BMS 524: Lecture 3 Purdue University Cytometry Laboratories Lecture 4 The Principles of Confocal...

BMS 524: Lecture 3Purdue University Cytometry Laboratories

Lecture 4

The Principles of Confocal Microscopy: Components of the microscope.

BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”

1 Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine

UPDATED October 27, 1998

J.Paul Robinson, Ph.D. Professor of Immunopharmacology

Director, Purdue University Cytometry Laboratories

These slides are intended for use in a lecture series. Copies of the graphics are distributed and students encouraged to take their notes on these graphics. The intent is to have the student NOT try to reproduce the

figures, but to LISTEN and UNDERSTAND the material. All material copyright J.Paul Robinson unless otherwise stated, however, the material may be freely used for lectures, tutorials and workshops. It may not

be used for any commercial purpose.

The text for this course is Pawley “Introduction to Confocal Microscopy”, Plenum Press, 2nd Ed. A number of the ideas and figures in these lecture notes are taken from this text.


Overview

• Components of a confocal microscope system

• Optical pathways

• Optical filters

• Resolution

• 3D basics

• Other components


Benefits of Confocal Microscopy

• Reduced blurring of the image from light scattering• Increased effective resolution• Improved signal to noise ratio• Clear examination of thick specimens• Z-axis scanning• Depth perception in Z-sectioned images• Magnification can be adjusted electronically


Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter


Confocal Principle

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter


Fluorescent Microscope

Objective

Arc Lamp

Emission Filter

Excitation Diaphragm

Ocular

Excitation Filter

Objective

Laser

Emission Pinhole

Excitation Pinhole

PMT

EmissionFilter

Excitation Filter

Confocal Microscope


MRC 1024 System

UV Laser

Kr-Ar Laser

Optical Mixer

ScanheadMicroscope


MRC 1024 SystemLight Path

PMT


Optical Mixer - MRC 1024 UV

Argon Laser

Argon-KryptonLaser

Fast Shutter

UV CorrectionOptics

FilterWheels

To Scanhead

UV Visible

Beam Expander


Optical Mixer - MRC 1024 UV

Argon Laser

Argon-KryptonLaser

Fast Shutter

UV CorrectionOptics

FilterWheels

To Scanhead

UV Visible

353,361 nm

488, 514 nm

488,568,647 nm


MRC 1024 Scanhead

From Laser

To and from Scope

32

1PMTGalvanometers

EmissionFilterWheel


To Scanhead

From Scanhead


Scanning Galvanometers

xy

Laser in

Laser out

Point Scanning

ToMicroscope


The Scan Path of the Laser Beam767, 1023, 1279

511, 1023

00Start

Specimen

Frames/Sec # Lines1 5122 2564 1288 6416 32


How a Confocal Image is Formed

CondenserLens

Pinhole 1 Pinhole 2

ObjectiveLens

Specimen

Detector

Modified from: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989


Fundamental Limitations of Confocal Microscopy

FromSource

To Detector

.x,y,z

2

n2 photons2

1

n1 photons

1

z

y

xVOXEL

PIXEL

From: Handbook of Biological Confocal Microscopy. J.B.Pawley, Plennum Press, 1989


Optical Resolution

• Gray Level

• Pixelation

• Aberrations


Gray Level & Pixelation• Analogous to intensity range

For computer images each pixel is assigned a value. If the image is 8 bit, there are 28 or 256 levels of intensity If the image is 10 bit there are 1024 levels, 12 bit 4096 levels etc.

• The intensity analogue of a pixel is its grey level which shows up as brightness.

• The display will determine the possible resolution since on a TV screen, the image can only be displayed based upon the number of elements in the display. Of course, it is not possible to increase the resolution of an image by attributing more “pixels” to it than were collected in the original collection!


Pixels

• Pixels & image structure

Hardcopy usually compromises pixel representation. With 20/20 vision you can distinguish dots 1 arc second apart (300 m at 1 m) so 300 DPS on a page is fine. So at 100 m, you could use dots 300 mm in size and get the same effect! Thus an image need only be parsimonius, i.e., it only needs to show what is necessary to provide the expected image.


Pixels

T


320x240 x 24

Zoom x 2Zoom x 8

Zoom x 4

Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.

Magnifying with inadequate information. This is known as “empty magnification” because there are insufficient data points.

The final image appears to be very “boxy” this is known as “pixilation”.

The final image appears to be very “boxy” this is known as “pixilation”.


541x600x8

361x400x8

180x200x8

Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!

Magnifying with adequate information. Here, the original image was collected with many more pixels - so the magnified image looks better!

Socrates?….well perhaps not...

Socrates?….well perhaps not...


320x240 x 24

1500x1125x24

Originals collected at high resolution - compared to a low resolution image magnified

Originals collected at high resolution - compared to a low resolution image magnified


Sampling Theory• The Nyquist Theorem

– Nyquest theory describes the sampling frequency required to represent the true identity of the sample.

– i.e., how many times must you sample an image to know that your sample truly represents the image?

• Nyquist claimed that the rate was 2f. It has been determined that in reality the rate is 2.3f - in essence you must sample at least 2 times the highest frequency.


Raman Scattering

• At an excitation line of 488 nm, Raman scatter will be at 584 nm or less with increased concentration of protein, etc.

• Is directly proportional to the power of the laser light


3D Image Reconstruction

z

x

y


z

x

y

z

yy



x

y

z

y

y

z



Fluorescent image of paper


Pine Tree pollen - collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories


Fly eye! - collected on a Bio-Rad MRC 1024 at Purdue University Cytometry

Laboratories


Collagen fibers collected using transmitted light and fluorescence [collected on a Bio-Rad MRC 1024 at Purdue University Cytometry Laboratories ]


Collagen fibers collected using transmitted light [collected on a Bio-Rad

MRC 1024 at Purdue University Cytometry Laboratories ]


• Top: Endothelial cells (live) cultured on a coverslide chamber. The cells were stained with stain that identified superoxide production (hydroethidine) and were color coded (red =high stain, green =low stain) then a 3D reconstruction performed and a vertical slice of the culture shown. Here, the original image was collected with many more pixels - so the magnified image looks better!

• Left: Same endothelial cells with hydroethidine stain (live cells) showing a fluorescence reconstruction - note fluorescence is only in nuclear regions - no cytoplasm is stained. Imaged on Bio-Rad MRC 1024 system.

• Top: Endothelial cells (live) cultured on a coverslide chamber. The cells were stained with stain that identified superoxide production (hydroethidine) and were color coded (red =high stain, green =low stain) then a 3D reconstruction performed and a vertical slice of the culture shown. Here, the original image was collected with many more pixels - so the magnified image looks better!

• Left: Same endothelial cells with hydroethidine stain (live cells) showing a fluorescence reconstruction - note fluorescence is only in nuclear regions - no cytoplasm is stained. Imaged on Bio-Rad MRC 1024 system.


Creating Stereo pairs

z

xy

Pixel shifting -ive pixel shift for left+ive pixel shift for right


SUMMARY SLIDE

• Components of a confocal system

• Optical pathways

• Optical filters

• Resolution

• 3D basics

• Light paths and lasers

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