MICROSOPY

Image of bacterium Beggiatoa

spp. (bacterial mat) in a
dissecting microscope at 30x
[Dr. Mark Martin]

Abesamis, Acosta, Agustin, Aquitania, Bagsican,

Cristobal, Dela Cruz [2EMT]
Microscopy
 Light Microscopy
 Bright field Microscopy
 Dark field Microscopy
 Fluorescence Microscopy
 Phase-Contrast Microscopy
 Differential Microscopy
 Confocal Microscopy
 Polarizing Microscopy

 Electron Microscopy
 Transmission Electron Microscopy
 Scanning Electron Microscopy
Bright Field Microscopy
 In phase mechanism
 Employs vertical light
 Light source: Illuminator (light)
 Stained preparation
 3 System of Lenses: Condenser,
Objective, Eyepiece
 Have several objective lenses
 Resolving power adds detail to
obtain the image (max. 0.2μm)
 Parfocal – objectives remain
focused when the objective is
switched Cross section of a frog’s ovary
[Pointed: Oogonia]
Zoology laboratory specimen
Components of a Bright Field Microscope

Components and light path

of a bright field microscope
[Mescher, 2009]
Dark Field Microscopy
 Out-of-Phase mechanism
 Employs strong oblique light
 With an opaque disk
 Observing unstained, living
specimen

Shed exo-skeleton of a Daphnia

magna (40x) [Howard Webb]
Fluorescence Microscopy
 Capable of imaging the distribution
of a single molecular species based
solely on the properties of
fluorescence emission
 Irradiated with UV light Three-color fluorescence
image of an endothelial cell
 Stained preparation showing the tubulin (blue),
endosomal pathway
 Optical system: Heat filter and components (green), and
Barrier filter actin cytoskeleton (red).

 Light source: Hg vapor lamp/Quartz

iodine lamp
Fluorescence Microscopy
Stained with DAPI (4’.6-diamino-2-
Stained with acridine orange phenylindole)

Kidney cells [Mescher, 2009]

Fluorescence Microscopy
Phase Contrast Microscopy and
Differential Inference Microscopy
Phase Contrast Microscopy
 In phase mechanism
 Examining biological tissues
 Combination of in-phase and
out-of-phase mechanism
 No staining preparation for
specimen
 Produces visible images from
transparent objects
 Has annular shape diaphragm
Differential Inference Microscopy
 Nomarski Microscopy or
Normarski Inference Contrast
(NIC)
 In phase mechanism
 Examining biological tissues
 Combination of 2 light
mechanism
 No staining preparation for
specimen
 Produces apparent three-
dimensional image
Phase Contrast Microscopy and
Differential Inference Microscopy
Phase Contrast Microscopy Differential Inference Microscopy

Cheek Cell (Spencer Diamond, 2007) Micrasterias radiata (DIC) [Wikipedia]

 Phase Contrast Microscopy and
Differential Inference Microscopy
Differential Inference
Bright Field Microscopy Phase Contrast Microscopy Microscopy

Unstained cells under three different types of light microscope [Mescher, 2009]
Confocal Microscopy
 Out-of-phase
 Creates clear 3-D image
 Avoids stray of light
 Great resolution:
1. A small point of high-intensity
light provided by a laser;
2. A plate with a pinhole
aperture in front of the image
detector
 Illumination: Computer-driven
mirror system (beam splitter)
Principle of confocal microscopy [Mescher, 2009]
 Confocal Microscopy
Confocal Laser Scanning Microscope

Organ of Corti (Cochlea and hair cells)

[Dr. Sonja Pyott of Department of Biology and Marine
Biology, University of North Carolina, Wilmington, NC, USA]
Confocal Microscopy

Fluorescent Microscopy Confocal Microscopy

Fluorescent and laser confocal images of a neuron in hippocampal slice gene gun transfected
with DCX DsRed and µ1A GFP. [Robert McNeil, 2009]
Polarizing Microscopy
 Recognize structures of highly
organized molecules
 Instrument measuring optical
properties of crystals and fibers
 Polaroid Filter (between the light
source and condenser)
 Analyzer (at the draw tube)
 Birefringence – capacity to
change the direction of the axis
of light

Tobacco Mosaic Virus

Polarizing Microscopy

Bright Field Microscopy Polarizing Microscopy

Collagen fibers [Mescher, 2009]

Electron Microscopy
Both uses electron beams and magnetic lens producing a high resolution image

Transmission Electron Microscopy Scanning Electron Microscopy

 Two-dimension image  Three-dimension image

 Resolution – 2.5nm  Resolution – 2.0nm
 Max. Magnification:  Max. Magnification:
1,000,000x 200,000x
 Preparation of
 Preparation of specimen: Thicker than
specimen: Thinly sliced the TEM
 Electron pathway: Pass  Electron pathway:
thru the specimen Reflected by a metal
Electron Microscopy
Both uses electron beams and magnetic lens producing a high resolution image

Transmission Electron Microscopy Scanning Electron Microscopy

Normal Human Blood (2007) [Photo by

Myelinated axon (2007) Bruce Wetzel, courtesy of the National
Cancer Institute]
HISTOLOGICAL
TECHNIQUES Image of human skeletal muscle
stained with H&E
Abesamis, Acosta, Agustin, Aquitania, Bagsican,
Cristobal, Dela Cruz [2EMT]
Histological Techniques

Numbering Blocking Microtomy

Fixation Trimming Staining

Dehydration Wax Mounting

Infiltration

Clearing Embedding Labeling

 Histological Techniques

Process in preparing a tissue specimen in the laboratory[Mescher, 2009]

Histological Light Microscope Electron Microscope
Techniques
Fixation Buffered isotonic solution of 37% (Double fixation) Buffered glutaraldehyde
formaldehyde + Buffered osmium tetroxide
Dehydration Water + Alcohol↑ (EtOH) Dioxane ↑ or Acetone ↑
Embedding Paraffin, Resin Epoxy, Resin
Staining H&E (Hematoxylin and Eosin) staining Phosphotungstic acid [Negative stain –
Papanicolaou staining [Pap smear] Viruses, Nerves, Polysaccharides]
Sudan stainig [Lipids] Osmium tetroxide [Lipids]
Mounting Canada Balsm, Glycerol
Histological Techniques
1. Direct observation of living tissues
2. Cell, Tissue, Organ culture
3. Mechanical micromanipulation and microdissection
4. Use of radiation probes
5. Cinematography
6. Differential centrifugation
7. Microincineration
8. Frozen section method
9. Freeze drying technique
10. Use of stain

ThinCert™ Cell Culture Inserts

Direct Observation of living tissues
 Microscopy (mechanism):
 In phase
 Out-of-phase

 Exterioration
 Observation: exterior of an organ
 Transillumination
 Major application of visible light
 Transmission of light through tissues of the A bright light is transmitted
body to the head showing the
 Transmission of light through fingers, body cavity or organ, such
producing a red glow due to red blood cells as the brain.
absorbing all other colors of the light.
Cell, Tissue, Organ culture
 “In vitro” cultivation
 Cell
 Cells are grown under a
controlled condition
 Tissue
 Growth of tissues and/or
cells separate from the
organism
Stem cell culturing
 Organ
 Accurate model functions of
an organ
 Study: Effects of drugs and
hormone
Mechanical micromanipulation and
microdissection
 A technique that
manipulates the
structure or composition
of a single cell under a
microscope using
pipettes or glass
needles
Intracytoplasmic sperm injection (ICSI).
[Zhang, J, Xu, K, Glob. libr. women's med.,
(ISSN: 1756-2228) 2008; DOI 10.3843/GLOWM.10369]
 Cinematography
 Motion picture of cell activity under objective of
microscope

Time-lapse movies of in vitro neuronal migration.

[Robert McNeil, 2009] Time-lapse movie of two
neuronal growth cones making
contact. [Robert McNeil, 2009]
Differential Centrifugation
 Procedure used to
separate certain
organelles from whole
cells for further analysis
of specific parts of cells
 A tissue sample is first
homogenised to break
the cell membranes and
mix up the cell contents.
 Analysis: Sedimentation
Equilibrium (molecular
weight determination) Differential Centrifugation Process
Microincineration and Radiation Probes

Microincineration Use of Radiation Probes

 Study of biological  Stained organelle is

chemistry of cell subjected to a high
power laser (radiation)
to assess the its effect
on the cell

Microincinerator
[Photo taken by Tami Port.
Retrieve from Science Prof Online educational website.]
Frozen Section Method and Frozen
Drying Method
Frozen Section Method Frozen Drying Method

 A method used in  Preparation is done

preparing a selected through freezing and
portion of tissue for dehydrating the tissue
pathologic for biological
examination observation
 Cryostat – device for sectioning frozen
block with tissue [freezing microtome]
Leica CM-3050S cryostat [Retrieve from NICHD -
The Eunice Kennedy Shriver National Institute of
Child Health and Human Development ]
Use of Stain
 Vital staining
 Biopsy (living organism)
 Trypan Blue: Distinguish viable
from nonviable cells (absorbs
dye)
 Intravital staining – tissue
inside the body (injected)
 Supravital staining – tissue Carcinoma in situ. Full-thickness atypia is
clinically observed as a red-velvet patch
outside the body (erythroplasia) and stains strongly with
supravital stain, such as toluidine blue O.
 Dead tissue [Retrieve from eMedicine – Medical
 H&E (Hematoxylin and Eosin) Refernce Website.]