LSM 510 in Live Cell Imaging Welcome

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Content

The Subject of Interest Scanning Strategies for fast Image Acquisition Multicolor Labeling Emission and Excitation Crosstalk Spectral dimension - Emission Fingerprinting - Excitation Fingerprinting - Online Fingerprinting Applications - Quantitative FRET Imaging - FRAP and FLIP - Photoactivation & Photoconversion Zeiss features for live cell imaging

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging The Subject of Interest

In fact Cells are highly dynamic Structures.

The decision of a cell to proliferate or to underwent cellular suicide depends on dynamic analyses of a complex information network.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Starting with the details – fundamental Questions

• Which Ligand binds to the receptor?

• Is molecule A changing the conformation of the complex of the molecules B and C?

• How fast can a membrane associated protein reach its target structure in the nucleus?

• Is there a binding secuence for that protein at DNA? What happens after binding?

• Which nerve cell is responsible to react after a visual stimulus?

• Which cells of an embryo are the progenitors liver cells?

• How can the malaria germ enter a blood cell?

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging What are the specimens?

Specimen Required equipment

Cell culture Cell culture lab, cell incubation equipment at the microscope Tissue culture, tissue co cultures or Tissue culture lab, advanced incubation combinations of cell and tissue cultures equipment at the microscope (O2 control), with anorganic materials micro manipulation Living animals Animal housing, surgery equipment, special holding frames, vital function monitoring, micro manipulation

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging What are the dyes?

Staining Principle Stained Components Direct or indirect Antibody Labeling Proteins, glycoproteins and polysaccarid structures at the cell surface Active uptake of fluorescent particles by Endosomes, lysosomes, phagosomes cellular transport mechanisms Dyes selective for the physical and Cellular membrane (DiI), DNA, endoplasmic chemical environment of a described reticulum, Golgi apparatus, mitochondria subcellular compartment Voltage dependent dyes Mitotracker red and green Ion sensitive dyes Ca++ imaging (Fura, Fluo4, Calcium Green) pH imaging (SNARF) Site specific expression of fluorescent Diverse number of proteins of interest proteins

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Scanning Strategies for fast Imaging Unidirectional Scan

Fly Back Blanking, Zoom 1 Fly Back Blanking, Zoom 0.7 Fly Back Blanking, Zoom 1, Fly Back Blanking, Zoom 2, Rotation 45° Rotation 45°, X,Y Offset DSP controls Pixel Synchronized

Ê AOTFs Ê Two Scanner Mirrors Ê Data Acquisition Bi-directional Scan

Bi-directional Scan, Zoom 1 Bi-directional Scan, Zoom 1 Bi-directional Scan, Multitrack Rotation 45° Configuration

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging DSP : Scanmode Flexibility

DSP controls Pixel Synchronized can

• AOTFs S • Two Scanner Mirrors

• Line Scan • Data Acquisition Spline

With a motorized scanning stage n single XY frames can

n patched together, for ROI Sca overview images of

Tile Sca specimen with are exceeding Selective Excitation, a single field of Bleaching, Uncaging and view Data Acquisition from User defined ROIs

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging

Scan Mode: Online Crop Function

• Use the Crop Function to adapt the scan field to the area of your interest • The scan field is free rotate able due to the two independent scanning mirrors • Any geometry from 1x4 to 2kx2k pixel is possible

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Scanning Strategies: Real Regions of Interest

• Scanning real Regions of Interest (ROIs) is a feature of the LSM 510 requiring a fast laser switcher (AOTF, or AOM). • Up to 99 ROIs of regular or user defined shapes can be defined. • While scanning ROIs, the scan mirrors cover a field, neglecting all lines not including ROI pixel. A smart configuration of scan field rotation and ROI arrangement can save scan time. • Advantages: save memory, scan faster, prevent photo bleaching in neighbored cells, minimize photo damage. • Applications: Photo bleaching experiments (FRAP and FLIP) Uncaging (localized excitation, release of caged compounds).

definition of ROIs full frame scan after for bleaching localized bleaching

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution

Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x512

Time resolution: 1s

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution

Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x100

Time resolution: 100 ms

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Time Series: Live Cell Imaging a high Time Resolution

Oregon Green BATPA-AM loaded Cardio- myocytes Frame: 512x32

Time resolution: 20ms

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Multifluorescence: The crosstalk problem

FITC / Rhodamin double labeling: while simultaneous excitation and detection of both dyes a part of the FITC emission is detected in the Rhodamin detection channel (Emission Crosstalk).

The problem can be solved by sequential excitation and emission detection. As a precondition the wavelength used for FITC excitation should not excite Rhodamin (no Excitation Crosstalk).

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Multifluorescence: Detection with Line and Frame wise Multi- Tracking

The fastest switching between two tracks is available in with line wise switching at bi- directional scanning mode

• The Configuration Control allows to define up to 4 different track for sequential dye excitation and emission detection. • The tracks can be switched line or frame wise. If line wise switching is active, no mechanical changes of optical elements between two tracks are allowed.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging New: MultiChannel unmixing

The solution: MultiChannel Unmixing

Works on all LSM 510 and LSM 5 PASCAL with LSM Software Rel. 3.2

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging New: MultiChannel unmixing

The problem: excitation Crosstalk

Crosstalk is the most often occurring problem in multifluorescence imaging. While emission crosstalk of dyes can easily be avoided by selective excitation with the Zeiss multitracking function, the use of dyes with overlapping excitation bands was hardly possible in non-META systems.

Now the Zeiss ACE software is also available for the channel based LSM systems and solves the problem of excitation crosstalk in multi-labelling experiments. Crosstalk No crosstalk

Æ Additional advantage: faster acquisition (single- instead of multitrack)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Channel unmixing – Wide Field II

FluoCells-Widefield

raw data

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Channel unmixing – Wide Field II

FluoCells-Widefield- unmixed

Unmixed with MultiChannel ACE

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging The new way of detection in the scan head

Based on the proven 510 concept META detector replaces one Single Detector PMT array with 32 elements Optical Grating for even dispersion Capture full emission spectra Fast electronic selection of PMT elements Truly confocal Adjustable pinhole (x,y, Ø) Field upgradeable

20.04.2004 -20- LSM 510 in Live Cell Imaging Emissipon Fingerprinting

Lambda Stack Acquisition Separated Channel Image

Linear Unmxing

Extraction of Reference Spectra

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 5

Sytox Green and FITC in cultured fibroblasts

Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 5

Sytox Green and FITC in cultured fibroblasts

FITC Sytox Green overlay

Sample: Mary Dickinson, PhD, Caltech, Pasadena, USA

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 6

CFP, CGFP, GFP and YFP Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively

Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Emission Fingerprinting - Example 6

CFP, CGFP, GFP and YFP Cultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively

CFP CGFP

GFP YFP

Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging

Emission Fingerprinting - Example 7

Separating Alexa532 and Cy3 from autofluorescence background in Drosophila Retina

Sample: Dr. Zimmermann, University Potsdam, Germany

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Excitation Fingerprinting

A lambda stack will be acquired. The Data Button displays the wavelengths used for excitation of each image.

Note: The Reuse Button must not be used, it can crash the SW. The Info Button is inactive.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Examples for Excitation Fingerprinting

Emission Fingerprinting Excitation Fingerprinting

Drosophila retina whole mount stained for actin with Alexa 568-Phalloidin (green); Autofluorescence (red)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Online Fingerprinting

Linear Unmixing of Lambda Stacks during image acquisition

Select Reference Spectra from Spectra Database Define spectral range of Lambda Stacks Start scanning Lambda Stacks are neither displayed nor saved Benefits: - visual analysis of results in z-stacks and time series experiments. - Reduction of data (xyzt-λ)

t = 0 t = 20 s t = 60 s

Example: Simultaneous monitoring of Ca2+ with Indo-1 and translocation of PKCa_YFP after histamine stimulation in HEK 293 cells

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Options for Emission Fingerprinting

Generate Channel with Residuals

- Generates an additional „Channel with Residuals“ -> pixel-by-pixel display the difference between fit and original data (for the channel of the Lambda Stack that shows the greatest deviation)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging

Binning in the META detector

no binning (10.7 nm)

0 350 0 300

500

2 channel binning (21.4 nm) y 2

i s

n 00

e 0

t 2 n I 0 150 0 100 500 0

3 channel binning (32.1 nm) M in 4 E b T 5 A 3 5 0 bin 2 0 d 5 0 5 4 e 2 6 0 te in 5 0 ] b 6 8 c 0 0 m t 6 0 [n o 2 h r in 6 0 ht b 4 g s no 6 0 n e 6 le 0 e tt av in w 4 channel binning (42.8 nm) g

Spectral resolution Sensitivity

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRET Analysis in Laser Scanning Microscopy

What is FRET ?

FRET (fluorescence resonance energy transfer) is the non-radiative transfer of photon energy from an excited fluorophore (the donor) to another fluorophore (the acceptor) when both are located within close proximity (1-10 nm).

FRET applications • Protein/protein interactions • Detection of conformational changes • Specialized FRET tools like yellow Chameleon for Ca++ Imaging

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Preconditions for FRET Analysis

Appropriate FRET pair with Specific staining of the Parallel orientation of the overlap between donor molecule (protein) of axis of interacting dye emission and acceptor interest molecules excitation

• EBFP & EGFP FRETz • ECFP & EYFP • EGFP & Rhodamine • FITC & Rhodamine •FITC & CY3

CFP Em YFP Ex

FRETy

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Quantitative Analysis using Filter FRET

Track 1 Excitation of CFP Detection of CFP

Excitation of YFP Detection of YFP

Excitation of CFP Detection of YFP ¾ FRET signal (not corrected)

CFP only YFP only CFP and YFP doing FRET

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Sensitized Emission – Calculation of Fc

capital letter : Specimen ⎡ Df ⎤ ⎡ Af ⎤ Fa Fc = Ff − ⎢ ⋅ Fd⎥ − ⎢ ⋅ Fa⎥ small letter : Filter set ⎣ Dd ⎦ ⎣ Aa ⎦

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Sensitized Emission – The 3 Methods

Method 1: Fc (FRETcorrected) D.C. Youvan et al. 1997

Fc is corrected for donor and acceptor contribution to the signal measured with the FRET filter set. Fc is not normalized for the donor acceptor concentration. High Fc numbers occur were high concentration of donor and acceptor are present.

⎡ Df ⎤ ⎡ Af ⎤ Fc = Ff − ⋅ Fd − ⋅ Fa ⎣⎢ Dd ⎦⎥ ⎣⎢ Aa ⎦⎥

Fc = Ff −[Donor corr.]−[Acc. corr.]

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Sensitized Emission – The 3 Methods

Method 2: Fn (FRET net) G.W. Gordon et al. 1998

Fn is corrected for donor and acceptor contribution to the signal measured with the FRET filter set as Fc. Fn is given as Fc divided by the multiplied concentrations of donor and acceptor. This emphasize FRET occurring at low concentrations of donor and acceptor.

Ff −[Donor corr.]− [Acc. corr.] Fn = G ⋅ Fd ⋅ Fa

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Sensitized Emission – The 3 Methods

Method 3: NF (normalized FRET) X. Xia et al. 2001

NF is corrected for donor and acceptor contribution to the signal measured with the FRET filter set as Fc. NF is given as Fc divided by the square root of the multiplied concentrations of donor and acceptor. This results in FRET values normalized for donor and acceptor concentration.

Ff −[Donor corr.]− [Acc.corr.] NF = G ⋅ Fd ⋅ Fa

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Quantitative FRET Analysis using Acceptor Bleach

Principle • Some donor (CFP) signal is transferred (FRET) to the acceptor (YFP) • The acceptor is bleached (chemically destroyed) • The donor signal increases (up to 30%) since no energy transfer to the acceptor is possible.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Quantitative FRET Analysis using Acceptor Bleach

100% YFP

∆ CFP in%

100% CFP X% YFP

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Quantitative FRET Analysis using Acceptor Bleach

Experimental Conditions ∆ CFPmax at total YFP bleaching • Use non bleaching laser intensities of 458 and 514nm for CFP and YFP imaging • Bleach YFP from 100 to 10% with 100% power of 514nm laser line • Apply linear regression analyses to yield values for CFP intensities without acceptor

(FCFP-max at YFP = 0) FCFP − max − FCFP − min E = = 30 % • Lit.: H.Amiri et al. in Cell FCFP − max Calcium (2003)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP and FLIP

What is FRAP?

FRAP (fluorescence recovery after photobleaching) is the recovery of fluorescence ater bleaching of an defined region due to fluorophores that move from the surrounding into the bleached area.

What is it good for?

FRAP is used to measure the dynamics of 2D or 3D molecular mobility e.g. diffusion, transport or any other kind of movement of fluorescent labeled molecules in membranes or in living cells.

What is FLIP?

FLIP (fluorescence loss in photobleaching is almost the same as FRAP with the only difference that bleached area is different from the area used for bleaching. In addition to that bleaching is applied not only once but repetitively. FLIP is also used to studying cellular dynamics.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP and FLIP – the Principles FLIP FRAP

1. The molecule of interest is labeled 1. The molecule of interest is labeled 2. Non bleaching laser intensities are used for 2. Non bleaching laser intensities are used for time lapse imaging time lapse imaging 3. A maximum laser dosage is applied in a 3. A maximum laser dosage is applied pixel precise defined Region Of Interest to repetitively in a pixel precise defined Region destroy the fluorescent dye (bleaching) Of Interest to destroy the fluorescent dye 4. After bleaching the time series is (bleaching) immediately at maximum speed 4. Between the bleaching cycles images are 5. The behavior of the recovery of fluorescence obtained from regions differing from the in the bleached area is a measure for bleach region mobility of the labeled molecule 5. The behavior of the loss of fluorescence in the observed areas is a measure for mobility of the labeled molecule

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Quantitative Imaging

Parameters obtained: • mobile fraction of fluorescent molecules • rate of mobility

Note: The speed of bleaching and measurement is absolutely critical for FRAP measurements in particular in rapidly moving systems.

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Definition of Bleach Area

Note: The laser beam will be turned off outside the bleach ROIs (AOTF) The pixel time is set by scan speed

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Definition of Bleach Procedure

setting of bleaching parameters for spot, line or frame bleaching

Bleach after number of scans and Bleach repeated after number of scans If activated, then bleaching procedure is automatically integrated in to a time series Example: - 5 control images - bleach routine - repeat bleach after 2 scans Note: The total number of imaging scans is set in the Time Series Control window

Different Z position: defines the current stage position as the position for bleach (useful bleaching with two photon lasers)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Quantitative FRET Analysis using Acceptor Bleach

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Time Series and Mean of ROI

• The bleach routine is automatically included in a time series experiment by pushing the Start B Button • Online mean of ROI is important to determine the end of fluorescence recovery

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Example 1

GFP-expressing myoblast cell line

ROI 2

ROI 2 ROI 1 ROI 1

A. Sporbert, MDC Berlin, Germany

Experiment Pixel-precise bleaching of the nucleus followed by slow GFP flow-back from the cytoplasm (note that the overall concentration of fluorescent GFP is reduced due to the bleaching event). Result GFP can pass the nuclear membrane (the nuclear pore complex).

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Combination with Emission Fingerprinting I

1 2

1. Obtain Reference Spectra from single labeled control

2. Acquisition of the complete emission spectrum (Lambda Stack: x([yzt]-l)

3. Linear Unmixing separates the emission signals 3

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Combination with Emission Fingerprinting II

Dual colour FRAP monitoring GFP and YFP using Emission Fingerprinting

GFP YFP overlay

Living cultured cells expressing GFP and a YFP-SHP fusion protein

F. Boehmer, Friedrich Schiller University Jena, Germany

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging FRAP – Combination with Emission Fingerprinting III

Sample: Dr. F. Boehmer, Friedrich Schiller University Jena, Germany ROI 1

ROI 2

ROI 1

ROI 2 Experiment: Pixel-precise bleaching of GFP and YFP in ROI 1only (note that the overall concentration of fluorescent FPs is reduced due to the bleaching event).

Result: Slow flow-back of GFP from the cytoplasm and adjacent nucleus into the bleached nucleus; free GFP passes the nuclear membrane

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Two new Members of the Family of Fluorescent Proteins

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Photoactivatable GFP (PA-GFP)

• Invented 2002 by George Patterson NIH Bethesda, USA 1 • Directed Mutation of Wt-GFP • Green Fl Increase after activation: 100-fold 2

Applications • Investigation of Cellular Dynamics • Developmental Biology

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Kaede – a Photoconvertable Fluorescent Protein

• Kaede (Japanese: Maple Leaf) invented 2002 by Atsushi Miyawaki , Riken Japan • Conversion from bright green ( ex. 488 nm ) to bright red (ex. 543 nm) via 405 nm laser irradiation (UV light) • Change in red/green ratio up to 2000 fold due to conversion • Conversion sensitivity is pH dependent, very high stability of the red form (Tetramer 4.33 x 26.75 kDa = 116.0 kDa, free diffusible:29 µm2/s in cytosol)

free KAEDE protein expressed in HeLa cells; Sample: Prof. Myawaki, EMBO course, Kobe, JAPAN, Nov. 2002

Stony Coral: • An excellent switchable marker for cells open brain • With LSM 510 META 405 nm pixel precise coral conversion can be performed (Trachyphyllia • With Emission Fingerprinting any red/green ratio geoffreyi) can be used as an unique marker

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Kaede: stepwise Photoconversion

Spectral Signatures...... Linear Unmixing of x,y,λ, t series

t=0s

t=90s

• Complete detection of emission shifts via Lambda Stack acquisition • Even and efficient conversion of KAEDE via repetitive 405 nm pulses • Very low power settings are efficient • Due to very high coupling efficency of the 405 nm diode laser into the LSM

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Define any red/green ratio as a new reference spectrum

• Any ratio between the two states of Kaede Reference spectra (green/red) can be used as a unique marker • Use the ratio as the reference for unmixing (online and offline Fingerprinting)

30% Conversion 50% Conversion 70% Conversion

Ch 1 (e.g green) Ch 2 (e.g red) Ch 3 (e.g blue)

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Zeiss Features for Live Cell Imaging

• Max. 5 frames/s at full flexibility of scan field geometry and orientation

• Bi-directional scan, line skip, spline scan

• Fast image acquisition with Axiocam integrated into LSM software

• No moving parts for lambda stack acquisition

• 8 channel parallel data acquisition

• Online Fingerprinting

• Time series integrated bleach routine

• Repetitive bleaching

• Bleaching at diff. Z position (NLO)

• Multiple time series

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004 LSM 510 in Live Cell Imaging Conlusion

Having the right equipment - live cell imaging can by a colorful adventure!

Specimen: cell culture, CFP/GFP/YFP labeled histone B2 Imaging: 16h time series acquisition of lambda / z stack Image processing: Linear Unmixing, maximum projection Curtsey Saiwaki San, Japan

Dr. Jörg Lindenau Training, Application and Support Center -TASC- 20.04.2004