COURSE GOALS:

Use appropriate descriptive language and

terms.*

Understand the appropriate use of techniques to

study material at the histological level. *

Identify tissues. (Epithelial, Nervous, Muscle, Connective Tissue)

Identify composite of tissues as an organ.

*We begin to address these goals TODAY!

Exercise: Learn the Language of Histology

Examine your image and write a description that can be used to identify it.

- Form groups of four--> same letter/numbers 1-4examples: group 1: A1, A2, A3, A4

group 2: E1, E2, E3, E4

- Mix up your group’s images and descriptions and exchange them with another group.

Yours 1-4 Theirs 1-4

- Work as a group to match the description with the image.

- Check results on slide that is coming up

Learn the Language of Histology

BEFORE:

“-looks like an abstract painting…..Looks like it was colored with colored pencils.”

“-looks like a bunch of worms on a pink background…..3 white areas- bigger one to the left of picture.”

“-tree bark with blue bugs crawling on it.”

“-looks like a yellow river with some kind of fish swimming upstream and the edges of the river are made up of big hunks of ice.”

“-the center has light thin dashes/stripes. One side of the slide has rectangular blocks. The other border is light.”

Learn the Language of Histology

After:

Cross section of nonkeratinized stratified squamous epithelium facing a lumen. Undifferentiated cells at basal end of epithelium are smaller with darker nuclei and are positioned on a loose connective lamina propria; likely esophagus.

ORGANISM

IMAGE

TISSUE PREPARATION

1) Fixation

2) Embedding

3) Sectioning

4) Staining

5) Imaging

Tissue Preparation

1) Fixation: halts cell metabolism, preserves cell/tissue structure

• Different fixatives- different degrees of protein denaturing

• Choice of fixative depends on level of analysis

– Light microscopy: formaldehyde, glutaraldehyde

– Electron Microscopy: glutaraldehyde, osmium

Tissue Preparation

1) Fixation

Mode of action:

- cross link proteins: glutaraldehyde/formalin

- precipitate proteins: methanol*

- react with membrane lipids: osmium tetroxide

- membranes become permeable

Produce different levels of tissue preservation

* Methanol often solubilizes membranes

Tissue Preparation

2) Embedding: infiltrate water-filled spaces with embedding medium

Series of soluble replacements

H2O/fix alcohol xylene embedding medium

• Dehydration: replace with ethanol, acetone• Clearing: replace with xylene• Embedding: replace with paraffin wax,

plastic resin

Tissue Preparation

3) Sectioning

3 dimensions --> 2 dimensions

Orientation: Planes of Section

- whole mount (unsectioned)- cross section- longitudinal section- random

Planes of Section

KIDNEY CORTEXBox #17, slide 51 (B), 52 (T)Nicole Monteiro – Wed, 03/25/2009

Kidney Tubules

Tissue Preparation3) Sectioning

Section thickness depends on imaging method.

-Microtome (Light microscopy) ~ 1-10 um -Cryostat - frozen tissues (Light microscopy) ~ 1-30um-Ultramicrotome (Electron Microscopy) ~ 0.1 um

HistoTip: For sharper images, cut thinner sections.

Tissue Preparation

4) Staining*

• Nonspecific: general

• Specific: identified molecules

* To be discussed in detail in a few days

Tissue Preparation

4) Imaging ----> Microscopy

• Compound light microscope - light

• Confocal microscopy - coherent light

• Electron microscopy- electron beam

Microscopy

Imaging Resources Websites: links are on course website- Review materials

NIKON-- recommended for clarityhttp://www.microscopyu.com/articles/optics/

ZEISS http://zeiss-campus.magnet.fsu.edu/ OLYMPUShttp://www.olympusmicro.com/primer/virtual/virtual.html

Compound microscope

Nikon E200

2 Sets of Conjugate Focal Planes:

1) Image-forming (field planes)

2) Illuminating (aperture planes)

The sets of focal planes are in focus and superimposed in properly aligned microscope

http://www.microscopyu.com/articles/formulas/formulasconjugate.html

Conjugate Planes:1) Focused at 1,

focused at all (pointers etc.)

2) Planes alternate in succession:

illumination / image-form

3) Poor image quality: dirt, dust, poor alignment

Objective lens

- gathers light from specimen

- projects a magnified, real image up into body tube.

Ocular lens

- produces a secondarily enlarged real image projected by the objective.

- can be fitted with scales, markers or crosshairs whose images can be superimposed on the image of the specimen.

Magnification:

MAGNIFICATION

Magnifying power of Ocular lens (Mocular)

Magnifying power of Objective lens (Mobjective)

Visual Magnification = Mocular X Mobjective

Compound microscope

Resolution= Resolving Power

-the smallest distance (d) at which two objects can be successfully distinguished.

Resolution (d): d = (0.61 x )/ NA

= wave length NA= numerical aperture

Compound Microscope

Quick Question: How can you make d smaller?

Numerical Aperture (NA): measure of objective’s

ability to collect light from specimen NA= n sin

n = refractive index of medium= one half of angular aperture

http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html

NA=0.22 NA=1.0

Resolution: d = 0.61 x NA

Refractive index (η) of different media Air=1.0003 Water=1.33 Immersion Oil=1.515

NA= n sin

Resolution versus Wavelength

Resolution: d= 0.61 x NA

Wavelength (nanometers) Resolution (micrometers)360 .19400 .21450 .24500 .26550 .29600 .32650 .34700 .37

Resolving Distance (d)

Human eye 0.2 mmLight Microscope 0.2 umScanning Electron Microscope 2.5 nmTransmission Electron Microscope 1.0 nm

Resolution: d= (0.61 x )/ NA

HistoTip: Avoid confusion when discussing resolution. Increased resolution or resolving power usually means a SMALLER value of d (distance).

PROBLEM:

Objective lens A:

Magnification = 40X

N.A. = 0.45

Objective lens B:

Magnification = 40X

N.A. = 0.80

-->Which objective lens would give the sharper image and why?

PROBLEM:

You photograph some liquid crystalline DNA using objective D and objective E. You then enlarge the images to the same size using Photoshop in the manner described below.

Image D : 20X objective, NA= 0.40, enlarged 10X

Image E : 4X objective, NA= 0.10, enlarged 50X

Which image would be sharper and why?

Empty Magnification: an image is enlarged, but no additional detail is resolved. A : 20X objective, NA= 0.40, enlarged 10X. Magnified 200B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200HistoTip: Maximum useful magnification=1000 X N.A.

HistoTip: Maximum useful magnification=1000 X N.A.