes not only a cells, but also a small amount of surrounding tissues Surgical Procedures performed to obtain tissue specimen Surgical Procedures performed to obtain tissue specimen  Incisional Biopsy  Removes only a portion  Excisional Biopsy  Generally removes the entire area Surgical Procedures performed to obtain tissue specimen Surgical Procedures performed to obtain tissue specimen  Punch Biopsy  Involves the use of a circular blade that is rotated down through the epidermis and dermis and into the subcutaneous fat  3-4 mm cylindrical core  Shave Biopsy  Small fragments are shaved from a surface (skin)  Curettings  Tissue is scooped or spooned to remove tissue or growths from body cavite Surgical Procedures performed to obtain tissue specimen Methods of FTE 1. Teasing or Dissociation 2. Squash Preparation (Crushing) 3. Smear Preparation  Streaking  Spreading  Pull-apart 4. Frozen Section Teasing or Dissociation  Process whereby a selected tissue specimen is immersed in a watch glass containing isotonic salt solution, carefully dissected or separated, and examined under the microscope. Squash Preparation (Crushing)  Process whereby small pieces of tissue not more than one mm. in diameter are placed in a microscopic slide and forcibly compressed with another slide or with cover glass Smear Preparation  Process of examining sections or sediments, whereby cellular materials are spread lightly over a slide  Streaking  Spreading  Pull apart  Touch prep Smear Preparation Streaking  Made using sterile swab or an applicator stick  Material is applied to the glass slide rapidly in a direct or zigzag line.  Used in gram staining, vaginal discharge and purulent wound Smear Preparation Spreading ▪ Small part of the material is place on a slide and gently teasing the mucus strands apart by using application stick ▪ Mucopurulent samples (sputum) Smear Preparation Pull apart ▪ Place a drop of specimen on a slide, cover by another slide, then the two slides are pulled apart ▪ Blood smears, bone marrow aspirate, enzymatic lavage Smear Preparation Touch preparation or Impression  Touching tissue or specimen using glass slide, allowing the cells to transfer directly to the slide for examination.  Urethral discharge, excised tissue, smear from skin slits