Microscopy Workshop

University Hospital of Wales, Cardiff Wales Heart Research Institute

10 November, 2011

Adam Westmacott

Introduction

• Light travels in straight lines

• In the form of a Sine wave

Introduction

• There are only 2 things that the eye can see

• Colour-Represented by Wavelength

Introduction

• Intensity-Represented by Amplitude

• So we can only see our specimen if it has a different Colour or Intensity than the background

Jargon

• Resolution- Resolution can be defined as the least distance between 2 points at which they can still be recognised as 2 separate entities

• For the eye, this is 70 microns, when the object is 250mm away

• For light microscopy, this is 0.24 microns

• Contrast-The phenomena that allows you to distinguish relevant information from irrelevant. Either by colour or intensity

• Contrast and Resolution are inversely proportional

Jargon

• Working Distance- The distance between the specimen and the front lens of the objective

• Depth of Field –Depth of field in a microscope is the area in front of and behind the specimen that will be in acceptable focus

• Field of View- This is the area of the specimen in view down the eyepieces. It is dependant on the magnification and the F.N.( field number) of the eyepiece.

Jargon

• Field of View-A F.N. of 22 indicates a F.O.V. diameter of 22mm when using a 1x objective. F.O.V. when using 20x objective = 1.1mm F.O.V. when using 100x objective = 0.22mm

• Numerical Aperture- This indicates the resolving ability of an objective. Larger N.A.= Greater resolution and also brighter fluorescence signal. However larger N.A.= less depth of field and shorter working distance. NA= nSinA, where n= refractive index of medium and A is the half angle at which light enters the objective.

Jargon

• Chromatic Aberration- When white light passes through a convex lens, the colours split and focus at different points causing colour fringing. Objectives have additional elements to overcome this problem.

• Apochromatic objectives are fully corrected and Achromatic objectives are corrected for red/blue.

• Plan objectives are designed, assuming a flat specimen, to provide a focused image across the whole field of view

Contrast Techniques

• Brightfield

• Darkfield

• Phase Contrast

• Differential Interference Contrast

• Hoffman Modulation Contrast

• Fluorescence

Contrast-Enhancing TechniquesSpecimen

TypeImaging

Technique

TRANSMITTED LIGHT

Transparent SpecimensPhase Objects

Bacteria, Spermatozoa,Cells in Glass Containers,

Protozoa, Mites, Fibers, etc.

Phase ContrastDifferential Interference Contrast

(DIC)Hoffman Modulation Contrast

Oblique Illumination

Light Scattering ObjectsDiatoms, Fibers, Hairs,

Fresh Water Microorganisms,Radiolarians, etc.

Rheinberg IlluminationDarkfield Illumination

Phase Contrast and DIC

Light Refracting SpecimensColloidal Suspensionspowders and minerals

Liquids

Phase ContrastDispersion Staining

DIC

Amplitude SpecimensStained Tissue

Naturally Colored SpecimensHair and Fibers

Insects and Marine Algae

Brightfield Illumination

Fluorescent SpecimensCells in Tissue Culture

Fluorochrome-Stained SectionsSmears and Spreads

Fluorescence Illumination

Birefringent SpecimensMineral Thin Sections

Liquid CrystalsMelted and Recrystallized

ChemicalsHairs and Fibers

Bones and Feathers

Polarized Illumination

Microscope

• 3 different types

• Stereo

• Inverted Compound

• Upright Compound

Stereo Microscopes

• 2 separate optical paths at an angle to each other

• Brain merges the 2 images to give a 3D image

• Magnification range 4x- 200x• Magnification should not exceed 1000x

NA• 2 principal types

Greenhough Optics

10 Degree Converging light path

Great depth of focus

Galilean Optics

Parallel Optics

Allows build up of other accessories

Stereo Microscopes

• Variety of Illumination options

• Transmitted Brightfield, Darkfield, Oblique and Polarised Light

• Fluorescence

• Reflected through the optics or by separate source

• Fibre optic goose neck or ring lights

Inverted Microscopes

• Predominately used for looking at specimens in suspension- Live cells

• Long working distance optics

• Whole variety of contrast techniques

• Environmental and temperature control

• Sample Manipulation/Injection

• Magnifications 40x – 1000x

Inverted Microscopes

Inverted Microscopes

Optical Workbenches

Upright Microscopes

• Samples on glass slides

• Variety of Contrast techniques

• Magnification range 10x – 1000x

Upright Microscopes

Student Teaching Microscope

Fully Motorised and computer controlled with advanced imaging system

The Microscope- What is Important

• Location• Avoid direct lighting - not next to a

window.

• Comfortable working area

• Adjustable chair

• Isolate external vibration

The Microscope- Illumination

• Ensure that the illumination is matched to the application

• 6v 30W, 12v 100W and now true colour LED

• Fluorescence- HG, XE, Metal Halide or LED

Condensers

• The condenser presents the light for the objective to do its work

• Remember that a compromise is just that

• A swing out condenser will not give as even an illumination at low power as a Ultra Low Condenser. You may not see it, but the camera will. Make sure software has shading correction.

Condensers

Microscopy Workshop

www.olympusmicro.com

Micro_Series_Part_5.pdf

Thank You