Why Confocal?

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

2 Why Confocal?

edu/tutorials/opticalsectioning/confocalwidefield/index.

3 Confocal vs Widefield Microscopy medulla muscle fiber pollen widefield confocal

4 Confocal vs Widefield Microscopy widefield confocal

5 Confocal vs Widefield Microscopy Axial resolution <0.2 µm ~0.6 µm >2 µm ~0.8 µm

6 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.

7 Principles of Confocal Microscopy

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

9 Confocal Microscope

Confocal Microscope Components What does the PMT see? http://micro.

10 Confocal Microscope Components What does the PMT see?

11 Confocal Microscope Components Movie

12 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

14 Gain and Offset correct settings gain too high offset too high 1.7 orrect settings gain too high gain too high offset too high offset too high gain too low offset too low 2.4 Martin Spitaler, 06/02/2008, <FILM GIP.doc>

15 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.

16 Spinning Disk Confocal Microscopy

Spinning Disk Confocal Microscopy http://zeiss-campus.magnet.fsu.

17 Spinning Disk Confocal Microscopy

18 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

19 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.

20 Advantage of Confocal Microscopy (2) 3D reconstruction of specimen

21 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).

22 Spectral Bleed-through Artifact (ECFP)

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

brightest and most photostable fluorophores for the least

24 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.

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

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

27 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

Spectral Imaging Fluorephore spectra are additive http://zeiss-campus.

28 Spectral Imaging Fluorephore spectra are additive

29 Spectral Bleed-through Remedy Spectral Imaging Lambda Stack

30 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

31 Studying Colocalization

32 Colocalization of Fluorophores vinculin actin

33 ImageJ

Colocalization of Fluorophores 255 0 255 Fluorogram: A scatterplot graphs the

image, or a selected region of interest, on a two-dimensional histogram.

34 Colocalization of Fluorophores 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

the 2 nd channel S1 aver = average intensityof pixels in the 1

35 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

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

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

38 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

39 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.

40 Example 1 of Colocalization Channel 1 Channel 2 Colocalization Fluorogram Rr = R = k1 = k2 =

41 Example 2 of Colocalization Channel 1 Channel 2 Colocalization Fluorogram Rr = R = k1 = k2 =

42 Artifacts in Colocalization bleed through correct Spectral bleed through

43 Artifacts in Colocalization Black level Intensity Alignment

Basic Properties of Digital Images

Basic Properties of Digital Images Cell Biology Application of Microscopy Class 13: Image Processing and Manipulation Pascale Lacor, Mature rat hippocampal neurons attacked by Alzheimer s related neurotoxins (100X) Basic Properties of Digital