Abesamis, Acosta, Agustin, Aquitania, Bagsican, Cristobal, Dela Cruz [2EMT]

Image of bacterium Beggiatoa

spp. (bacterial mat) in a

dissecting microscope at 30x

[Dr. Mark Martin]MICROSOPY

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

Com

pone

nts

of

a B

rig

ht F

ield

Mic

rosc

op

e

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

Stained preparation

Optical system: Heat filter and Barrier filter

Light source: Hg vapor lamp/Quartz iodine lamp

Three-color fluorescence

image of an endothelial cell

showing the tubulin (blue),

endosomal pathway

components (green), and

actin cytoskeleton (red).

Fluorescence Microscopy

Stained with acridine orangeStained with DAPI (4’.6-diamino-2-

phenylindole)

Kidney cells [Mescher, 2009]

Fluo

resc

enc

e M

icro

scopy

Phase Contrast Microscopy and

Differential Inference 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

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 Differential Inference Microscopy

Phase Contrast Microscopy and

Differential Inference Microscopy

Phase Contrast Microscopy Differential Inference Microscopy

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

Phase Contrast Microscopy and

Differential Inference Microscopy

Phase Contrast MicroscopyDifferential Inference

MicroscopyBright Field 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

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 Laser Scanning Microscope

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

Two-dimension image

Resolution – 2.5nm

Max. Magnification: 1,000,000x

Preparation of specimen: Thinly sliced

Electron pathway: Pass thru the specimen

Three-dimension image

Resolution – 2.0nm

Max. Magnification: 200,000x

Preparation of specimen: Thicker than the TEM

Electron pathway: Reflected by a metal

Transmission Electron Microscopy Scanning Electron Microscopy

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

Electron Microscopy

Transmission Electron Microscopy Scanning Electron Microscopy

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

Myelinated axon (2007)

Normal Human Blood (2007) [Photo by

Bruce Wetzel, courtesy of the National

Cancer Institute]

HISTOLOGICAL

TECHNIQUESAbesamis, Acosta, Agustin, Aquitania, Bagsican, Cristobal, Dela Cruz [2EMT]

Image of human skeletal muscle

stained with H&E

Histological Techniques

Numbering

Fixation

Dehydration

Clearing Embedding

Wax Infiltration

Trimming

Blocking Microtomy

Staining

Mounting

Labeling

Histological Techniques

Histological

Techniques

Light Microscope Electron Microscope

Fixation Buffered isotonic solution of 37%

formaldehyde

(Double fixation) Buffered glutaraldehyde

+ Buffered osmium tetroxide

Dehydration Water + Alcohol↑ (EtOH) Dioxane ↑ or Acetone ↑

Embedding Paraffin, Resin Epoxy, Resin

Staining H&E (Hematoxylin and Eosin) staining

Papanicolaou staining [Pap smear]

Sudan stainig [Lipids]

Phosphotungstic acid [Negative stain –

Viruses, Nerves, Polysaccharides]

Osmium tetroxide [Lipids]

Mounting Canada Balsm, Glycerol

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

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 body

Transmission of light through fingers, producing a red glow due to red blood cells absorbing all other colors of the light.

A bright light is transmitted

to the head showing the

body cavity or organ, such

as the brain.

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

Organ

Accurate model functions of an organ

Study: Effects of drugs and hormone

Stem cell culturing

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

Study of biological

chemistry of cell

Stained organelle is

subjected to a high

power laser (radiation)

to assess the its effect

on the cell

Microincineration Use of Radiation Probes

Microincinerator

[Photo taken by Tami Port.

Retrieve from Science Prof Online educational website.]

Frozen Section Method and Frozen

Drying Method

A method used in

preparing a selected

portion of tissue for

pathologic

examination

Preparation is done

through freezing and

dehydrating the tissue

for biological

observation

Frozen Section Method Frozen Drying Method

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

outside the body

Dead tissue

H&E (Hematoxylin and Eosin)

Carcinoma in situ. Full-thickness atypia is

clinically observed as a red-velvet patch

(erythroplasia) and stains strongly with

supravital stain, such as toluidine blue O.

[Retrieve from eMedicine – Medical

Refernce Website.]