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 1-4 examples: 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 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 H 2 O/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 CORTEX Box #17, slide 51 (B), 52 (T) Nicole Monteiro – Wed, 03/25/2009 Kidney Tubules

Tissue Preparation 3) 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 clarity ZEISS OLYMPUS

Optical Components - Light source -Diaphragm -Condenser -Lenses -objectives - oculars 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

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 (M ocular ) Magnifying power of Objective lens (M objective ) Visual Magnification = M ocular X M objective 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

NA=0.22 NA=1.0 Resolution: d = 0.61 x NA

Refractive index (η) of different media Air= Water=1.33 Immersion Oil=1.515 NA= n sin 

Resolution versus Wavelength Resolution: d= 0.61 x NA Wavelength (nanometers) Resolution (micrometers)

Resolving Distance (d) Human eye 0.2 mm Light Microscope 0.2 um Scanning Electron Microscope 2.5 nm Transmission 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. = >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 200 B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200 HistoTip: Maximum useful magnification=1000 X N.A.

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