imaging introduction - stanford universityotolic.stanford.edu/documents/imaging-introduction.pdf ·...

Imaging Introduction

September 24, 2010

Friday, March 25, 2011

What is a microscope?

• Merriam-Webster: an optical instrument consisting of a lens or combination of lenses for making enlarged images of minute objects; especially: compound microscope (a microscope consisting of an objective and an eyepiece mounted in a drawtube)


Why is it important to know how a microscope works?



• Interpreting your images correctly



• Interpreting your images correctly• Take the best images possible



• Interpreting your images correctly• Take the best images possible• Work independently



• Interpreting your images correctly• Take the best images possible• Work independently• Properly use it and not damage it

($35,000 - $70,000)



• Interpreting your images correctly• Take the best images possible• Work independently• Properly use it and not damage it

($35,000 - $70,000)• Push the limits of the microscope and

use it for new applications


Components of microscope


Why do we care about this?

Not Aligned Aligned w/ Smallest Aperture

Aligned w/ Largest apertureAligned w/ Medium Aperture

20x Objective on Upright Zeiss


63x ObjectiveNot Aligned Aligned w/ Smallest Aperture

Aligned w/ Largest apertureAligned w/ Medium Aperture


Elements of transmitted light path

Adapted from Sluder & Nordberg 2007

Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser


From Jeff LichtmanFriday, March 25, 2011

Illumination Cone Diagram


Aperature Diaphram applethttp://www.microscopy.fsu.edu/primer/java/kohler/condensercones/index.html

What would happen if the field diaphram was vared?


http://www.microscopy.fsu.edu/primer/java/kohler/condensercones/index.html

http://www.microscopy.fsu.edu/primer/java/kohler/condensercones/index.html

Benefits of Varying Field and Aperture Diaphragms

• Field Diaphragm– Affects the “field” of view– Stopping it down limits the area of view– Stopping it down useful when trying to prevent loss of contrast due

to scattered light-the field diaphragm thus can improve image contrast

– Does not affect the intensity of light in the illuminated region

• Aperture Diaphram– Decreasing can increase contrast– Decreasing increases depth of focus– Increasing, increases resolution


Transmitted Light MethodsBrightfield

Pros:– Does not require high intensity light– Ideal for measurements because no

distortionCons:– Contrast at the expense of

resolution

From FSU MicroscopyBright field Phase DIC

Our eyes see only changes in intensity, they cannot see changes in the phase of light, and light passing through unstained tissue does not change intensity very much because of only slight changes in the index of refraction.

Ways to enhance contrast:• Stain Tissue• Phase Contrast• DIC


Phase Contrast• Pros

– Can get contrast without losing resolution– On living tissue, dead cells are dark, live cells are

bright

• Cons– Phase images are usually surrounded by halos

around the outlines of details– Requires special objective– Phase annuli do limit the working numerical aperture

thus resolution– Not good with thick specimens– Requires much more light than brightfield

From FSU MicroscopyFriday, March 25, 2011

Differential Interference Contrast (DIC)

From FSU Microscopy

• Pros– Can get contrast without losing resolution– Prism does not need to be in the back focal plane of

the objective– Psuedo-3D shadow casting effect– Good for thicker specimens because of optical

sectioning– Smaller specimen features are not obscured by

adjoining regions having large optical gradients

• Cons– Cost– Sensitive only to gradients in a specific direction, so

the image of a given object can change as it is rotated.

– Analyzer must be removed in fluorescence imaging– Requires much more light than brightfield


Next Up: Objective


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser


Next Up: Objective


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser

Objective


How do you choose an objective?

From Zeiss BrochureFriday, March 25, 2011

Objective Innerds – Why so many lenses?


We don’t live in an ideal worldSpherical Aberrations

Chromatic Aberrations

Field Curvature


Main Classes of Objectives

• Achromats– 2 λ color & 1 λ spherical corrections

• Fluorite– 3 λ color & spherical corrections

• Apochromats– 4 λ color and spherical correction

• Plan– Flat field corrected

Sluder & Nordberg 2007 Friday, March 25, 2011

Main Classes of Objectives

• Achromats– 2 λ color & 1 λ spherical corrections

• Fluorite– 3 λ color & spherical corrections

• Apochromats– 4 λ color and spherical correction

• Plan– Flat field corrected

Sluder & Nordberg 2007

IR and UV is always special and need to pay particular attention


What do the corrections depend on?


Why do corrections matter? Co-localization

North 2006

Beads Fission Yeast


Image Sharpness

Improper Coverslip thickness

North 2006; FSU MicroscopyFriday, March 25, 2011

Numerical Aperature


Numerical Aperture Effects


Resolution: 1.22 λ / (NAobj + Nacondenser)

More Next Week!


Tube Length


Ray Optics


Infinity Corrected Microscope


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser

Objective


Infinity Corrected Microscope


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser

Objective

Tube Lens


Benefits of Infinity Corrected Microscopes

• Flexibility: Inserting pieces of glass in Infinity space do not cause a lot of chromatic aberrations

• More correction lenses can go in the objective, so less aberrations


Elements of reflected light path


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser

Objective

Tube Lens


Elements of reflected light path


Collector Lens

Field diaphragm

Aperture Diaphragm

Condenser

Objective

Tube Lens

Filter Cube

Light manipulation are the same just comes from above!Field diaphragm in the incident light path-really useful to remove background!!!


Reflected Köhler Illumination


Reflected Köhler Illumination

Why can we come from above in Fluorescence?


Spectral Separation

Alexa 488

Excitation Emission

This spectral separation of the excitation wavelengths and emission wavelengths allows us to use the same light path through the objective and separate emission from excitation when needed.


Why is there spectral separation?Jablonski Diagram


Where does the spectral spread come from?


The Filter Cube:How we separate light paths

From FSU Microscopy

To DetectorEmission

Filter

FromIlluminatorDichroic Mirror

ExcitationFilter


Dichroic Mirror

DM 565Designed to pass light above a certain wavelength and reflect light below it

Normal Mirror


Dichroic Mirror Reality%

Tra

nsm

ittan

ce

IdealReality


Filter Cube Specs%

Tra

nsm

ittan

ce

Excitation EmissionDichroic


2-photon considerations


Light Source

Fluorescent Specimen

Excitation lightFluorecence

To Detector

How the Cube works


Filter Design


Fluorescent Lamps


4 Major Fluorescent Lamp Sources• Mercury• Metal halide Xenon

LED


Binocular/Trinocular head


Function of EyepiecesImage formation



EyepiecesEver get your specimen into focus using your eyes and then you take the image with the computer and it is blurry or you see nothing (for confocal)?



EyepiecesEver get your specimen into focus using your eyes and then you take the image with the computer and it is blurry or you see nothing (for confocal)? Parfocality!


Eyepieces


Questions?

• Next Time– Confocal Microscopy– PSF– 2-photon Microscopy


References• Sluder, Greenfield, and Joshua J Nordberg. 2007. Microscope basics. Methods in

cell biology 81, no. 06: 1-10. doi:10.1016/S0091-679X(06)81001-0. http://www.ncbi.nlm.nih.gov/pubmed/17519159.

• North, Alison J. 2006. Seeing is believing? A beginners' guide to practical pitfalls in image acquisition. The Journal of cell biology 172, no. 1: 9-18. doi:10.1083/jcb.200507103. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2063524&tool=pmcentrez&rendertype=abstract.


http://www.ncbi.nlm.nih.gov/pubmed/17519159




Imaging Path Illumination Path


Numerical Aperature

1.4NA Oil Lens1.4 = (1.5) sin(u)u = 69 degrees


Inverted Microscope


What is the main difference between these two?

Olympus BX51$35,000

Barska Compound Microscope$300


What is the main difference between these two?

Olympus BX51$35,000

Barska Compound Microscope$300

Infinity Correction!Friday, March 25, 2011

Locations

Field stop Neutral Density FiltersAlternate field stop location

on other microscopes


Aperature Diaphragm Condenser

Turret

Polarizer


imaging introduction - stanford universityotolic.stanford.edu/documents/imaging-introduction.pdf ·...

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