vital imaging

Lecture 11Live Cell Imaging Applications in Confocal Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”1Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine : 

Lecture 11Live Cell Imaging Applications in Confocal Microscopy BMS 524 - “Introduction to Confocal Microscopy and Image Analysis”1Credit course offered by Purdue University Department of Basic Medical Sciences, School of Veterinary Medicine UPDATED February 2000 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.

Applications : 

Applications Organelle Structure Probe ratioing Conjugated antibodies DNA/RNA Cytochemical Identification Oxidative Metabolism Exotic Applications

Applications : 

Applications Organelle Structure & Function Mitochondria (Rhodamine 123) Golgi (C6-NBD-Ceramide) Actin (NBD-Phaloidin) Lipid (DPH)

Example of DIC and Fluorescnece : 

Example of DIC and Fluorescnece Human cheek epithelial cells (from JPR!) stained with Hoechst 33342 - wet prep, 20 x objective, 3 x zoom (Bio-Rad 1024 MRC) Giardia (DIC image) (no fluorescence) (photo taken from a 35 mm slide and scanned - cells were live when photographed)

Slide 5: 

Fluorescence Microscope image of Hoechst stained cells (plus DIC) Image collected with a 470T Optronics cooled camera

PI - Cell Viability : 

PI - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead PI PI PI PI PI PI PI PI PI PI PI PI PI PI Viable Cell Damaged Cell

Specific Organelle Probes : 

Specific Organelle Probes BODIPY Golgi 505 511 NBD Golgi 488 525 DPH Lipid 350 420 TMA-DPH Lipid 350 420 Rhodamine 123 Mitochondria488 525 DiO Lipid 488 500 diI-Cn-(5) Lipid 550 565 diO-Cn-(3) Lipid 488 500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH - diphenylhexatriene TMA - trimethylammonium

Organelle Function : 

Organelle Function Mitochondria Rhodamine 123 Endosomes Ceramides Golgi BODIPY-Ceramide Endoplasmic Reticulum DiOC6(3) Carbocyanine

Oxidative Reactions : 

Oxidative Reactions Superoxide Hydroethidine Hydrogen Peroxide Dichlorofluorescein Glutathione levels Monobromobimane Nitric Oxide Dichlorofluorescein

Slide 10: 

DCFH-DA DCFH DCF

Exotic Applications of Confocal Microscopy : 

Exotic Applications of Confocal Microscopy FRAP (Fluorescence Recovery After Photobleaching) Release of “Caged” compounds Lipid Peroxidation (Parinaric Acid) Membrane Fluidity (DPH)

4D Imaging : 

4D Imaging Time 1 2 3 4 5 This could also be achieved using an X-Z scan on a point scanner.

GFP technology : 

GFP technology

Diseases associated with A-type lamin mutations : 

Diseases associated with A-type lamin mutations Emery Dreifuss muscular dystrophy Dilated cardiomyopathy Limb-girdle dystrophy Dunnigan-type lipodystrophy

Emery Dreifuss muscle dystrophyEM chromatin : 

Emery Dreifuss muscle dystrophyEM chromatin muscle wasting and weakness Contractures of joints Cardiomyopathy associated with conduction defects

Tools for investigating lamin organisation : 

Tools for investigating lamin organisation Different clones of cell line CHO-K1 transfected with DNA constructs encoding chimeric proteins consisting of Green Fluorescent Protein fused to normal and muted lamins Confocal microscopic techniques Standard biochemical techniques

Slide 18: 

Methods Construction of A-type lamin-GFP plasmids Transfection of CHO-K1 cells Selection for neomycin resistance Subcloning at single cell density Growing cells at 30°C for several days Analysis of living or fixed cells by confocal microscopy

Slide 19: 

Green Fluorescent Protein (GFP) Deep sea jellyfish Aequora victoria Absorption: wt GFP 395 nm EGFP/ 480 nm pS65T-C1 Emission: wt 509 nm EGFP 540 nm Other variants: GFPuv,CFP, BFP, YFP,DsRed(2)

Slide 20: 

pS65T-C1 vector

Slide 21: 

A-type lamin-GFP chimeric proteins expressed in CHO-K1 cells -COOH NH2- -COOH NH2- -COOH Lamin A-GFP Lamin Adel10-GFP Lamin C-GFP

Slide 22: 

Confocal laser scanning analysis Make Z-series (0.2-0.4 µm slices) and perform 3D-reconstruction Scan living cells every minute at fixed level using low laser intensity (1% of 15 mW)

CHO-K1 cells stably transfected with lamin A-GFP : 

CHO-K1 cells stably transfected with lamin A-GFP

Confocal laser scanning analysis : 

Live imaging of lamin-(GFP) 3-dimensional reconstruction of cells FRAP and FLIP Confocal laser scanning analysis

Lamina reformation during mitosis : 

Lamina reformation during mitosis

Slide 26: 

Z-series of lamin A-GFP transfected CHO

Slide 27: 

Z-series slices Viewing 3-D reconstruction

Slide 28: 

3-D Projection of Z-series CHO Lamin A-GFP

Slide 29: 

3-D Projection of Z-series CHO Lamin Adel10-GFP

Slide 30: 

Projection of z-series of a lamin Adel10-GFP transfected cell

Bleaching of fluorescence in living cells : 

Bleaching of fluorescence in living cells Fluorescence Recovery After Photobleaching (FRAP) Fluorescence Loss of Intensity after Photobleaching (FLIP) Bleaching of fluorescent molecules and monitoring recovery from bleaching in a sample gives information about molecular organisation

FRAPFluorescence Recovery After Photobleaching: : 

FRAPFluorescence Recovery After Photobleaching: Record fluorescence intensity of a structure Bleach a well-defined area Record speed of fluorescence recovery

Ideal recovery curve FRAP : 

Ideal recovery curve FRAP Intensiteit (%) Tijd (sec.) 80 60 100 40 20 0 0

Cross section bleached area : 

Cross section bleached area

Slide 35: 

I(x,t) van onder naar boven: t = 5 / 50 / 150 / 500 / 5000 / 15000 / 50000 ms. A = 2 m  breedte ROI = 4 m .

Slide 36: 

Fluorescence recovery after bleaching Lamina Tubule Nucleoplasm

Slide 37: 

GFP fluorescence recovery after bleaching (FRAP) analysis GFP intensity

Fluorescence loss of intensity after photobleaching (FLIP) : 

Fluorescence loss of intensity after photobleaching (FLIP) Record fluorescence intensity in a sample Bleach repetitively in a small area until no recovery can be observed Record loss of fluorescence in unbleached areas

Measuring loss of fluorescence : 

Measuring loss of fluorescence

Wild-type lamin A-GFP : 

Wild-type lamin A-GFP R386K lamin A mutation Before After After Before Bleaching

Vital Imaging using fully automated imaging system : 

Vital Imaging using fully automated imaging system Hardware Camera Mac G3 Objective heater Light source

Slide 42: 

Macro for vital imaging system

Slide 43: 

Extendend macro

Vital Imaging : 

Vital Imaging Structures Fluorochromes DIC Spatial resolution Binning Stacksize Temporal resolution Viability

Cells in culture : 

Cells in culture Imaging morphologic and molecular changes in time and space (4D) Vital Imaging

Slide 46: 

DIC resolution sec44 1frame/sec (1hr)

Moleculair : 

Moleculair Survival and Fluo 17 hrs

Vital imaging : 

Vital imaging Fluorescence Time lapse

Proliferation : 

Proliferation Survival 6 days

15/30 IR : 

15/30 IR Ischemia and reperfusion:mouse model

New developments : 

New developments

Real Time Confocal Imaging : 

Real Time Confocal Imaging

UltraVIEW confocal systemWhat is a confocal image? : 

UltraVIEW confocal systemWhat is a confocal image? Main parts: Confocal scanner Detector Light source

How to create a Confocal Image ? : 

How to create a Confocal Image ? Move the Sample - Not convenient especially using biological samples - Very Slow Move the Point source illumination - Laser & Galvo Mirrors - Slow, most popular Move the Pinholes - The basis of using a Nipkow Disk - Fast Scanning

Galvo Mirror Laser Scanners : 

Galvo Mirror Laser Scanners Single laser beam The most common method of scanning Advantages: Good spatial resolution and confocality Disadvantages: Slow and high level of photobleaching and phototoxicity

The Nipkow Disk - Petran 1968 : 

The Nipkow Disk - Petran 1968 Multi-point scanner Pinholes The first proposed method of confocal scanning Advantages: Fast, real time confocal Disadvantages: Historically - Poor light efficiency through the disk

Technology behind the UltraVIEW system : 

Technology behind the UltraVIEW system Novel patented scanning principle CCD detection

Slide 58: 

Sample Objective lens Dichroic Pinhole CCD Camera Laser beam Microlens Collector disc Pinhole disc UltraVIEW Scanner

Slide 59: 

Detector: Digital cooled CCD e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - Si + SiO 2 + 22µm 22µm hv e - e - e - e - e - e - e - e - e - e - e - CCD = Charge Coupled Device Semiconductor devices similar to computer RAM chips Incoming photons produce electrons which are trapped in “potential wells” CCD pixel

UltraVIEW concept : 

UltraVIEW concept Benefits ( i ) improved image quality due to higher S/N ( ii ) less photobleaching and phototoxicity ( iii ) faster image capturing

Slide 61: 

Large area and high lateral resolution Cardiac myocyte stained with RyR2 antibody

3D reconstruction of fluo-3 microinjected neurone : 

3D reconstruction of fluo-3 microinjected neurone UltraVIEW in combination with its 3D visualisation program allows true 3D construction with time useful for looking at structures within thick sections such as brain slices.

Temporal resolution : 

Temporal resolution Time (s) t1 t2 t3 t4 t5 t6 The fastest confocal without compromising confocality (up to 100 frames/s)

Fluo3AM loaded cardiac myocytes : 

Fluo3AM loaded cardiac myocytes

Slide 65: 

GFP-P58 a marker for Gogi-ER Trafficking of GFP-tagged protein

Slide 66: 

Phototoxicity Time (s) Very low phototoxicity

Slide 67: 

GFP-tagged histone in Drosophila embryo

Did you get all key information about the technology? : 

Did you get all key information about the technology? Lateral resolution (x-y) Axial resolution (z) Temporal resolution (t) Photobleaching Phototoxicity Superior resolution and speed with reduced photoblecahing and phototoxicity makes UltraVIEW idealy suited for live work.