Why Confocal?

Cell Biology Application of Microscopy Class 10: Confocal Fluorescence Microscopy Fatima Santos, Mouse blastocyst (epigenetic marks) (630X)

Why Confocal?

Confocal vs Widefield Microscopy medulla muscle fiber pollen widefield confocal http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/index.html http://www.olympusconfocal.com/theory/confocalintro.html

Confocal vs Widefield Microscopy widefield confocal

Confocal vs Widefield Microscopy Axial resolution <0.2 µm ~0.6 µm >2 µm ~0.8 µm http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

Confocal Microscopy The principle of confocal imaging was developed in 1950s by Marvin Minsky and aims to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of widefield microscopes. Marvin Minsky

Principles of Confocal Microscopy http://www.olympusconfocal.com/theory/confocalintro.html

Confocal Microscope Intense light source (laser) Laser combiner Confocal module Microscope (upright) Computer Vibration isolation table

Confocal Microscope http://www.olympusconfocal.com/theory/confocalintro.html

Confocal Microscope Components What does the PMT see? http://micro.magnet.fsu.edu/primer/java/digitalimaging/photomultiplier/endonpmt/index.html

Confocal Microscope Components Movie

Confocal Microscope Settings PMT gain Gain ~1000 V may produce noisy images. PMT offset The higher the offset, the higher the pixel intensity on screen will be displayed as black. Pinhole size Ideally the pinhole should be set at (or near) 1 Airy unit. Scan speed The higher the speed, the higher the noise. Laser power

Gain and Offset correct settings 2.9 3.7 1 9.6 1 2.3 - gain too high offset too high 1.7 orrect settings.7 1 2.9 9.6 1 2.3-2.9 1 2 2.3 1 2.4 1.7 gain too high gain too high 2.9 1 2.9 1 2 2 offset too high offset too high 2.3 1 2.4 2.3 1 gain too low offset too low 2.4 Martin Spitaler, 06/02/2008, <FILM GIP.doc>

Single- and Multiple-beam Scanning Single-beam scanning: LSCM (laser scanning confocal microscope) Multiple-beam scanning Spinning disk confocal microscope Tandem scanning confocal microscope

Spinning Disk Confocal Microscopy http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

Spinning Disk Confocal Microscopy http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/spinningdiskfundamentals/index.html http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

Spinning Disk Confocal Microscopy Advantage: 1. Fast 2. Less photo-damage 3. Don t have to use laser 4. Don t have to use PMT Disadvantage: 1. Fixed pin hole size 2. Lower axial resolution 3. Synchronization issue http://zeiss-campus.magnet.fsu.edu/articles/spinningdisk/introduction.html

Advantage of Confocal Microscopy (1) Optical sectioning Produce thin (~0.6 µm) optical sections through thick (50 µm or more) specimen. The optical sections can be used to re-construct the specimen in 3D space.

Advantage of Confocal Microscopy (2) 3D reconstruction of specimen https://vimeo.com/sciinstitute

Advantage of Confocal Microscopy (3) More Color, less bleed-through: Because the images are detected by a computer rather than by eye, it is possible to distinguish more colors (spectral unmixing).

Spectral Bleed-through Artifact (ECFP)

Spectral Bleed-through Remedy 1. Selecting fluorophores with minimal spectrum overlap

Spectral Bleed-through Remedy 2. Balancing the degree of labeling for different dyes Use the brightest and most photostable fluorophores for the least abundant cellular targets. Examining one fluorophore using the filter set for another fluorophore.

Spectral Bleed-through Remedy 3. Careful laser and filter selection / management

Spectral Bleed-through Remedy Sequential scanning (scan with one laser first, and the second one)

Spectral Bleed-through Remedy 4. Spectral imaging and linear unmixing Original Linearly unmixed and pseudo-colored SYTOX Green (nucleus) Alexa 488-phalloidin Oregon Green-antibody against mitochondrion http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/linearunmixing/index.html

Spectral Imaging Fluorephore spectra are additive http://zeiss-campus.magnet.fsu.edu/tutorials/spectralimaging/additiveproperties/index.html

Spectral Bleed-through Remedy Spectral Imaging Lambda Stack

When Do You Need to Use LSCM? Analyzing the expression level of a cytosolic protein No Analyzing the intracellular localization of a protein Yes Analyzing the localization of a protein in the lamellipodium No Analyzing protein level in phenol red-containing medium Yes Fluorescent live cell imaging No Analyzing protein co-localization Yes Analyzing fluorescent protein dynamics in ms scale No

Studying Colocalization

Colocalization of Fluorophores vinculin actin

ImageJ http://rsbweb.nih.gov/ij/index.html

Colocalization of Fluorophores 255 0 255 Fluorogram: A scatterplot graphs the intensity of one pseudocolor (or channel) versus another for each pixel in the image, or a selected region of interest, on a two-dimensional histogram. Rat hippocampus Neurofilament GFAP Fibroflast F-actin Vinculin Epithelial cell EYFP DsRed

Pearson s Correlation Coefficient S1 i = signal intensity of pixels in the 1 st channel S2 i = signal intensity of pixels in the 2 nd channel S1 aver = average intensityof pixels in the 1 st channel S2 aver = average intensityof pixels in the 2 nd channel

Overlap Coefficient S1 i = signal intensity of pixels in the 1 st channel S2 i = signal intensity of pixels in the 2 nd channel

Overlap Coefficient S1 i = signal intensity of pixels in the 1 st channel S2 i = signal intensity of pixels in the 2 nd channel

Colocalization Coefficient S1 i = signal intensity of pixels in the 1 st channel S2 i = signal intensity of pixels in the 2 nd channel S1 i,coloc equals S1 i if S2 i is greater than zero, and vice versa for S2 i,coloc

Colocalization Coefficient S1 i = signal intensity of pixels in the 1 st channel S2 i = signal intensity of pixels in the 2 nd channel S1 i,coloc equals S1 i if S2 i lies within the region of interest thresholds and equals zero if S2 i represents a pixel outside the threshold levels.

Example 1 of Colocalization Channel 1 Channel 2 Colocalization Fluorogram Rr = 0.4233592 R = 0.9566072 k1 = 0.58164597 k2 = 1.5732893 http://www.olympusfluoview.com/java/colocalization/index.html

Example 2 of Colocalization Channel 1 Channel 2 Colocalization Fluorogram Rr = -0.14634663 R = 0.83284664 k1 = 0.9546193 k2 = 0.7266074

Artifacts in Colocalization bleed through correct Spectral bleed through

Artifacts in Colocalization Black level Intensity Alignment