Carl Zeiss MicroImaging, LLC.

Spectral Unmixing for

LSM 710 in ZEN 2009 A Carl Zeiss How-To Guide

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This document does not contain or represent a warranty by Carl Zeiss with regard to the technical

processes described in the documentation or to certain reproduced product characteristics. Furthermore,

Carl Zeiss shall not be held liable for any possible errors, omissions or other inaccuracies in this

documentation. This document only contains a general description of the technical processes and

information, the implementation of which in any individual case may not be appropriate in the form

described here. In cases of doubt, we recommend contacting Carl Zeiss Applications Support. Carl Zeiss

draws attention to the fact that the information and references contained in these documents may be

subject to technical modifications, in particular due to the continuous further development of Carl Zeiss's

products.

Carl Zeiss MicroImaging, LLC.

Applications Support

One Zeiss Drive

Thornwood, NY 10594

Telephone: 800-509-3905 E-Mail: support@zeiss.com

www.zeiss.com/support

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Table of contents Introduction ............................................................................................................................................ 4

Step 1: Acquire Spectral Data ................................................................................................................. 4

Step 2: Adjust Acquisition Settings for Balanced Image......................................................................... 5

Step 3: Define Spectra to use for Unmixing ............................................................................................ 5

1. Automatic Component Extraction (ACE) ...................................................................................... 5

2. Individual measurements with biological controls ......................................................................... 8

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Introduction

Zeiss LSM 700, 710 or 780 confocal systems allow spectral data can be acquired and

processed in the ZEN software. This data can be used to separate multiple fluorophores with a

high degree of overlap in emission spectra. This manual illustrates how to setup, acquire and

unmix spectral data on your Zeiss LSM using ZEN 2009.

Step 1: Acquire Spectral Data

In order to collect high resolution spectral data in ZEN make sure that lambda mode is used for data

collection. In the light path menu make sure that the middle button “Lambda Mode” is depressed.

Running the system in lambda mode allows for the collection of spectral data over a user defined

wavelength range as well as a user defined spectral resolution.

1. Select “Lambda Mode” in Light Path menu

2. Select spectral collection range

3. Define spectral resolution

a. For simultaneous acquisition use resolution of 9.7 nm or greater (LSM 710 and 780 only)

b. All spectral acquisition on the LSM 700 is sequential

4. Select lasers with appropriate MBS

1. 1.

2.

3.

4.

2.

3.

4.

LSM 710 and 780 LSM 700

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Step 2: Adjust Acquisition Settings for Balanced Image

Use the gallery view of the lambda image to adjust the detector gain, offset, scan speed and laser powers

to create well balanced image. It is important to eliminate any saturated and under exposed pixels in the

image to ensure proper unmixing.

Select range indicator palette to view underexposed pixels (in blue) and saturated pixels (in red). Adjust

imaging parameters to rid image of blue and red pixels.

Step 3: Define Spectra to use for Unmixing When performing an unmixing experiment, a reference spectrum from each individual fluorophore is

needed. There are two different ways to acquire the reference spectrum in ZEN:

1. Automatic Component Extraction (ACE)

2. Individual measurements with biological controls

1. Automatic Component Extraction (ACE)

The automatic component extraction function, ACE, will automatically try to detect the individual

spectrum of each component in the image. This function will only work if each component is not

completely co-localized with any other components (i.e. there are pixels in the image with only one

fluorophore present). After the components have been automatically identified with ACE, the image may

be unmixed.

1. Go to the Unmixing tab on the left side of the image container

2. Select the Auto find/ACE button

3. Select number of fluorophores in the sample

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4. Identify background ROI using selection tools if needed

5. Unmix image

1.

2.

3.

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4.

5.

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The resulting unmixed image will be created in a new image tab with the suffix “_unmixed”. The

unmixed Image contains a channel for each component with a reference spectrum

2. Individual measurements with biological controls

In some cases, creating biological controls with only one fluorophore is necessary to generate a reference

spectrum for that fluorophore. Typically this is required when the fluorophores in the sample have a high

degree of spectral overlap (i.e., GFP and Alexa488) and if the sample has a high degree of spatial

colocalization between the different fluorophores. In this scenario, the individual reference spectrum

must be collected and saved for use with the data of interest. As with ACE, it is important to acquire data

without any underexposed or saturated pixels.

Note: It is important that the reference spectrum and data are collected over the same wavelength range.

It is also important to keep the imaging configuration the same for both the reference sample and the

experimental sample. In other words, laser lines, beam splitters, detection range and objective lens should

not be changed when acquiring spectra. Optics such as the MBS and/or emission filters can affect the

Unmixed image is created in new

image tab

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shape of the collected spectral data. The objective lens should also be kept the same as different lenses

have different transmission properties for some areas of the light spectrum.

Image sample with single reference fluorophore

1. Go to the unmixing tab on the left side of the image container

2. Select an ROI shape from unmixing control tab below image

3. Save reference spectra

Repeat steps 1-3 until all reference spectrums have been collected

Image experiment

4. Load reference spectrum

5. Identify background ROI using selection tools if needed

6. Unmix image

1. 2.

3.

10

3.

4.

11

5.

5.

6.